The Role And Mechanism Of IL-6 In LPS/D-GalN Induced Acute Liver Injury

ObjectionIn recent years,the incidence of acute liver injury in China was increasing year by year.This is a serious threat to human health.IL-6 has a variety of biological function and it plays a complex role in the inflammatory response.IL-6 has pro-inflammatory and anti-inflammatory effects.Most of the pro-inflammatory effects of IL-6 are due to trans-signal transduction pathways,whereas IL-6 classical transduction pathways mediate anti-inflammatory and regenerative signaling.Previous reports have shown that IL-6 is relevant to acute liver injury,but the role of IL-6 in acute liver injury is not well defined.Therefore,the purpose of our study is to clarify the role of IL-6 in acute liver injury,and to study the relevant mechanisms.Hoping to enrich the new theory,to provide new methods for the diagnosis and treatment of acute liver injury.MethodsWe chose C57BL/6 mice as experimental mice.IL-6 recombinant protein was pretreated ahead.LPS/D-GalN together intraperitoneal injection was used to induce acute liver injury model in mice.Then we collected plasma and liver tissues at different time windows(2h,6h).We observed the hepatic histological changesusing the HE staining assay in each group.We used microplate method to detect plasma alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels in each group.We detected mRNA expression of inflammatory factors in liver by real time PCR,and analyzed the protein expression levels of inflammatory cytokines by enzyme-linked immunosorbent assay(ELISA).We determined the triglyceride content in the liver by a TG enzymic method.After pretreatment with IL-6,we used different concentrations of LPS to stimulate primary hepatocytes,RAW264.7 and primary macrophages.We used RT-PCR to detect the expression of iNOS,TNF-a,RANTES.We used enzyme-linked immunosorbent assay(ELISA)to detect the expression of inflammatory proteins in macrophage culture media.We used the macrophage supernatant to culture the primary hepatocytes.We used RT-PCR to detect the differences in mRNA expression levels of inflammatory factors.Result1.We explored the best concentration of intraperitoneal injection of LPS/D-GalN to induce acute injury is LPS(50 ?g/kg)/D-GalN(400 mg/kg).2.At 6 hours,LPS/D-GalN-induced-group compared to the control group,the liver appearance was obvious swelling and congestion,liver index significantly increased.In the model group given IL-6 in advance,the liver swollen and congestion was less obvious and IL-6 significantly reduced liver index.3.At 2 hours,LPS/D-GalN-induced-group compared control group mice,HE staining results showed that the liver slightly congestive.There was no significant difference between the two groups.At 6 hours,LPS/D-GALN induced severe liver injury(liver tissue fragmentation and necrosis,and visible a large number of inflammatory cell infiltration).In the model group given IL-6 in advance,liver injury reversed.4.At the time of 2 hours and 6 hours,LPS/D-GalN-induced-group was significantly higher in plasma ALT and AST than that of control group.In the model group mice given IL-6 in advance,the levels of ALT and AST in the plasma were significantly lower.5.At 2 hours and 6 hours,the mRNA and protein expression of M1 inflammatory factors significantly increased in the model group.After pretreatment with IL-6,the mRNA and protein levels of inflammatory factors were significantly lower.6.LPS caused a weak inflammatory response to the liver parenchymal cells.After IL-6 pretreatment,there was no significant difference in inflammatory factors mRNA.7.We used different concentrations of LPS stimulate different macrophages.The mRNA and protein expression of M1 inflammatory factors significantly increased.After pretreatment with IL-6,the mRNA and protein expression of M1 inflammatory factors decreased significantly.We used a certain concentration of LPS to stimulate macrophages.The mRNA expression of inflammatory factors were decreased in a dose-dependent manner after pretreatment with different concentration gradient IL-6.8.The levels of inflammatory mRNA expression in hepatocytes were increased in a dose-dependent manner compared with the supernatant of macrophages cultured LPS.After IL-6 pretreatment,the expression of inflammatory mRNA levels decreased significantly.9.Compared with PBS group,IL-4 or IL-13 stimulated RAW264.7 cells,the anti-inflammatory mRNA expression significantly increased.The expression of IL-4R and IL-10 mRNA in IL-6 group were significantly higher.ConclusionWe induced acute liver injury by injecting different concentrations of LPS and D-GalN in C57BL/6 mice at different time points(2h,6h),and identified the optimal dose.Basis on this model,IL-6 pretreatment significantly relieved LPS/D-GalN-induced acute liver injury in mice,mainly manifested in the reduction of liver congestion,the reversal of hepatic lobular structural disorder and the expression of ALT and AST in plasma.In the acute liver injury model,the occurrence of inflammation plays an important role.LPS stimulate the kupffer cells to promote the release of inflammatory factors,which promote liver cell apoptosis and necrosis,and ultimately cause liver injury.Our results showed that IL-6 significantly reduced the mRNA and protein levels of inflammatory cytokines,and inhibited inflammatory response.In vitro,we found LPS causing a weak inflammatory response to the liver parenchymal cells,and IL-6 did not show a regulatory role on the inflammatory response.IL6 inhibited the expression of inflammatory factors induced by LPS in macrophages.IL-6 also promoted IL-4 or IL-13-induced macrophage M2-type polarization.LPS pretreatment in macrophage supernatant,caused hepatocellular inflammation,and IL-6 and LPS simultaneously pretreated significantly reduced the hepatocellular inflammation.This indicated that LPS stimulated macrophages to release inflammatory factors,further acting on hepatocytes,leading to the occurrence of hepatocellular inflammation,and IL-6 could inhibit hepatocellular inflammation development through inhibiting LPS-induced macrophage of M1-type polarization.