The Effect Of The Tripterygium Wilfordii Hook F. On Diabetic Liver Injury

Objective:The liver is the metabolism hub of sugar, fat and protein, and has a important role for the development of type 2 diabetes. Diabetic liver injury is mainly caused by fatty infiltration of the liver leading to non-alcoholic fatty liver disease(NAFLD). Since the pathogenesis is not clear, there is no specific treatments or drugs. The liver is an important natural immune organ, and also is an important target organ of inflammatory mediators. The immune and inflammatory pathways play a central role in the pathogenesis of diabetic liver injury. Tripterygium wilfordii Hook F., a traditional Chinese medicine, has potent anti-inflammatory, antioxidant, immunosuppressive and other pharmacological properties. Celastrol(a pentacyclictriterpene extract derived from Tripterygium wilfordii Hook F.), is also the main active ingredient. So far, there is no evidence regarding the effect and mechanism of innate immune alterations of Tripterygium wilfordii Hook F. on fatty liver injury. The present study was aimed to investigate protective effects of Tripterygium wilfordii polyglucosides on the liver injury in diabetic rats, and to elucidate the possible mechanism involved in vivo; We interfered the expression of TLR4 gene in steatosis HepG2 cells by siRNA and detected the expressions of TLR4 and downstream inflammatory factors, and to elucidate the possible target and involved mechanism of celastrol in vitro. Methods:1. In vivo studies75 SD rats were randomly assigned to normal control group(NC group, n = 15), which were fed with standard diet, and high fat diet group(HFD group, n = 60), which were fed with high-fat diet for 8 weeks. The HFD rats were administered a single tail intravenous injection of 30 mg/kg STZ to induced type 2 diabetes models. Type 2 diabetic rats were randomly assigned to four groups: diabetes mellitus(DM group, n=15), and diabetic rats treated with low-dose TWP(DM+TL group, 1 mg/kg·d, n= 15), and diabetic rats treated with medium-dose TWP(DM+TM group, 3 mg/kg·d, n=15), and diabetic rats treated with high-dose TWP(DM+TH group, 6 mg/kg·d, n=15). TWP was given by daily gastric gavage for 8 weeks. We analyzed the liver histopathological and biochemical changes, and the serum levels of inflammatory factors by ELISA. We also analyzed the gene and protein expressions of TLR4/MyD88/NF-κB signaling pathway and downstream inflammatory factors IL-1β and TNFα by immunohistochemistry, RT-PCR and Western Blotting, respectively.2. In vitro studiesWe induced steatosis in human hepatoma cell line(HepG2 cells) with free fatty acids(FFAs, palmitic acid: oleic acid = 1: 2), and determined lipid accumulation by oil red O staining and cell survival by CCK8 assay. We synthesized chemically TLR4 siRNA in vitro and transfected HepG2 cells by Lipofectamine 2000, and screened effective silencing sequence. The cells were divided into 7 groups: normal control group, FFAs group, FFAs + negative siRNA group, FFAs + TLR4 siRNA group, FFAs + celastrol group, FFAs + celastrol + negative siRNA group and FFAs + celastrol+ TLR4 siRNA group. We analyzed the gene and protein expressions of TLR4/MyD88/NF-κB signaling pathway and downstream inflammatory factors IL-1β and TNFα by RT-PCR and Western Blotting, respectively. Results:1. In vivo studies(1) Compared with the NC group, the levels of blood lipids, insulin, HOMA-IR and HOMA-β were significantly increased in the HFD group after 8 weeks high-fat diet(P<0.05), which indicating that hyperinsulinemia and insulin resistance were emerged; blood glucose was significantly higher accompanied by polydipsia, polyuria and polyphagia after injection of STZ, which indicating that the model of type 2 diabetic rats was induced successfully.(2) General indicators:(1)Biochemical indexes: The levels of blood glucose, TG and TC in the DM group were significantly increased compared with the NC group(P<0.05); however, no significant changes were observed in TWP-treatment groups compared with the DM group(P>0.05); the parameters of liver, renal function and blood routine had no difference among the five groups(P>0.05).(2)Liver index: Compared with the NC group, the level of LW/BW was increased significantly in the DM group(P<0.05), and was decreased in the DM+TH group(P<0.05).(3) Histological changes of hepatic tissues in rats: There was clear structure of hepatic lobule and hepatocytes, no obvious inflammatory cell infiltration in the NC group in H&E staining sections. Hepatic lobular structure generally disappeared, and diffuse large bubble-like hepatocyte steatosis with obvious inflammatory cell infiltration in the DM group. Lesions in TWP treatment groups were less than that in the DM group, particularly in the TH group. Masson’s trichrome staining showed no obvious coloration appeared along the vascular wall of the portal vein and portal area in the NC group. However, diabetic rats showed more fibrosis changes around the portal area. The degree of fibrosis observed in hepatic tissues was much lower in TWP-treated rats than diabetic rats.(4) Serum levels of inflammatory factors: ELISA analysis showed serum NF-κB,IL-1β and TNFα levels in the DM group were significantly increased compared with those in the NC group(P<0.05), and serum levels in TWP-treatment groups were decreased in a dose-dependent manner(P<0.05).(5) Effects of TWP on expressions of TLR4/MyD88/NF-κB signaling pathway of hepatic tissues in rats: RT-PCR and Western Blotting analysis showed TLR4,MyD88,NF-κB,p-IκBα and downstream inflammatory factors IL-1β and TNFα mRNA and protein levels in the hepatic tissues in the DM group were significantly increased compared with those in the NC group(P<0.05). Additionally, gene and protein expressions were decreased in TWP- treatment groups in a dose-dependent manner(P<0.05).2. In vitro studies(1) Best FFAs concentration: 0.5mM FFAs treatment showed significant steatosis in HepG2 cells, and did not affect cell viability compared with the normal control group(P>0.05). Therefore, 0.5mM FFAs is optimal induction concentration.(2) Best celastrol concentration: 0.5μM celastrol treatment showed significantly decreased of red lipid droplets in steatosis HepG2 cells, and did not affect cell viability compared with the control group(P>0.05). Therefore, 0.5μM celastrol is optimal intervention concentration.(3) Transfection efficiency by fluorescence microscopy and flow cytometry: there were visible green fluorescence by fluorescence microscope after TLR4 siRNA transfection, and transfection efficiency reached 90% by flow cytometry. We successfully transfected siRNA genes into HepG2 cells, and reached high transfection efficiency.(4) Effective silencing sequence screening: The group of TLR4 siRNA3 transfection showed the lowest gene and protein expression in steatosis HepG2 cells(P<0.05). Therefore, TLR4 siRNA3 was the most effective silence sequence.(5) Effects of celastrol on expressions of TLR4/MyD88/NF-κB signaling pathway in HepG2 cells: The expressions of TLR4, MyD88, NF-κB and downstream inflammatory factors IL-1β and TNFα mRNA and protein in the HepG2 cells by RT-PCR and Western Blotting analysis were significantly increased in the the FFAs group compared with those in the NC group(P<0.05); and were significantly decreased(P<0.05) in TLR4 siRNA and celastrol intervention group compared with the FFAs group. TLR4 siRNA and celastrol intervention together could decrease the expressions furthermore(P<0.05). The inhibition degree of NF-κB and downstream inflammatory cytokines IL-1β and TNFα were less than TLR4. Conclusion:We firstly found that TWP treatment could delay the progression of diabetic liver disease in type 2 diabetic rats via inhibition of TLR4/MyD88/NF-κB signaling cascade pathways, and its downstream inflammatory effectors. Celastrol partially blocked the TLR4-mediated immune and inflammatory signaling pathways and had a synergistic effect with TLR4 siRNA. Celastrol regulated the expression of downstream inflammatory mediators possibly through others pathways in addition to blocking TLR4 signaling pathway.