Mechanism Of Sini Powder And Its Representative Component Paeoniflorin In Improving Non-alcoholic Fatty Liver Disease

ObjectiveNon-alcoholic fatty liver disease(NAFLD)is a progressive liver disease,including steatosis,non-alcoholic steatohepatitis,fibrosis,cirrhosis and hepatocellular carcinoma.The main culprit of NAFLD is the imbalance between energy intake and energy expenditure,which leads to ectopic fat accumulation.Sinisan is a commonly used liver-regulating compound recipe,which has the effects of removing pathogenic factors,relieving depression,soothing the liver and regulating the spleen.Clinical applications and experimental studies have proved that Sinisan has hepatoprotection,but its potential mechanism of prevention and treatment is still unclear.The pathogenesis of NAFLD is the accumulation of free fatty acids in the liver,resulting in hepatic steatosis,increasing the sensitivity of liver to oxidative stress and inflammation,and promoting the process of NAFLD.Therefore,this paper simulated the formation process of NAFLD,used free fatty acid to induce NAFLD cell model,and studied the therapeutic effect and mechanism of Sinisan on NAFLD.Inflammation and oxidative stress are important components of almost all acute and chronic liver diseases.In Sinisan,the single drug with the strongest immunoregulatory effect is Radix Paeoniae Alba.Paeoniflorin is one of the main active components of Radix Paeoniae Alba,which has anti-inflammatory,immunomodulatory and hepatoprotective effects.Paeoniflorin may be one of the effective substances of Sinisan.Exploring the effective substances are of great significance to the research and development of new drugs and the quality control of traditional Chinese medicine.It is also the basis of optimizing and simplifying the traditional Chinese medicine compound.Therefore,we used cell models of inflammation and oxidative damage induced by lipopolysaccharide and hydrogen peroxide respectively to explore the effect and mechanism of paeoniflorin on inflammation and oxidative stress,and to explore the role of paeoniflorin in the treatment of NAFLD with Sinisan.Methods1.HepG2 and LO2 cells were treated with different concentrations(0,0.25,0.5,1,1.5 and 2 mM)of free fatty acid(FFA)for 24 h.CCK-8 method was used to detect the effect of different concentrations of FFA on cell proliferation.Then the cells were stimulated with different concentrations(0,0.25.0.5 and 1 mM)of FFA and corresponding concentrations(0.0.25%.0.5%and 1%)of bovine serum albumin(BSA)for 24 h.The formation of lipid droplets in cells was detected by oil red O staining,and the absorbance at 490 nm was measured.Triglyceride(TG)content in cells after treatment by FFA at different concentrations and at different time points was determined.Western blot was used to detect the protein expression of ADRP and SREBP1,and immunofluorescence was used to detect ADRP expression.2.LO2 cells were treated with Sinisan decoction of different concentrations(0.0.01,0.1,1,10 and 100 mg/mL)and different concentrations of compound C(0,1,5,10 and 20 μM)for 24 h.CCK-8 method was used to detect the effect of different concentrations of Sinisan and compound C on cell proliferation.Different concentrations(0,0.1,1,10 mg/mL)of Sinisan and 1 mM FFA were used to stimulate the cells for 24 h.The ultrastructure of the cells was observed by transmission electron microscope.SOD activity,ROS staining,MDA and TG content were detected.The formation of lipid droplets in 2D and 3D cells was observed by oil red O staining.The expression of CD36,ABCA1,CPTla and IP3R1 in 2D and 3D cells were detected by RT-PCR.The levels of TNFα and IL6 in cell culture supernatant were detected by ELISA.The protein expression of AQP4,Cx43,TNFα,RhoA,NLRP3,caspase 1,IL1β,LC3II,Bax and Bcl2 were detected by Western blot.In addition,the cells were divided into control group,model group,Sinisan group,compound C group and compound C plus Sinisan group.The protein expression of TNFα,RhoA,LC3II,Bax and Bcl2 were detected by Western blot.3.Cells were stimulated with different concentrations of lipopolysaccharide(0,0.01,0.1,1 and 10 μg/mL)and paeoniflorin(0,25,50,100 and 200 μM)for 24 h.The effects of different concentrations of lipopolysaccharide and paeoniflorin on cell proliferation were detected by CCK-8 method.The levels of TNFa and IL6 in cell culture supernatant were detected by ELISA.The cells were pretreated with 50 and 100 μM paeoniflorin for 2 h,and then treated with paeoniflorin and lipopolysaccharide for 6 h.The expression of AQP4,Cx43,TNFα,MMP9,RhoA,NLRP3,ASC,caspase 1 and IL1β were detected by Western blot.4.Cells were stimulated with different concentrations of hydrogen peroxide(0,200,400,600 and 800 μM)for 24 h.CCK-8 method was used to detect the effect of different concentrations of hydrogen peroxide on cell proliferation.The cells were pretreated with 50 and 100 μM paeoniflorin for 2 h,and then treated with paeoniflorin and hydrogen peroxide for 8 h.The protein expressions of RhoA,ROCK1,NLRP3,C-PARP1 and CK18 were detected by Western blot.Results1.In HepG2 cells,1-2 mM FFA significantly inhibited cell viability,but other concentrations had no significant effect on cell viability.In LO2 cells,2 mM FFA significantly inhibited cell viability,but other concentrations had no significant effect on cell viability.BSA did not affect the formation of lipid droplets.With increasing FFA concentration,the fluorescence of oil red O staining was increasing,the red part is increasing under light microscope and the absorbance is increasing.The results of HepG2 and LO2 were similar.Different concentrations of BSA had no significant effect on the content of TG.FFA of different concentrations could increase the content of TG,which was positively correlated with the concentration.In 1 mM FFA treatment,TG content increased with the increase of treatment time,there was significant statistically difference from 6 h.When FFA was prepared with BSA without free fatty acid,there was no significant difference in SREBP1 protein expression and activation,while the abundance of ADRP expression in HepG2 cells was higher than that in LO2 cells.When FFA was prepared with BSA containing free fatty acids,the expression of SREBP1 protein was significantly different.After 12 h of treatment,there was no significant difference between the two cells.After 24 h of treatment,SREBP1 protein was over-activated in HepG2 cells,and GAPDH was significantly decreased,but there was no significant change in the expression of SREBP1 protein in LO2 cells.LO2 cells are suitable for establishing a benign and chronic steatosis cell model.2.Sinisan at the concentration of 0.01-1 mg/mL did not affect cell proliferation,while Sinisan inhibited cell proliferation at the concentration of 10 mg/mL and 100 mg/mL.Transmission electron microscopy showed that Sinisan inhibited the formation of lipid droplets,which was consistent with oil red O staining.Sinisan reduced the content of TG at the concentration of 0.1 mg/mL and 1 mg/mL,and the effect of Sinisan on 3D cell level is better than that on 2D cell level.Sinisan could improve the mRNA levels of CD36,CPT1 a and IP3R1 in 2D and 3D cell levels.In 2D cell level,Sinisan could not improve the decrease of ABCA1 level induced by FFA,but in 3D cell level,Sinisan could up-regulate ABCA1 mRNA level.According to the ultrastructure of organelles,autophagy was ubiquitous,and FFA induced mitochondrial damage and endoplasmic reticulum swelling.Sinisan can significantly reduce the damage of mitochondria and swelling of endoplasmic reticulum.Sinisan reduced MDA content and ROS expression,increased SOD enzyme activity,decreased the production of TNFα and IL6,decreased the protein expression of AQP4,Cx43,TNFα,RhoA,NLRP3,caspase 1,IL1β,LC3Ⅱ and Bax,and up-regulated the protein expression of Bcl2.Compound C had obvious cytotoxicity.Compound C inhibited the protein expression of TNFα,RhoA,LC3II and Bax,and increased the protein expression of Bcl2.3.Lipopolysaccharide and paeoniflorin did not affect cell viability.Paeoniflorin reduced the production of TNFα and IL6,and down-regulated the protein expression of AQP4,Cx43,TNFα,MMP9,RhoA,NLRP3,ASC,caspase 1 and IL1β.600 μM hydrogen peroxide inhibited cell proliferation,and 800 μM hydrogen peroxide significantly promoted the apoptosis of cells.Paeoniflorin could inhibit the protein expression of RhoA,NLRP3,C-PARP1 and CK18,and had no obvious effect on ROCK1.Conclusion1.Sinisan can reduce the accumulation of lipid,improve lipid metabolism,improve the ultrastructure of cells,inhibit inflammation and oxidative stress,and have a preventive effect on NAFLD.2.Sinisan inhibits ROS level and activation of RhoA and NLRP3 inflammasome,and regulates ROS/RhoA/NLRP3 signaling pathway.Therefore,anti-inflammation and anti-oxidation are one of the mechanisms.Sinisan inhibits autophagy and apoptosis,which is one of its mechanisms.3.Paeoniflorin inhibits cell swelling and cell apoptosis by regulating RhoA/NLRP3 pathway,and plays an important role in anti-inflammatory and antioxidation.It has little effect on RhoA/ROCK1 pathway.Under the same dosage of paeoniflorin,the traditional Chinese medicine compound is better than monomer.However,paeoniflorin can be used as one of the effective substances of Sinisan.