Effect of black raspberry seed oil on inflammation and lipid metabolism in high-fat diet-induced obese mice and db/db mice

이희재 Seoul National University 2016 국내박사

Increased interest in beneficial health effects of raspberries has raised the production of raspberries in Korea in recent years. The processing of black raspberry fruits for juice, wine, and puree typically produces the seeds as a byproduct. Black raspberry seed (BRS) oil, which can be obtained from black raspberry product pomace, contains about 30% α-linolenic acid (ALA), an n-3 fatty acid, and other beneficial phytochemicals such as tocopherols. The ultimate objective of the study was to evaluate whether BRS oil has beneficial effects on inflammatory status and lipid metabolism in high-fat diet-induced obese mice and db/db (type 2 diabetes) mice. The objective of the first part of the study was to determine the effect of BRS oil on inflammation and lipid metabolism in high-fat diet-induced obese mice. Five-week old C57BL/6 mice were divided into two groups: 1) mice fed high-fat diet consisting of 50% calories from lard, 5% from soybean oil, and 5% from corn oil (control group), and 2) mice fed high-fat diet consisting of 50% calories from lard and 10% from BRS oil (BRS oil group). The BRS oil used in the study comprised 57.0% linoleic, 29.4% α-linolenic, and 9.80% oleic acids. Mice were fed the experimental diets for 12 weeks ad libitum. The content of ALA was significantly (P<0.001) higher in the BRS oil diet than the control diet. There were no significant (P>0.05) differences in initial body weight, final body weight, weight gain, food intake, and food efficiency between the two groups. The weights of liver, spleen, kidney, and epididymal adipose tissue of the BRS oil group were higher than those of the control group with no significant (P>0.05) differences. After 12 weeks, ALA in the liver of the BRS oil group accounted for 1.84% of the total fatty acids, which was significantly (p<0.01) higher than that of the control group (0.45%). Inflammation-involved proteins such as TLR4, NF-κB, phospho-NF-κB, COX2, I-κBα, and phospho-I-κBα were lower in the liver of the mice fed the BRS oil than those in the control. mRNA levels of pro-inflammatory markers including NF-κB, TNFα, IL-1β, IL-6, iNOS, COX2, and MCP1 in the liver and epipidymal adipose tissue of the BRS oil group were lower than those of the control. On the other hand, mRNA levels of anti-inflammatory markers including IL-10, arginase1, Chi3l3, and Mgl1 were higher in the liver and epipidymal adipose tissue of the BRS oil group than those of the control. Leptin level in serum was lower in the mice fed BRS oil diet than in the control diet without significant (P>0.05) difference. mRNA level of leptin was lower in the epididymal adipose tissue of the mice fed BRS oil than in the control without significant (P>0.05) difference. mRNA level of adiponectin was significantly (P<0.01) higher in the epididymal adipose tissue of the BRS oil group than in the control. Whereas, adiponectin level in serum was higher in the mice fed BRS oil diet than in the control diet with no significant (P>0.05) difference. Levels of TG in serum and liver of the mice fed BRS oil were 14.2% and 12.1%, respectively, lower than those of the control group. Serum non-esterified fatty acids (NEFA) and total cholesterol levels were 42.1% and 13.0%, respectively, lower (P<0.05) in the mice fed BRS oil diet than in the control. Serum HDL-C level was 4.10% higher in the BRS oil group than in the control group without significant (P>0.05) difference. Total lipid content in the liver of the BRS oil group was 13% lower than that of the control group without significant (P>0.05) difference. NEFA and total cholesterol levels in the liver of the BRS oil group were 25.7% (P<0.05) and 53.2% (P<0.001), respectively, lower than those of the control group. The mRNA levels of lipogenic markers such as CD36, FABP1, SREBP-1c, FAS, and SLC25A1 were lower in the liver of the BRS oil group than those of the control group. Whereas, mRNA levels of fatty acid oxidation markers including CPT1A, ACADL, HADHα, and ACOX were higher in the liver of the mice fed BRS oil than in the control mice. PPARα significantly increased both in mRNA (P<0.001) and protein (P<0.01) levels in the liver of the mice fed BRS oil diet compared with the control group. However, PPARγ mRNA and protein levels showed no significant (P>0.05) differences between the two groups. PPARα mRNA level was significantly (P<0.05) higher in the epididymal adipose tissue of the mice fed BRS oil than in the control. However, there were no significant (P>0.05) differences in PPARα protein and PPARγ mRNA and protein expression levels between the two groups. The objective of the second part of the study was to evaluate the status of the markers related to inflammation and lipid metabolism in db/db mice fed diets containing different concentrations of BRS oil. Mice were divided into four groups: 1) C57BL/6 mice fed 16% calories from soybean oil (normal CON); 2) C57BL/KsJ-db/db mice fed 16% calories from soybean oil (CON); 3) C57BL/KsJ-db/db mice fed 8% calories from soybean and 8% calories from BRS oil (BRS 50%); and 4) C57BL/KsJ-db/db mice fed 16% calories from BRS oil (BRS 100%). Mice were fed the experimental diets for 10 weeks ad libitum. There were no significant (P>0.05) differences in the food intake and initial body weight among the three db/db groups. The food intake and body weight of the normal CON were significantly (P<0.05) lower than those of the db/db groups. The final body weight of the BRS 50% was significantly (P<0.05) lower than that of the CON. Final body weight of the BRS 100% was lower than that of the CON without significant (P>0.05) difference. The weights of the liver, epididymal adipose tissue, spleen, and kidney were not significantly (P>0.05) different among the db/db mice. There was no significant (P>0.05) difference in blood glucose level among the db/db mice. Insulin level was significantly (P<0.05) lower in the serum of the BRS 50% and BRS 100% than in the CON. After 10 weeks, n-6 to n-3 fatty acid ratios were significantly (P<0.05) lower in the livers and epididymal adipose tissues of the BRS 50% and BRS 100% mice than in the CON. Whereas, ALA and total n-3 fatty acids contents were significantly (P<0.05) higher in the livers and epididymal adipose tissues of the BRS oil-treated mice than in the normal CON and CON. Serum TNFα and IL-6 were significantly (P<0.05) lower in the BRS 50% and BRS 100% than in the CON. Serum IL-10 was significantly (P < 0.05) higher in the BRS 100% than in the CON. Protein expression levels involved in inflammation such as TLR4, NF-κB, and COX2 were lower in the epididymal adipose tissues of the BRS 50% and BRS 100% than those of the CON. In the liver and epididymal adipose tissue, mRNA levels of pro-inflammatory markers including TLR4, TNFα, IL-1β, IL-6, iNOS, COX2, MCP1, and CCR2 in the BRS 50% and BRS 100% were lower than in the CON. On the other hand, anti-inflammatory markers including IL-10, arginase1, Chi3l3, and Mgl1 were higher in the epididymal adipose tissues of the BRS 50% and BRS 100% than in the CON. In the epididymal adipose tissue, macrophage infiltration markers (F4/80 and CD68) and leptin mRNA were significantly (P<0.05) lower in the BRS 50% and BRS 100% than in the CON. Levels of TG, NEFA, and total cholesterol in the serum were significantly (P<0.05) lower in the BRS oil-treated groups than in the CON. Serum HDL-cholesterol level was significantly (P<0.05) higher in the BRS 50% than in the CON. Levels of total lipid, TG, NEFA, and total cholesterol in the liver were significantly (P<0.05) lower in the BRS oil-treated groups than in the CON. The mRNA levels of lipogenesis markers including CD36, FABP1, SREBP-1c, FAS, and SLC25A1 in the livers of the BRS oil groups were significantly (p<0.05) lower than in the CON. On the other hand, fatty acid oxidation markers were significantly (CPT1A and ACOX; P<0.05) or without significance (ACADL and HADHα; P>0.05) higher in the BRS oil groups than in the CON. PPARα mRNA level was significantly (P<0.05) higher in the liver of the mice fed BRS 50% diet than in the CON, whereas PPARα protein expression was significantly (P<0.05) higher in the liver of the mice fed BRS 100% diet than in the CON. PPARγ mRNA level was significantly (P<0.05) lower in the liver of the mice fed BRS 50% diet than in the CON, whereas PPARγ protein expression was significantly (P<0.05) lower in the liver of the mice fed BRS 100% diet than in the CON. These results are in agreement with the PPARα results. PPARα mRNA level was significantly (P<0.05) higher in the epididymal adipose tissue of the BRS 50% and BRS 100% than in the normal CON and CON. PPARα protein was significantly (P<0.05) higher in the epididymal adipose tissue of the BRS 100% than in the normal CON and CON. There were no significant (P>005) differences in PPARγ mRNA levels between the BRS oil groups and CON. To detect the effect of BRS oil on hepatic lipid accumulation, Oil Red O staining was performed. The Oil Red O staining showed that the lipid droplets in the livers of the BRS oil-treated mice were smaller and fewer than those of the CON, indicating reduced hepatic lipid accumulation in the BRS oil groups. This study demonstrated that dietary BRS oil treatment reduced pro-inflammatory markers and promoted anti-inflammatory markers in liver and epididymal adipose tissue of high-fat diet-induced obese mice and db/db mice. BRS oil also lowered lipogenesis markers and raised fatty acid oxidation markers in the livers of high-fat diet-induced obese mice and db/db mice. Results of this study suggest that BRS oil may be a good source of ALA, an n-3 fatty acid, with anti-inflammatory effects on high-fat diet-induced obese mice and obese diabetic mice by ameliorating inflammatory responses. Also, BRS oil may improve lipid metabolism by inhibiting lipogenesis and promoting fatty acid oxidation in high-fat diet-induced obese and db/db mice. These results would promote utilization of BRS, a byproduct of beverage processing of black raspberries, and ultimately help black raspberry producers and manufacturers.

Dual Roles of Nrf2 in Adaptive Survival Response to Oxidative and Inflammatory Stresses in Normal vs Cancer Cells

Hee Geum Lee 서울대학교 대학원 2014 국내박사

Inflammation has been implicated in multi-stage carcinogenesis. In effect of helping chronic inflammation to develop an incipient neoplasia is contributed with largely of the innate immune system which is demonstrated functionally important in inflammation status. Prostaglandins play a key role in inflammation response in inflammation-associated carcinogenesis. COX-2-derived prostaglandins are highly elevated in inflamed tissue, and it provokes cardinal signs of acute inflammation, proliferation, apoptosis, immunity, angiogenesis, invasion, metastasis and surveillance. Intratumoral levels of PGE2 depend not only upon the rate of production but also on the rate of its degradation. The nicotinamide adenine dinucleotide positive-dependent catabolic enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) metabolizes PGE2 to the biologically inactive 15-keto-prostaglandins. To take account of strong link between inflammation and its associated carcinogenesis, it is important to control the 15-PGDH expression in inflammation status. In present study, we show that the regulation of COX-2 on 15-PGDH expression both in colitis and in its-related carcinogenesis. To consider the logic of 15-PGDH as a function of prostaglandins degradation, it should be happened more severe inflammation in 15-PGDH knockout conditions. Contrary to our expectation, there were less inflammatory protein expression and similar pathological appearances in 15-PGDH knockout inflamed tissues compared with wild type one on mouse colitis and gastritis model. From these data, we hypothesized that accumulated prostaglandins in 15-PGDH knockout conditions may conduct an adaptive response and be ready for protect the tissues or cells from exogenous stimulus. As a cellular deference mechanism against various exogenous stresses, certain molecules are involved in its adaptive response. Among them, nuclear factor-erythroid 2p45 (NF-E2)-related factor (Nrf2), a master regulator of the basal and inducible expression of diverse cytoprotective proteins is mainly important on cellular adaptive response. Nrf2 and its target protein HO-1 were highly elevated in chemical- or bacterial-induced inflammation tissues on 15-PGDH knockout mouse than wild type mouse. These findings suggest that Nrf2/HO-1 upregulation was resulted from accumulated prostaglandins in blocking of degrading pathway and this signal transduction was prepared to respond to the oxidative stress and capable of coping with it by inducing an adaptive response to maintain homeostasis or even to stimulate ourselves to promote a healthy state. Multiple lines of evidence support those Helicobacter pylori infection is one of the primary causes of gastritis and peptic or duodenal ulcer, which are provoked by oxidative stress(Hahm, Kim et al. 2003). In spite of the intimate relationship between Helicobacter pylori infection and gastritis (or its related gastric carcinogenesis), not all infected individuals developed clinical diseases(Uemura, Okamoto et al. 2001). This is a report about 10 year’s follow-up of Helicobacter pylori-infected individuals (Uemura, Okamoto et al. 2001), and according to the results of this seminal paper, only 2.9% of Helicobacter pylori positive patients developed the gastric cancer, while the remaining subjects were asymptomatic. The other 97% of individuals did not developed gastric cancer. It reflects some of defense mechanism under Helicobacter pylori infected condition, and several host and environmental determinants are important in Helicobacter pylori-related disease. Helicobacter pylori infection induced Nrf2 and concomitant HO-1 expression in both of cells and animals. And Helicobacter pylori also stimulated nuclear accumulation of Nrf2, as well as its interaction with antioxidant response elements located in the PIAS2 promoter and regulating degradation of STAT3 signaling. PIAS2 is one of STAT3 negative regulator, E3 SUMO-protein ligase, binding to STAT3 dimer and leading its degradation. PIAS2 expression was also lowered in Nrf2 knockout MEF cells, lending further support to Nrf2-dependent PIAS2 expression. To evaluate the protective role of Nrf2 in Helicobacter pylori induced gastritis in mice, nrf2 knockout mice were utilized. Helicobacter pylori induced gastritis was much severe in nrf2 knockout mice compared with its wild type littermate. The Nrf2 and HO-1 expression levels were coordinately increased in Helicobacter pylori infected human stomach. These observations suggest that Helicobacter pylori infection can trigger the cellular defense mechanism through Nrf2-mediated upregulation of PIAS2, and consecutively related with STAT3 degradation. Although a wide body of evidence indicates that activation of Nrf2 protects against a variety of toxicants and diseases, the prolonged activation of Nrf2 has been shown to favor the progression of several types of cancers. Nrf2 has been shown to be constitutively elevated in lung, breast, head and neck, ovarian, and endometrial carcinomas. Abnormally enhanced Nrf2 ability resulted cancer cell proliferation and promote chemoresistance and radioresistance. Survival of nearly all types of tumor cells depend on glycolysis, which proceeds at a much higher rate in tumor cells than that in the noncancerous tissues. Thus, tumor cells are exposed frequently and maybe constantly to glucose deprived conditions. In particular, the microenvironments of solid tumor tissues are often confronted with unfavorable conditions such as hypoxia and hypoglycemia(Warburg 1956, Weber 1977, Weber 1977). Thus, cancer cells tend to develop self-defense mechanisms to survive such adverse conditions. Although prolonged glucose deprivation induced apoptotic cell death in HepG2 cells, there was upregulated HO-1 expression before apoptosis manifested. We found that the induction of Nrf2-mediated HO-1 upregulation in glucose deprived HepG2 cells confers an adaptive survival response before the manifestation of cell death. Taken together, these observations suggest that the Nrf2 might protect the cells and tissues from Helicobacter pylori-induced gastritis or DSS-treated colitis, particularly regulating inflammatory environment through inhibiting the IL6/STAT3 activated signaling. While activated Nrf2 in cancer cells obtained the survival response under hypoglycemia. Thus, better understanding of the mechanisms by which inflammation mediators cause Nrf2 activation and induce HO-1 expression will be useful in the development of novel therapeutic strategies for effective treatment or prevention of cancer.

The anti-inflammatory effect of zerumbone on THP-1 cell-derived macrophages

김민주 전남대학교 2018 국내석사

Inflammation plays an important role in causing human disease by a body defense mechanism caused by various external stimuli such as diabetes, cancer, asthma, rheumatoid arthritis and psoriasis. Toll-like receptors (TLRs) are pattern-recognition receptors (PRRs) that are expressed on the surface of innate immunity cells, including antigen-sensing cells. TLR activates antigen-presenting cells by activating genes that produce inflammatory cytokines, chemokines, and by stimulating the production of key co-stimulatory molecules in T lymphocyte activation by binding to the pathogen-associated molecular pattern (PAMP) of invading microorganisms. Thus, TLRs play an important role in innate immunity and adaptive immunity by activating T lymphocytes. Lipopolysaccharide (LPS) is a constituent of the cell wall of gram-negative bacteria and is known to be a factor that triggers the inflammatory response by macrophages. LPS stimulates toll-like receptor 4 (TLR4) in macrophages or monocytes and promotes secretion of pro-inflammatory molecules including nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukins (ILs). These inflammatory mediators are known to cause fatal consequences to the host if acute or excessive reactions occur, and they play an important role in biological defense by producing NO and cytokines in the early stages of inflammation. Macrophages have been used as in vitro models for evaluating anti-inflammatory effects. Zerumbone is a major component of the essential oil of Zingiber zerumbet Smith, a kind of wild ginger. There have been many studies on cancer, inflammatory diseases, and reduction of free radicals, including hematologic malignancies. It is also known to have effects on the proliferation of immune cells, cell cycle progression, cytokine production and expression. However, the specific mechanism of anti-inflammatory effects of zerumbone in differentiated human monocyte THP-1 cells were not fully understood. In this study, THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA, 1 μM) for 48 h to differentiated into macrophages. Differentiated THP-1 cells were cultured for 48 h in the presence or absence of zerumbone (1-10 μM) and then treated with 500 ng/mL LPS for 6 h before harvesting. In this study, we showed that zerumbone inhibits the secretion of cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), tumor necrosis factor-α and the induction of nuclear factor-𝛋B (NF-𝛋B) under LPS-induced inflammation of THP-1 cell-derived macrophages. Zerumbone treatment significantly also inhibited Toll-like receptor 2/4 protein and mRNA expression and MyD88 gene expression under LPS-induced inflammation of THP-1 cell-derived macrophages. Histone deacetylase (HDAC) removes acetyl groups from lysine residues in transcription factors and histones. Therefore we investigated HDAC3 and SIRT1 activity in the presence or absence of zerumbone (1-10 μM) under LPS-induced inflammation of THP-1 cell-derived macrophages using by enzyme-linked immunosorbent assays, Real-time PCR, Western blotting and immunofluorescence analysis. LPS only treatment reduced HDAC3 expression under LPS-induced inflammation of THP-1 cell-derived macrophages. However, zerumbone treatment increased SIRT1 and inhibited HDAC3 under LPS-induced inflammation of THP-1 cell-derived macrophages. H3K9ac, H3K27ac and H3K4me2, which are regulated by histone acetylation and metylation enzymes, has an open chromatin structure that activates gene expression. LPS only treatment upregulated H3K9ac, H3K27ac and H3K4me2 under LPS-induced inflammation of THP-1 cell-derived macrophages. However upregulated H3K9ac, H3K27ac and H3K4me2 were decreased by zerumbone treatment under LPS-induced inflammation of THP-1 cell-derived macrophages. Taken together, zerumbone is a phytochemical useful for the treatment and prevention of chronic inflammatory diseases.

뇌 손상 동물모델에서의 신경교세포와 혈액 염증세포들의 반응 양상과 역할에 대한 연구

정혜경 The Graduate School, Ajou University 2011 국내석사

Brain inflammation is accompanied by brain injury. For several decades, brain inflammation has been considered toxic to the brain. However, recent studies also suggest beneficial roles of inflammation in injured brain. In this study, I analyzed patterns and roles of brain inflammation in injured brain in vivo from the early stage of the injury to have answers to the question whether brain inflammation is harmful and beneficial. In the first part of this study, acute neuronal damage was induced by stereotaxic injection of ATP into the substantia nigra pars compacta (SNpc) and the cortex of the rat brain. ATP acutely caused death of microglia as well as neurons in a similar area within 3 h, and monocytes filled the damaged core after neurons and microglia died. Neither activated microglia nor monocytes expressed iNOS, a major neurotoxic inflammatory mediator. Monocytes rather expressed CD68, a marker of phagocytic activity. Importantly, the total number of dopaminergic neurons in the SNpc at 3 h (~80% of that in the contralateral side) did not decrease further at 7 d. Similarly, in the cortex, ATP-induced neuron-damage area detected at 3 h did not increase for up to 7 d. Thus, the inflammatory responses of microglia and monocytes in response to ATP-induced acute injury may not be neurotoxic. In the second part, I examined behavior and roles of infiltrated monocytes in LPS-injected SNpc. Monocytes vigorously infiltrated at 3 d and thereafter gradually decreased. Some of infiltrated monocytes died, and the remaining monocytes changed morphologically from round to ramified ones. Monocytes exert alternatively activated phenotypes: strong expression of phagocytic activity but negligible expression of pro-inflammatory mediators such as TNF-α, IL-1β and iNOS. Accordingly, recovery of impaired astrocytes, endothelial cells, and neuronal cells occurred after monocytes infiltrated. These results indicate that monocytes play important roles to repair of the injured brain. In the third part, it has been examined how systemic inflammation (SI) affected brain inflammation. After the induction of SI, in the SN region, microglia were morphologically activated, neutrophils infiltrated, and mRNA/protein expression of inflammatory mediators increased within 4-8 h, and subsided within 1-3 days. However, dopaminergic neuronal loss was not detectable for up to 8 d after induction of SI. These results indicate that acute systemic inflammation causes brain inflammation, but this is not sufficiently toxic to induce neuronal injury. Taken together, these results in the three models suggest that brain inflammation functions to isolate damaged sites, remove dead cells and debris, and repair microenvironment of the brain rather than to produce cytotoxic proinflammatory mediators. Therefore, brain inflammation appears to be beneficial rather than detrimental to injured brain. 뇌염증반응은 뇌에 존재하는 microglia에 의해 주도적으로 일어나며 뇌손상을 악화시키는데 주요하게 작용하여 파킨슨병과 같은 퇴행성 뇌질환을 유발한다고 알려져 왔다. 그러나 최근의 연구 결과들로부터 neutrophil이나 monocyte와 같은 혈액 염증세포들이 뇌염증반응에 관여할 가능성과 뇌염증반응이 손상부위의 재생을 도와줄 가능성이 제시되었다. 뇌염증반응이 유해한지 아니면 유익한지에 대한 서로 다른 결과는 in vivo 실험과 in vitro 실험의 차이 또는 결과를 분석하는 시간에 따라 나타날 수 있으므로, 본연구에서는 손상받은 뇌에서 microglia와 neutrophil, monocyte들이 어떻게 작용을 하며, 신경세포 손상에 어떤 영향을 미치는지를 확인하여 뇌염증반응의 유해성과 유익성을 분석하였다. 첫째 과제에서는 쥐의 대뇌피질 (cortex)과 흑질 (substantia nigra)에 ATP를 주입하여 급성뇌손상 모델을 유도하였다. ATP가 주입된 중심부위에서는 신경세포뿐만아니라 microglia가 3시간내에 사멸되었으며, 그 주변지역의 microglia는 형태적으로 활성화되어 손상 부위를 둘러싸는 양상이 확인되었다. 손상 유도후 2일째부터 CD45^(+) monocyte가 손상주변부위로부터 유입되기 시작하여, 7일에 손상부위 전체에 유입되었으며, 14일에 점차로 그 수가 줄어드는 양상을 보였다. 그러나, neutrophil의 유입은 거의 관찰되지 않았다. 흥미롭게도 세포독성인자로 알려진 iNOS의 발현은 손상 유도후 초기 (3 시간)부터 회복시기 (28일)까지 거의 확인되지 않았으며, 그에 반해 식균작용의 표지로 알려진 CD68이 monocytes에서 높게 발현됨을 확인하였다. 더욱 중요한 것은, 손상 후 3시간에 측정한 도파민성신경세포의 수가 7일 후에도 감소를 나타내지 않았다. 이 결과들은 microglia가 손상받은 부위를 격리시키고, 혈액에서 유입된 monocyte는 손상받은 세포와 잔해들을 제거하는 식균작용에 관여하지만 신경세포나 주변조직의 손상을 유도하지 않는다는 것으로 생각할 수 있다. LPS를 흑질에 주입한 두번째 모델에서는, LPS 주입후 1일부터 monocyte의 유입이 시작되어, 3일에 가장 많은 세포가 확인 되었으며, 5-7일이 지나면서 일부가 세포사멸을 일으키고 일부는 microglia의 형태로 분화되었다. Microarray와 RT-PCR, 면역염색 결과, monoyctes는 iNOS 등의 classical activation에 관련된 염증매개물질을 발현하지 않는 대신, alternative activation에 관련된 식균작용에 관여하는 mannose receptor와 lysosomal enzyme들을 발현하였다. 이와 더불어, 도파민성 신경세포의 회복은 확인하지 못하였으나, astrocyte, 혈관내피세포, NeuN^(+) 신경세포, MAP-2^(+) dendrite, myelin의 회복을 확인하였다. 이러한 실험결과는 LPS에 의한 염증반응으로 유입된 monocytes가 손상받은 뇌에서 세포치유와 세포재생에 기여할 가능성을 시사한다. 세번째 모델에서는, 전신성 염증반응이 뇌염증반응을 유도하여 파킨슨병과 같은 퇴행성 뇌질환의 발병의 위험인자가 될 가능성을 조사하였다. LPS를 쥐의 꼬리정맥으로 주입하여 전신성 염증을 유도하면, 대뇌피질, 흑질, 해마 등에서 4-8시간에 micorglia의 형태적인 활성화와 neutrophil의 유입, 염증매개물질의 발현이 증가되었으며, 1-3 일 이내에 진정되었다. 그러나, 전신성 염증 유도후 8일이 지나도 흑질에서 도파민성 신경세포사멸은 확인되지 않았다. 이러한 연구결과는 단기적인 전신성 염증반응이 뇌염증 반응을 유도하지만, 신경세포의 손상을 유도할 정도로 독성이 크지 않음을 의미한다. 이러한 세가지 모델에서의 실험결과는 뇌염증반응이 손상된 뇌조직을 격리시키거나 죽은 세포들을 제거하고 뇌미세환경을 재생시키는 일을 하는 것으로 보아, 유해하기보다는 유익한 작용을 위해 일어난다는 것을 강력하게 시사하고 있다.

Study on the role of 4-1BB/4-1BBL interaction in obesity-induce adipose inflammation and metabolic dysfunctions

투 티 히엔 울산대학교 2015 국내박사

Obesity-induced inflammation plays a crucial role in the development of metabolic diseases such as insulin resistance and type 2 diabetes, cardiovascular diseases, and certain types of cancers. Adipose macrophages accumulation/activation and increased levels of inflammatory cytokines are a hallmark of obesity-induced inflammatory phenotype, and they play a pivotal role in the development of metabolic derangement in obesity. In obese adipose tissue, the interaction of cell with cell (adipose cell-macrophage) is likely crucial for the adipose inflammation and the development of metabolic diseases. However, molecules that mediate the cell-cell crosstalk remain unclear. 4-1BB (tumor necrosis factor receptor superfamily 9, TNFRSF9), a costimulatory receptor, modulates inflammatory processes through interaction with its ligand 4-1BBL on immune cell and/or non-immune cell surfaces. Our previous study using 4-1BB knockout obese mice has strongly suggested a critical role of 4-1BB and 4-1BBL interaction in the development of adipose tissue inflammation and metabolic disorders, but the underlying molecular mechanisms are unknown. This study aims to clarify the potential role of 4-1BB/4-1BBL interaction and the mechanisms involved in promoting obesity-induced adipose inflammation and metabolic derangements. The findings are as follows. First, the involvement of interaction between 4-1BB/4-1BBL in obesity-induced adipose inflammation was investigated. 4-1BB was expressed on adipocytes and was upregulated by obesity-related factors, which also enhanced 4-1BBL expression in macrophages. 4-1BB and/or 4-1BBL agonists, respectively, activated inflammatory signaling molecules (MAPK/IκBα and MAPK/Akt) in adipocytes and macrophages, and enhanced the release of inflammatory cytokines (MCP-1, TNF-α, IL-6). Disruption of the 4-1BB/4-1BBL interaction decreased the release of inflammatory cytokines from contact co-cultured adipocytes/macrophages. Moreover, 4-1BB/4-1BBL-mediated bidirectional signaling in adipocytes/macrophages promoted adipose inflammation. Second, the role of 4-1BB/4-1BBL interaction in obesity-induced macrophage proliferation and metabolism was examined. 4-1BBL stimulation on monocytes/macrophages enhanced reprogramming of glucose and lipid metabolism in the cells, and this was accompanied by cell proliferation. 4-1BBL stimulation on macrophages increased glucose uptake, transcript/protein levels of glucose transporter 1 and glycolytic enzymes, and lactate production. It also enhanced transcript levels of genes involved in the pentose phosphate pathway and lipogenesis. The 4-1BBL-induced metabolic reprogramming was mediated by Akt/mTOR signaling. Furthermore, the 4-1BB/4-1BBL interaction enhanced lipolysis in adipocytes followed by reduction of lipid droplets. Similar patterns of lipid alterations were observed by stimulating adipocytes/macrophages with 4-1BB/4-1BBL agonists, respectively. Thus, 4-1BB/4-1BBL-mediated bidirectional signaling in adipocytes/macrophages promotes adipose metabolic dysfunction. Thirdly, 4-1BB expression was examined in obese human patients. Levels of 4-1BB transcripts and soluble 4-1BB (s4-1BB) were correlated with BMI and expression of inflammatory markers, as well as with serum metabolic parameters. Moreover, s4-1BB was released from human adipocytes, and elicited chemotactic responses from human monocytes/T cells as well as enhancing their inflammatory activity, indicating that it may promote human adipose inflammation in obese conditions. Overall, these findings suggest that 4-1BB and 4-1BBL play crucial roles in obesity-induced adipose inflammation, and these molecules could serve as therapeutic targets and/or novel biomarkers for obesity-induced inflammation and metabolic complications. Phytochemicals/ food components targeting control of 4-1BB and/or 4-1BBL expression and their signaling pathways may be beneficial in protecting against obesity-induced inflammation and metabolic complications.

혈액에서 유입된 염증세포와 신호전달분자 Rac, SOCS에 의한 뇌염증반응의 조절에 관한 연구

지경애 아주대학교 2006 국내박사

Brain inflammation is a process of host defense against infection of injury. During this process, excessive inflammation could be harmful to surrounding tissue and consequently aggravate brain injury. Thus, to understand how brain inflammation occurs and how its duration and extent are controlled is important to limit neuronal damage in the brain. The first part of this thesis showed that activation of Rac1, a small G-protein, is involved in IFN-g-signaling in astrocytes. Compared to levels in control cells, IFN-g-induced GAS promoter activity and expressions of several IFN-g-responsive genes were markedly reduced in both cells expressing RacN17, a well-characterized Rac1 negative mutant. Thus, Rac1 may contribute to maximal activation of IFN-g responsive inflammatory signaling in rat astrocytes. The second part showed that thrombin, an inflammatory stimulator, increased expression of full name of cytokine-induced SH2 protein (CIS), one of SOCS family proteins. Since CIS reduced IFN-g-induced GAS-luciferase activity and tyrosine phosphorylation of STAT1 and STAT3, I concluded that thrombin could control duration and extent of inflammation by inducing expression of negative regulators of inflammation as well as pro-inflammatory mediators. In the third part, I showed regional differences in the extent of brain inflammation and neuronal damage in the substantia nigra pars compacta (SNpc) and the cortex, and underlying mechanisms that cause these differences. Microinjection of lipopolysaccharide (LPS) induced transient inflammatory responses and reversible neuronal damage in the cortex, but relatively severe and long-lasting inflammation and neuronal death in the SNpc. I found that the differential extent of neutrophil infiltration in these two areas could cause differential extent of inflammation. In the SNpc, cells labeled with two markers of microglia, OX-42 and ionized calcium binding adaptor molecule (Iba-1) showed different behaviors: The number of OX-42-ip cells increased at 12 h after LPS injection while the number of Iba-1-ip cells were dead. Since the OX-42 antibody detects CD11b that is expressed in neutrophils as well as in microglia, infiltration of neutrophils was detected using myeloperoxidase (MPO) as a specific marker of neutrophils. At 12 h following LPS injection, there was a dramatic increase in the number of MPO-ip neutrophils in the SNpc while relatively sparsely in the cortex. The MPO-ip cells were co-labeled with OX-42 and these cells expressed inducible nitric oxide synthase (iNOS). In agreement with these results, in leukopenic rats, MPO-ip cells were not detected for up to 3 days after LPS injection, and the loss of dopaminergic neurons were significantly attenuated. These results indicate that the extent of infiltrated neutrophil could determine the severity of inflammation in the cortex and the SNpc, and this event could be linked to the severity of neuronal damage in these two brain areas.

The role of miR-122-SOCS1 in allergic inflammation : 알러지 염증반응에서 miR-122-SOCS1의 역할 연구

Kyeonga Noh 강원대학교 일반대학원 2016 국내석사

Contributory role of leukotriene B4 receptors in neutrophilic inflammatory diseases

DongWook Kwak 고려대학교 대학원 2023 국내박사

Neutrophils have long been known to participate in acute inflammation because they are normally the early responder to inflammation stages and contribute to the resolution of inflammation. However, the roles of neutrophils in various chronic inflammatory diseases are recently emerging. These chronic diseases that involve neutrophilic inflammation are regulated by a network of mutually interacting inflammatory lipid mediators. The inflammatory lipid mediators include leukotriene B4 (LTB4) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) which are highly expressed in patients of neutrophilic asthma or inflammatory bowel diseases can induce another inflammatory cytokine interleukin-17 (IL-17) and interleukin-1 beta (IL-1β) that can act as neutrophils chemoattractant. I examined whether these inflammatory lipid mediators’ receptors, BLT1 and BLT2, play a critical role in regulating IL-17 or IL-1β in neutrophilic asthma and IBDs animal model. Although, I examined whether 5-/12-lipoxygenase, upstream of BLT1/2, are also critical for pathology in and an animal model of neutrophilic asthma and IBDs. As a result, I demonstrated that the ‘5-/12-LO-BLT1/2’ linked cascade regulates the inflammatory cytokines and therefore significantly contributes to the development of neutrophilic asthma and IBDs pathology. 1. Increased levels of neutrophils in bronchoalveolar lavage fluid (BALF) were associated with asthma severity. As leukotriene B4 (LTB4) is a principal chemoattractant molecule for neutrophils, its receptors, BLT1 and BLT2, may contribute to neutrophil-dominant airway inflammation. In the present study, I established a mouse model of steroid-resistant, neutrophil-dominant airway inflammation by house dust mite (HDM)/lipopolysaccharide (LPS) sensitization and HDM challenge, and I investigated whether BLT1/BLT2 signaling was associated with the development of neutrophilic airway inflammation. Blockade of BLT1 or BLT2 significantly suppressed airway inflammation and IL-17 production in this mouse model. The 5-LO and 12-LO enzymes, which catalyze the synthesis of BLT1/BLT2 ligands, were also critically associated with neutrophil-dominant airway inflammation and the synthesis of IL-17. Collectively, my results suggest that the 5-/12-LO-BLT1/BLT2-linked cascade significantly contributes to neutrophil-dominant severe airway inflammation via IL-17 synthesis in HDM-induced neutrophilic asthma. 2. The stimulation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and IL-1β synthesis are associated with chronic respiratory diseases such as neutrophil-dominant severe asthma. Leukotriene B4 (LTB4) is a principal chemoattractant molecule for neutrophil recruitment, and its receptors BLT1 and BLT2 have been suggested to contribute to neutrophil-dominant asthmatic airway inflammation. However, the relationship between BLT1/2 and NLRP3 in neutrophil-dominant asthmatic airway inflammation has not been previously studied. In the present study, I investigated whether BLT1/2 play any roles in stimulating the NLRP3 inflammasome and IL-1β synthesis. The blockade of BLT1 or BLT2 clearly suppressed the stimulation of the NLRP3 inflammasome and IL-1β synthesis in house dust mite (HDM)/lipopolysaccharide (LPS)-induced neutrophilic airway inflammation. The enzymes 5-lipoxygenase and 12-lipoxygenase, which catalyze the synthesis of BLT1/2 ligands [LTB4, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), and 12-hydroxyheptadecatreinoic acid (12-HHT)], were also critically associated with the stimulation of NLRP3 and IL-1β synthesis. Together, my results suggest that the 5-/12-LOX-BLT1/2-linked cascade are necessary for the simulation of the NLRP3 inflammasome and IL-1β synthesis, thus contributing to HDM/LPS-induced neutrophil-dominant airway inflammation. 3. Recent studies have reported that the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3)-dependent IL-1β and neutrophils have important roles in inflammatory bowel disease such as Crohn’s disease or ulcerative colitis. I previously reported that the leukotriene B4 receptor 2 (BLT2) has important role to mediate neutrophil recruitment and NLRP3 inflammasome stimulation in neutrophilic severe asthma. However, the association between BLT2 and NLRP3 inflammasome in chronic inflammatory bowel diseases has not been previously studied. In the present study, I observed that BLT2 plays critical mediatory role in stimulating the NLRP3 inflammasome and IL-1β synthesis in dextran sulfate sodium (DSS)-induced colitis. Furthermore, I also observed the roles of enzymes 5-lipoxygenase (5-LO) and 12-lipoxygenase (12-LO) which catalyze the synthesis of BLT2 ligands [LTB4, 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), and 12 hydroxyheptadecatreinoic acid (12-HHT)] in DSS-induced colitis. Taken together, my studies suggest that the 5-/12-LO-BLT2-linked cascade are necessary for the simulation of the NLRP3-dependent IL-1β synthesis, thus contributing to DSS-induced colitis.

들깨 (Perilla frutescens) 의 잎과 종자유가 비만 및 비만으로 유도된 염증성 대장질환에 미치는 영향

토마스샬롬사라 전북대학교 일반대학원 2020 국내박사

Background: The incidence of obesity in developed countries seems to slow down in recent years, while that of many developing countries continues to rise. Along with genetic and environmental factors, changing food habits awing to the availability of cheap but less-nutrient foods and lack of physical activity are also major risk factors of obesity. Obesity or overweight alone itself may not appear as a threat to life, but it leads to significant health problems that can result in chronic diseases. Diseases such as hypertension, cardiovascular disease, Type 2 diabetes, non-alcoholic fatty liver disease, and some forms of cancer, including colon cancer, are reported to be prevalent more in obese people, making it a significant cause of premature death. Treating obesity or its associated diseases with synthetic drugs raises concern due to their side effects. Also, some of the treatment methods are extremely costly, making it less accessible to people with low economic backgrounds. Effective dietary interventions are the cost-effective and most practical method to manage obesity and other lifestyle diseases. This work aimed to evaluate the potential role of Perilla frutescens, a medicinal herb belonging to the family of Lamiaceae, in managing obesity and obesity-associated colon inflammation. Firstly, the leaves of the purple perilla were analyzed for its anti-obesity effects. In the second set of studies, the oil extracted from the seeds of green perilla was investigated for its effect on colon inflammation. Through this work, we aimed to provide scientific evidence to claim the health benefits of including perilla oil and perilla leaves in the daily diet. Aim: Objective 1. To evaluate the potential effect of purple perilla leaves on high-fat diet-induced obesity and to elucidate the possible mechanism of action. Objective 2. To evaluate the potential effect of perilla oil on high-fat diet-induced colon inflammation (chronic colon-inflammation mice model) and to illustrate the mechanism of action. Objective 3. fat-1 transgenic mice can endogenously produce omega-3 fatty acids. Perilla oil contains omega-3 fatty acids as its primary component. In this study, we aimed to investigate the effect of omega-3 fatty acids in ameliorating high-fat diet-induced colon inflammation, using the fat-1 transgenic mice and dietary supplementation of perilla oil to wild type mice. Objective 4. To evaluate the potential effect of perilla oil in DSS-induced colitis mice (acute colitis model) challenged with a high-fat diet. Materials and Methods: Study 1. Five-week-old, C57BL/6J male mice were divided to four groups and were fed with a normal diet, high-fat diet, and high-fat diet supplemented with two different concentrations (100 mg/kg and 400mg/kg body weight) of perilla leaf extract for a period of 12 weeks, and obesity-related parameters were analyzed. Study 2. Five-week-old, C57BL/6J male mice were divided into five groups and were fed with normal diet, high-fat diet, and high-fat diet supplemented with three different oils (fish oil, perilla oil, and olive oil) for 16 weeks, and colon-inflammation related markers were evaluated. Study 3. Five-week-old, male fat-1 transgenic (TR) mice and their wild type (WT) littermates were divided into five groups and were fed with normal diet (WT and TR), high-fat diet (WT and TR), and high-fat diet supplemented with perilla oil (WT only) to compare the effects of omega-3 fatty acids in perilla oil and endogenously produced omega-3 fatty acids against high-fat diet-induced colon inflammation. After 16 weeks of the experiment, serum and colon inflammatory markers were analyzed. Study 4. Five-week-old, C57BL/6J male mice were divided into five groups and were fed with a high-fat diet (high-fat diet control and DSS- control) and high-fat diet supplemented with fish oil, perilla oil and olive oil for five weeks. DSS was administered through water for seven days, starting from week five. After a day of removal of DSS administration, the samples were collected and analyzed for colon inflammatory makers. Results: Study 1. Treatment with purple perilla leaf extract (PE) significantly prevented the high-fat diet-induced increase in body weight, improved serum triglyceride (TG) and total cholesterol (TC), decreased leptin, and increased adiponectin levels. Also, PE reduced hepatic TG and TC. Furthermore, in the adipose tissue and liver, expression levels of genes related to lipolysis and fatty acid β-oxidation such as ATGL, HSL, PPARα, and acyl CoA oxidase were upregulated in PE-treated mice. Thus, these results suggested that PE exerts anti-obesity effects, possibly by increasing β-oxidation. Study 2. Supplementation of all three oils significantly ameliorated the HD-induced colon inflammation, as indicated by the increased colon length and low macroscopic score. Perilla oil and fish oil increased the number of Bifidobacteria and reduced the number of Enterobacteriaceae, which in turn resulted in the lowering of endotoxin levels. In contrast, olive oil did not affect the amount of Bifidobacteria but reduced Enterobacteriaceae and endotoxin levels. The supplementation of the three oils also reduced the proinflammatory cytokines in serum and colon, such as IL-1β, IL-6, and TNF-α. Also, perilla oil suppressed the expression of COX2 and iNOS and inhibited the activation of NF-кB in the colon while increasing the expression of the tight junction protein, ZO-1. In conclusion, perilla oil exhibited noticeable improvement in HD-induced colon inflammatory conditions compared to olive oil. Study 3. Perilla oil supplemented WT mice, and fat-1 TR mice suppressed high-fat diet-induced body weight and improved serum lipid levels. Furthermore, high-fat-fed perilla oil group and fat-1 transgenic group exhibited increased colon length, lower macroscopic score, and reduced levels of proinflammatory cytokines in serum and colon tissue compared to wild type high-fat-fed group. The mRNA expression of proinflammatory markers was reduced, and colon epithelial barrier proteins were increased in high-fat-fed perilla oil and fat-1 transgenic groups. GPR120 is a membrane receptor protein activated by omega-3 fatty acids. The expression of GPR120 was increased in the perilla oil group, similar to that of the fat-1 transgenic group. These results suggested that perilla oil could attenuate high-fat diet-induced colon inflammation by suppressing inflammatory markers associated with NF-кB signaling pathway. Study 4. Administration of DSS resulted in the shortening of colon and increased disease activity index score compared to the HD group. These symptoms were significantly reversed in all the oil-treated groups. The body weight gain after DSS administration was significantly higher in perilla oil and olive oil supplemented groups compared to DSS- administered group. Also, perilla oil markedly attenuated the levels of TNF-α, IL-6, and IL-1β in serum and colon. The mRNA expression levels of proinflammatory markers in the colon were reduced, whereas tight junction proteins and epithelial defense barrier-associated markers were increased by the perilla oil treatment. Conclusion: The results showed that perilla leaf extract exhibited anti-obesity effects, possibly by increasing β-oxidation suggesting that consumption of perilla leaf could improve obesity. Furthermore, the supplementation of perilla oil ameliorated chronic and acute colitis conditions, and the effects were more pronounced than fish oil and olive oil. To summarize, the results of these studies suggested that consumption of different yields of Perilla frutescens, including the leaves and seed oil, could contribute to the management of lifestyle diseases. Notably, the results strongly supported the health benefits of using perilla oil in Korean cuisine, and perilla oil, which is a rich source of omega-3 fatty acids, could be a possible alternative for the omega-3 supplement. 연구배경: 선진국의 비만 발병률은 최근 몇 년간 감소한 반면, 많은 개발도상국의 비만 발병률은 계속 증가하고 있다. 유전적, 환경적 요인뿐 아니라 저렴하고 영양이 적은 음식의 섭취증가와 신체 활동의 부족이 비만의 주요 위험 요인이다. 비만은 자체로 생명의 위협이 되지 않을 수 있지만 만성질환을 초래할 수 있는 중대한 건강문제로 이어진다. 고혈압 및 심혈관질환, 제2형 당뇨병, 비알코올성 지방간질환, 대장암을 포함한 일부 형태의 암이 비만인 사람들에게 더 많이 발병하고 있다고 보고되는데 이는 조기 사망의 주요 원인이 된다. 비만이나 관련질환을 합성약물로 치료하는 것은 부작용의 우려가 많다. 또한 일부 치료법은 높은 비용으로 경제적 최약 계층의 사람들이 접근하기 어렵다는 단점이 있다. 적절한 식이요법은 비만과 다른 생활습관 질환을 관리하는데 비용과 효과면에서 실용적인 방법이 될 수 있다. 본 연구의 목적은 식품으로 쓰이고 꿀풀과에 속하는 들깨의 효과중 비만과 비만 관련 대장염에 대한 잠재적효과를 평가하는 것이다. 첫번째 연구에서 자주색 들깻잎의 항비만에 대한 효과를 검증하였다. 두 번째 연구에서는 녹색 들깨의 씨앗에서 추출한 기름이 대장염에 미치는 영향을 연구하였다. 본 연구를 통해 일상 식단에서의 들기름과 들깻잎의 건강기능성을 과학적으로 검증하고자 하였다. 연구목적: 연구목적 1. 고지방 식단으로 유도된 비만에 보라색 들깻잎이 미치는 항비만 효과를 평가하고 이러한 결과를 가져오는 가능한 매커니즘을 설명한다. 연구목적 2. 고지방 식단으로 유도된 대장염증(만성대장염 mouse 모델)에서 들기름의 잠재적 항염증 영향을 평가하고 그 메커니즘을 설명한다. 연구목적 3. 본 연구는 오메가-3 지방산을 내인적으로 생산할 수 있는 fat-1 유전자 변형 mouse 모델과 wildtype mouse에서 들기름의 첨가식이가 고지방 식이로 유도된 항염증을 개선하는데 있어 오메가-3 지방산의 역할을 평가하는 것을 목적으로 하였다. 연구목적 4. 고지방 식이와 함께 DSS로 유도된 대장염 mouse(급성 대장염 모델)에서의 들기름의 잠재적 효과를 평가한다. 재료 및 방법: 연구 1 : 생후 5주 된 C57BL/6J 수컷 mouse를 4개 그룹; 정상 식이, 고지방 식이, 저농도 들깻잎추출 첨가 및 고농도 들깻잎추출 첨가으로 나누어 12주 동안 섭취시키고 비만 관련 지표를 분석했다. 연구 2 : 생후 5주 된 C57BL/6J 수컷 mouse를 5개 그룹; 정상식이, 고지방식이, 3가지 다른 기름(어유, 들기름, 올리브유)으로 나누어 16주 동안 고지방식을 섭취시키고 대장염 관련 지표를 평가하였다. 연구 3. 생후 5주 된 수컷 fat-1 유전자변형 (TR) mouse와 wildtype (WT) mouse를 5개 그룹으로 나누어 정상식이 (WT와 TR), 고지방식이 (WT와 TR), 들기름을 처리한 고지방식 (WTPO)이를 섭취하도록 하였다. 실험은 16주 동안 진행되었으며 대장염 지표도 분석하였다. 연구 4. 생후 5주 된 C57BL/6J 수컷 mouse를 5개 그룹으로 나누어 5주 동안 고지방식이 (고지방식단 대조군, DSS-대조군)와 어유, 들기름, 올리브유를 보충식이 고지방식이를 섭취하도록 하였다. DSS는 5주부터 7일간 식수로 공급하였다. DSS 투여를 중단하고 시료들을 수집하고 대장염 지표를 분석하였다. 결과: 연구 1. 자주색 깻잎 추출물(PE)의 보충식이는 고지방 식이군에서 체중의 증가를 억제하였으며, 혈청 중성지방(TG)과 총콜레스테롤(TC) 개선, 렙틴 감소, 아디포넥틴 수치 증가를 나타냈다. 그리고 PE는 간의 TG와 TC를 감소시켰다. 또한 지방조직과 간에서는 ATGL, HSL, PPARα, acyl CoA 산화효소와 같은 지방산 β-산화 및 지방분해와 관련된 유전자의 발현 수준이 PE 처리된 mouse에서 고지방식이군에 비하여 상승되었다. 따라서, PE가 β-산화를 증가시킴으로써 항비만 효과를 나타낸다는 것을 시사한다. 연구 2. 어유, 들기름, 올리브유 모두고지방식이군에 비하여 증가된 대장길이와 감소된 Macroscopic score로 알 수 있듯이 고지방식이로 유도된 대장염증을 유의적으로 감소시켰다. 들기름과 어유는 Bifidobacteria 생균수를 증가시키고 Enterobacteriaceae 생균수를 감소시켰으며, 결과적으로 endotoxin 수치가 낮아지는 결과를 보였다. 이와 대조적으로 올리브유는 Bifidobacteria 생균수에 영향을 미치지 않고 Enterobacteriaceae 생균와 endotoxin 수치를 감소시켰다. IL-1β, IL-6, TNF-α와 같은 혈청 및 대장에 함유된 염증 유발 사이토카인 또한 3가지 오일의 섭취 의해 감소되었다. 또한 들기름은 COX2와 iNOS의 발현을 억제하고 대장 내 NF-кB의 활성화를 억제하는 동시에 접합단백질인 ZO-1의 발현을 증가시켰다. 결론적으로 들기름은 올리브유에 비해 HD군의 대장 염증 상태가 확연히 개선된 것으로 나타났다. 연구 3. 들기름의 섭취는 WT mouse와 fat-1 TR mouse 체중증가를 억제하고 혈청 지질 수치를 개선하였다. 또한, 고지방식이 들기름군과 fat-1 TR군은 야생형 고지방식이군에 비해 대장 길이가 증가하고, macroscopic score의 감소, 혈청 및 대장 조직 내 친염증 사이토카인 농도 감소 등의 결과를 보였다. 들기름을 함유한 고지방 식이와 fat-1 TR군에서 염증유발 지표의 mRNA 발현을 줄이고결장 상피 장벽 단백질 (colon epithelial barrier proteins) 지표로 증가시켰다. 오메가-3 지방산에 의해 활성을 나타내는 세포막 수용단백질인GPR120의 발현은 fat-1 TR군과 같이 들기름군에서도 증가하였다. 이는 들기름이 NF-кB 신호 경로와 관련된 염증 인자들을 억제하여 대장염을 약화 시킬수 있다는 것을 시사한다. 연구 4. 고지방식이군과 비교하여 DSS의 투여는 대장의 길이가 단축되었고, 질병활성도점수(DAS)는 증가한 것을 보여주었다. 그러나 이런 현상은 어유, 들기름, 올리브유를 섭취한 군에서 모두 유의적으로 역전되였다. DSS 투여 후 체중 변하는 들기름과 올리브유 섭취가DSS 투여군에 비하여 유의적으로 적어았다. 또한, 들기름은 혈청과 대장내의 TNF-α, IL-6 and IL-1β의 수치를 유의미하게 감소시켰다. 한편, 대장 내의 염증유발 지표의 mRNA발현을 감소되였나, 접합단백질과 상피 방어 장벽 관련 지표는 들기름보충식이에 의해 증가하였다. 결론: 결론적으로, 들깻잎 추출물은 β-산화를 증가시켜 항비만 효과를 나타내 깻잎 섭취가 비만을 개선할 수 있음을 시사한다. 또한 들기름의 보충은 만성 및 급성 대장염 상태를 개선시키고 그 효과는 어유, 올리브유보다 두드러지게 나타났다. 요약하면, 이 연구의 결과는 잎과 종자유를 포함한 들깨의 다른 부분을 섭취하는 것도 생활습관질환 관리에 기여할 수 있음을 시사한다. 특히 이 결과는 한식 요리에 들기름을 사용함으로써 얻을 수 있는 건강상의 이점을 적극 뒷받침하고 오메가-3 지방산의 풍부한 공급원인 들기름이 오메가-3 보충제의 대안이 될 수 있음을 시사한다.

Mediatory role of BLT2 in lung inflammation

Donghwan Park 고려대학교 대학원 2023 국내박사

류코트리엔, 프로스타글란딘 등이 속한 에이코사노이드는 염증성 질환에 관여하고 있다고 발표되어 잘 알려져 있는 지질대사 산물이다. 그 중에서도, LTB4는 아라키돈산이 5-LO를 통해서 대사되어 만들어지는 염증성 지질 대사 산물이다. 이러한 LTB4는 BLT1과 BLT2 두 종류의 수용체를 통해 생물학적 기능을 수행하게 되는데, 현재까지 대부분의 연구는 LTB4에 대한 높은 결합 친화성을 가지고 있고, 백혈구 등의 면역세포에서 발현되어 있는 BLT1에 집중되어 있었다. 반면에, LTB4에 대해 낮은 결합 친화성을 가진 BLT2는 체내에서 편재하여 발현되고 있으며, 상피세포, 비만세포, 암세포 등의 염증성 질환에서의 역할에 대해서 연구된 바가 있다. BLT2는 또한 아라키돈산이 12-LO를 통해서 대사되어 만들어지는 염증성 지질 대사 산물인 12(S)-HETE에 대한 수용체로서 작용한다. 이번 연구의 목적은 BLT2가 폐 염증 질병에서 어떠한 역할을 하고 있는지를 규명하고자 하였다. 본 연구에서는 맹장 결찰 및 천공 (CLP) 수술에 의한 패혈증 모델, 집먼지 진드기 (HDM)에 의한 호산구성 폐 염증 모델, LPS/OVA에 의한 호중구성 폐 염증 모델이 사용되었다. 1. 본 연구는 감염에 의해 발생하는 전신성 염증 반응 증후군인 패혈증에서 BLT2의 역할을 규명하고자, CLP 수술을 통한 패혈증 모델이 사용되었다. 패혈증이 유도된 생쥐 복강 세척액과 혈청에서 BLT2의 리간드인 LTB4와 12(S)-HETE의 발현 증가를 확인하였다. 또한, 이들을 형성하는 효소인 5-LO, 12-LO 단백질과 BLT2 단백질의 발현이 폐와 간조직에서 증가된 것을 확인하였다. 그리고 패혈증이 유도된 생쥐에 BLT2 억제제를 투여하였을 때, 패혈증과 연관된 사이토카인 (IL-6, TNF-, IL-1, IL-17)과 폐 염증이 감소된 것을 관찰하였다. 종합하여 보았을 때, 위 결과는 CLP로 인한 패혈증 모델에서 BLT2가 염증성 사이토카인의 조절을 통해 폐 염증을 유발함을 보여주었다. 이러한 발견은 패혈증 환자에 대한 잠재적인 치료 타겟으로써 BLT2를 제시하고 있다. 2. 앞선 연구에서, BLT1와 BLT2가 OVA에 의해 유도된 알레르기성 기도 염증에 대한 조절 인자로서 작용하고 있음을 밝혀낸 바 있다. 하지만, HDM이 알레르기성 천식에서 잘 알려진 위험 인자이고, 가장 일반적인 알레르겐으로 작용함을 고려하였을 때, 본인은 BLT1과 BLT2가 HDM으로 인한 알레르기성 기도 염증 반응에서 어떤 역할을 가지고 있는지에 대하여 규명하고자 하였다. 본 연구에서, HDM을 처리한 생쥐의 기도세척액에서 LTB4와 12(S)-HETE의 발현이 증가한 것을 확인하였다. BLT1, BLT2, 5-LO, 12-LO의 억제제를 각각 투여하였을 때, HDM에 의해 유도된 호산구성 염증 모델에서 발견되었던 TH2 사이토카인 (IL-4, IL-5, IL-13)의 발현량 증가, 폐 염증 및 점액 분비 증가 등의 현상이 모두 완화됨을 확인할 수 있었다. 이러한 결과들을 종합하면, 5-/12-LO-BLT1/2 연속단계가 TH2 사이토카인의 발현을 조절함으로써 HDM에 의해 유도된 기도 염증을 유발함을 알 수 있다. 본 연구 결과는 HDM에 대한 알레르기성 천식 환자들에게 잠재적인 치료 타겟으로써 BLT1/2를 제시하고 있다. 3. 과립구집락자극인자 (G-CSF)는 호중구성 천식 발병에 기여하고 있다고 알려져 있다. 하지만, 호중구성 천식에서 G-CSF 발현 조절 인자에 대한 연구가 부족한 상황이다. 앞선 연구에서는 BLT2가 호중구성 기도 염증에서 중요한 역할을 하고 있다는 사실을 밝혀냈다. 하여, 본 연구에서는 BLT2가 LPS/OVA에 의해 유도된 스테로이드 저항성, 호중구성 천식에서 G-CSF의 발현에 BLT2가 연관되어 있는지 확인하고자 하였다. 실험을 통해, 호중구성 기도 염증에서 G-CSF의 발현이 증가된 것을 확인하였고 이를 BLT2가 조절하고 있음을 확인하였다. 그리고 12-LO 또한 G-CSF 발현에 필수적임을 확인하였다. 따라서, 위 결과는 12-LO-BLT2 신호전달 네트워크가 G-CSF 발현을 조절하고, 이를 통해 호중구성 기도 염증 발달에 기여하고 있음을 나타낸다. 이러한 발견은 중증 천식 환자에게 새로운 잠재적 치료 타겟을 제시하고 있다. Eicosanoids such as leukotrienes and prostaglandins are well-defined lipid metabolites which were reported to be associated with inflammatory diseases. Among them, leukotriene B4 (LTB4) is a lipid mediator of inflammation that is generated from arachidonic acid (AA) via the 5-lipoxygenase (5-LO) pathway. LTB4 exerts biological functions by binding to two distinct receptors, BLT1 and BLT2. Majority of studies have focused on BLT1, which is the high-affinity receptor for LTB4 and expressed mainly on immune cells, such as leukocytes. On the other hand, BLT2, a low-affinity receptor for LTB4, is studied in inflammatory diseases and cancer and expressed ubiquitously, including airway epithelial and mast cells. BLT2 is also known to interact with 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), a lipid mediator derived from AA by the enzymatic action of 12-lipoxygenase (12-LO). The aim of this study was to investigate whether BLT2 plays any role in lung inflammation. Cecal ligation and puncture (CLP)-induced septic lung inflammation model, house dust mite (HDM)-induced eosinophilic lung inflammation model and lipopolysaccharide/ovalbumin (LPS/OVA)-induced neutrophilic lung inflammation model were used to evaluate the mediatory role of BLT2 in lung inflammation. 1. I investigated whether BLT2 played any role in sepsis, a systemic inflammatory response syndrome caused by infection. A murine model of CLP-induced sepsis was used to evaluate the role of BLT2 in septic inflammation. In the present study, I observed that the levels of ligands for BLT2 (LTB4 and 12(S)-HETE) were significantly increased in the peritoneal lavage fluid (PF) and serum from mice with CLP-induced sepsis. I also observed that the levels of BLT2 as well as 5-LO and 12-LO, which are synthesizing enzymes for LTB4 and 12(S)-HETE, were significantly increased in lung and liver tissues in the CLP mouse model. Blockade of BLT2 markedly suppressed the production of sepsis-associated cytokines {IL-6 (interleukin-6), TNF- (tumor necrosis factor-alpha), and IL-1 (interleukin-1) as well as IL-17 (interleukin-17)} and alleviated lung inflammation in the CLP group. Taken together, my results suggest that BLT2 cascade contributes to lung inflammation in CLP-induced sepsis by mediating the production of inflammatory cytokines. These findings suggest that BLT2 may be a potential therapeutic target for sepsis patients. 2. Previous studies have reported that the receptors of LTB4, BLT1 and BLT2 play mediatory roles in the allergic airway inflammation induced by OVA. However, considering that HDM is the most prevalent allergen and well-known risk factor for asthmatic allergies, I was interested in elucidating the contributory roles of BLT1/2 in HDM-induced allergic airway inflammation. The aim of this study was to investigate whether BLT1/2 play any roles in HDM-induced allergic airway inflammation. In the present study, I observed that the levels of ligands for BLT1/2 (LTB4 and 12(S)-HETE) were significantly increased in bronchoalveolar lavage fluid (BALF) after HDM challenge. Blockade of BLT1 or BLT2 as well as 5-LO or 12-LO markedly suppressed the production of TH2 cytokines {IL-4 (interleukin-4), IL-5 (interleukin-5) and IL-13 (interleukin-13)} and alleviated lung inflammation and mucus secretion in HDM-induced eosinophilic airway inflammation mouse model. Together, these results indicate that the 5-/12-LO-BLT1/2 cascade plays a role in HDM-induced airway inflammation by mediating the production of TH2 cytokines. My findings suggest that BLT1/2 may be a potential therapeutic target for patients with HDM-induced allergic asthma. 3. Granulocyte colony-stimulating factor (G-CSF) has been suggested to be closely associated with neutrophilic asthma pathogenesis. However, little is known about the factors regulating the production of G-CSF in neutrophilic asthma. I previously reported that a leukotriene B4 receptor 2, BLT2, played an important role in neutrophilic airway inflammation. Therefore, in the current study, I investigated whether BLT2 plays a role in the production of G-CSF in LPS/OVA-induced steroid-resistant neutrophilic asthma. The data showed that BLT2 critically mediated G-CSF production, contributing to the progression of neutrophilic airway inflammation. I also observed that 12-lipoxygenase (12-LO), which catalyzes the synthesis of the BLT2 ligand, 12(S)-HETE, was also necessary for G-CSF production. Together, these results suggest that the 12-LO-BLT2-linked signaling network is critical for the production of G-CSF, contributing to the development of neutrophilic airway inflammation. My findings can provide a potential new target for the therapy of severe neutrophilic asthma.