| Nonalcoholic fatty liver fibrosis is the key pathological process of nonalcoholic steatohepatitis(NASH)progressing to cirrhosis and the key to the long-term prognosis of NASH.Its effective prevention and treatment can prevent the occurrence of end-stage liver disease.Therefore,it is of great significance to find natural drugs that are safe,efficient,low toxic and have little side effects for the treatment of NASH/fibrosis.Hepatic stellate cells(HSC)are the main cell type in the progress of NASH/fibrosis.Its activation and proliferation lead to the excessive production and deposition of extracellular matrix(ECM),leading to the formation of liver fibrosis.The mechanism of NASH/fibrosis has not been fully understood,in which inflammatory response and oxidative stress are the main pathological mechanisms.Many inflammatory factors released by inflammation not only affect the activation,proliferation and differentiation of HSCs,but also regulate the migration and localization of HSCs,thus affecting the progress of liver fibrosis.Abnormal oxidative stress produces excessive peroxides,enhances liver inflammation,causes liver cell damage,and promotes the activation and proliferation of HSC.Therefore,inhibition of hepatic inflammatory response and oxidative stress can alleviate NASH/fibrosis.PPAR-γ is a type Ⅱ nuclear receptor and a major regulator in the process of glucose and lipid metabolism.Recent studies have found PPAR-γ can maintain the cell phenotype and biological activity of HSC at rest.PPAR-γ agonists inhibited TGF-β1 expression,thus blocking HSC proliferation and driving activated HSC apoptosis to improve fatty liver fibrosis.In addition,PPAR-γ can inhibit inflammation and oxidative stress.Therefore,PPAR-y may be an effective target for the treatment of nonalcoholic fatty liver fibrosis.Tanshinone ⅡA(Tan ⅡA)is the most well studied compound with the strongest pharmacological activity among fat soluble tanshinone.It has a variety of biological activities,including reducing lipid oxidation and reactive oxygen species production,anti-apoptosis,anti-inflammatory and anti-fibrosis.Studies have found that Tan IIA could play a therapeutic role in many diseases,but the specific efficacy and mechanism of Tan IIA on NASH/liver fibrosis have not been fully studied.And whether Tan IIA plays an anti-fibrosis role by regulating PPAR-y related signal pathways has not been studied.Objectives1.To investigate whether Tan ⅡA inhibits the progression of nonalcoholic fatty liver fibrosis by inhibiting inflammation and oxidative stress.2.To explore the specific molecular mechanism of t Tan ⅡA inhibiting nonalcoholic fatty liver fibrosis in vivo and in vitro.Methods1.Animal experimentMale C57BL/6 mice were randomly divided into the following 3 groups(n=20)(1)control(Control):conventional diet,intraperitoneal injection of olive oil(5ml/kg.bw)every other day;(2)Model(HFD+CCL4):high fat diet,intraperitoneal injection of 10%CC14 olive oil solution(5ml/kg.bw)every other day;(3)Tan IIA intervention(TAN):high fat diet,intraperitoneal injection of 10%CC14 olive oil solution(5ml/kg.bw)every other day,subcutaneous injection of 10 mg/kg Tan IIA every day.The patients were intervened continuously for 8 weeks.2.Cell experiment 1Rat stellate cell line HSC-T6 cells were divided into four groups:(1)Control(Control):cells were cultured for 24h and added with DMSO.(2)Model(TGF-β1):cells were cultured for 24h and 10ng/ml TGF-β1 was added.(3)Low dose intervention(TAN-L):cells were cultured for 24h,and 10ng/mL TGF-β1 was added.After incubation for 24h,cells were treated with 5μL Tan IIA for 24h.(4)High dose intervention(TAN-H):cells were cultured for 24h,and 10ng/mL TGF-β1 was added.After incubation for 24h,cells were treated with 20μL Tan ⅡA for 24h.3-Cell experiment 2HSC-T6 cells were divided into four groups:(1)Control(Control):cells were cultured for 24h and added with DMSO.(2)Model(TGF-β1):cells were cultured for 24h and 10ng/ml TGF-β1 was added.(3)Tan IIA intervention(TAN):cells were cultured for 24h,and 1Ong/mL TGF-β1 was added.After incubation for 24h,cells were treated with 20μL Tan IIA for 24h.(4)PPAR-y Inhibitor(T0070907):cells were cultured for 24h,and l0ng/mL TGF-β1 and 10μL T0070907 were added.After incubation for 24h,cells were treated with 20μL Tan ⅡA for 24h.Results1.Tan ⅡA inhibits nonalcoholic fatty liver fibrosis by inhibiting inflammatory response and oxidative stress(1)TanIIA inhibits inflammation induced by HFD combined with CCl4 in miceCompared with the Control group,the serum proinflammatory factors TNF-α,IL-6 and IL-1β in the Model group were significantly increased.Additionally,the inflammatory signal pathway TLR4-NF-κB was activated,and the mRNA and protein levels of TLR4,MyD88,NF-κB and IKK-βwere significantly enhanced.After the intervention of TanⅡA,the concentrations of serum TNF-α,IL-6 and IL-1β were decreased,and the expression of TLR4-NF-κB signaling pathway related proteins also were inhibited significantly.(2)Tan ⅡA inhibits oxidative stress induced by HFD combined with CCl4 in miceCompared with the Control group,the levels of SOD and GSH in the Model group decreased significantly,but the concentration of MDA increased.However,after Tan IIA treatment,the levels of SOD and GSH increased,while the level of MDA decreased.The same phenomenon was found in liver tissue.The CAT concentration in liver tissue was detected by ELISA kit.The results showed that compared with the Control group,the CAT activity in the Model group decreased significantly,which was increased significantly in liver tissue of Tan IIA group(P<0.05).(3)Tan ⅡA inhibits fibrosis induced by HFD combined with CCl4 in miceMasson staining results showed that compared with the Control group,extensive blue collagen deposition could be observed in the liver lobules of the Model group,and obvious fibrous tissue proliferation could be detected in the portal area and around the central vein.In the Tansh ⅡA group,the proliferation of fibrous tissue around the portal area and central vein was significantly improved,and the deposition of blue fiber collagen was reduced.The Sirius red staining results showed that a large amount of red collagen was distributed in the liver tissue of the Model group compared with the Control group,and Tan IIA intervention could significantly reduce the formation of red collagen.The results of immunofluorescence showed that compared with the Control group,the Model group expressed increased number of α-SMA and collagen-1 positive cells.The results of Western blotting also showed that the protein expressions of α-SMA and collagen-1 were increased significantly.After Tan ⅡA treatment,the number of α-SMA and collagen-1 positive cells were decreased,and the protein expressions of α-SMA and collagen-1 were reduced significantly.2.Tan ⅡA regulates PPARy-Smad signaling pathway in vitro and in vivo to improve nonalcoholic fatty liver fibrosis(1)Tan ⅡA up-regulates PPARy in fibrotic miceCompared with the Control group,both mRNA and protein levels of the liver PPARy of the Model group were significantly decreased,while in tan ⅡA group,both mRNA and protein levels of the PPAR-y were significantly elevated.The similar results were obtained in the immunofluorescence.Tan ⅡA intervention significantly increased the expression of PPAR-y.(2)Tan ⅡA regulates JAK2/STAT5/PPAR-γ/Smad2/3 signal pathway in fibrotic miceCompared with the Control group,the phosphorylation levels of JAK2 and STAT5 in the liver tissue of the Model group were significantly reduced,while the phosphorylation levels of smad2/3 were significantly enhanced.After Tan IIA intervention,the phosphorylation levels of JAK2 and STAT5 were increased,while the phosphorylation levels of smad2/3 decreased.(3)Tan ⅡA inhibits TGF-β1 induced HSC-T6 cell inflammationCompared with the Control group,the secretion level of inflammatory factors(IL-6 and TNF-α)in HSC-T6 cells was significantly increased.Tan IIA(5μL,20μL)can significantly reduce the secretion level of inflammatory factors.The same trend was found in the mRNA expression of cytokines.(4)Tan ⅡA inhibits TGF-β1 induced oxidative stress in HSC-T6 cellsCompared with the Control group,the ROS production in TGF-β1-activated HSC-T6 cells increased significantly.Compared with the TGF-β1 group,both high and low doses of Tan IIA could reduce the production of ROS,with significant differences.In addition,after the activation of HSC-T6 by TGF-β1,the levels of SOD and GSH decreased significantly,but the concentration of MDA increased.After the intervention of Tan IIA,the levels of SOD and GSH increased,while the levels of MDA decreased.(5)Tan ⅡA inhibits TGF-β1 induced HSC-T6 cell fibrosisWestern blotting showed that the protein expression of α-SMA and collagen-1 in TGF-β1 induced HSC-T6 cells were significantly increased.Both high and low doses of Tan ⅡA could reduce the protein expression of α-SMA and collagen-I,but only the high dose group had significant difference.Immunofluorescence showed similar results.(6)Tan ⅡA up-regulates TGF-β1 induced PPAR-γ of HSC-T6 cellsCompared with the Control group,the activated HSC cells expressed lower protein and mRNA levels of PPAR-γ,while 20 μL Tan IIA significantly up-regulated the expression of PPARy.(7)Tan ⅡA regulates JAK2/STAT5/PPARγ/Smad2/3 signal pathway in HSC-T6 cellsCompared with the Control group,the phosphorylation levels of JAK2 and STAT5 in activated HSC-T6 cells were significantly decreased,while the phosphorylation levels of Smad2/3 were increased.After the intervention of Tan ⅡA(20 μL),the phosphorylation levels of JAK2 and STAT5 increased,while the phosphorylation levels of Smad2/3 decreased.(8)Apply PPAR-y inhibitor weakens the anti-fibrotic effect and mechanism of Tan IIATan IIA combined with PPAR-y inhibitor(T0070907)reversed the inhibitory effect of Tan IIA on inflammation and oxidative stress.Compared with Tan ⅡA group,the expression of inflammatory factors in Tan IIA+T0070907 group were significantly increased,the levels of SOD and GSH were decreased,but the concentration of MDA was increased.In addition,compared with Tan ⅡA alone,Tan IIA+T0070907 combined intervention significantly increased the protein expression of a-SMA and collagen-I.After the application of PPAR-γ inhibitor,the expression of mRNA and protein of PPAR-y were inhibited.Additionally,western blotting showed that Tan ⅡA+T0070907 intervention could inhibit Smad2/3 phosphorylation levels.Conclusions(1)Tan ⅡA inhibits the progression of fibrosis in nonalcoholic fatty liver fibrosis mice,including inhibition of inflammatory response,oxidative stress and fibrosis.(2)Tan ⅡA inhibits TGF-β1 induced fibrosis in HSC-T6 cell.(3)Tan ⅡA inhibits nonalcoholic fatty liver fibrosis by regulating JAK2/STAT5/PPARγ/Smad2/3 signaling pathway. |
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