Effect And Mechanism Of Matrine On Improving Calcium Balance Of Endoplasmic Reticulum In The Treatment Of Nonalcoholic Fatty Liver

Background Nonalcoholic fatty liver disease(NAFLD)is a metabolic disease with a high incidence and increasing year by year.The pathogenesis of the disease is complex and closely related to hepatic steatosis,insulin resistance,oxidative stress,and inflammatory response.Recent studies have shown that continuous endoplasmic reticulum stress(ERS)is the main cause of NAFLD,and the destruction of calcium homeostasis in the endoplasmic reticulum(ER)is the key to persistent endoplasmic reticulum stress response.Sarcoplasmic/endoplasmic reticulum calcium ATPase(SERCA)is a core pathway involved in the regulation of ER calcium reuptake.Under the pathological condition of NAFLD,calcium ion reuptake disorder caused by the inhibition of SERCA activity can lead to ER calcium disorder,which could induce continuous ERS and then promote the continuous deterioration of NAFLD.The active ingredients or monomers extracted from traditional Chinese medicine have been used in the treatment of various clinical diseases,and their effectiveness is significant and the toxicity is low.Matrine(Mat)is the main active ingredient of Sophora flavescens,and has various pharmacological effects such as anti-inflammatory,anti-tumor and anti-fibrosis.Studies have found that Matrine can attenuate liver fatty lesions induced by carbon tetrachloride and reduce serum aminotransferase levels in rat models with hepatitis.The previous study showed that the extract of Sophora flavescens can effectively improve the liver fatty lesions of NAFLD mice and regulate the calcium ions in liver cells.At the same time,the existing literature reports that Matrine can improve fibrosis and glucose disorder through inhibiting ERS,and can exert anti-cancer effect through inducing ERS.Its bidirectional regulation in ERS is still controversial.Therefore,the protective effect of Matrine on NAFLD/ nonalcoholic steatohepatitis(NASH)and its influence and molecular mechanism on ERS in this disease need to be clear.Objectives 1.To clarify the protective effect of Matrine on NAFLD and NASH through animal experiments.2.To clarify the regulation effect of Matrine on endoplasmic reticulum stress in hepatocytes through cell experiments.3.To preliminarily clarify the mechanism of Matrine on endoplasmic reticulum stress through calcium fluorescence and molecular docking experiments.Methods 1.100 C57BL/6J mice were randomly divided into 10 groups.Five groups of NAFLD model induced by high-fat diet(Con group,HFD group,HFD+Mat L group,HFD+Mat M group,HFD+Mat H group)were treated with Matrine for 5-12 weeks.Five groups of NASH model induced by methionine deficiency(Con group,MCD group,MCD+Mat L group,MCD+Mat M group,MCD+Mat H group)were treated with Matrine for 3-6 weeks.The mice were killed after weighing.The blood of mice was collected and the serum levels of TG,TC,ALT,AST,TNF-?,IL-6 and IL-10 were detected.The liver tissues of mice were stained with H&E,oil red O and the content of the related proteins were determined.2.L02 human hepatocytes were divided into normal control group(Con group),palmitic acid model group(PA group)and palmitic acid + low,medium and high dose Matrine treatment group(PA+Mat L group,PA+Mat M group,PA+Mat H group)with palmitic acid as model reagent.The changes of lipid droplets in cells were detected by oil red O staining,the oxidative stress status in cells was detected by ROS fluorescence,the mitochondrial potential was detected by mitochondrial JC-1 fluorescence,endoplasmic reticulum stress was detected by immunohistochemistry,and apoptosis was detected by Annexin V-FITC/PI staining.Moreover,the expression of lipid metabolism,apoptosis and endoplasmic reticulum stress-related proteins were detected by Western Blotting.3.L02 cells were divided into Con group,PA group,PA+Mat L group,PA+Mat M group,PA+Mat H group and Matrine group with different doses(Mat L group,Mat M group,Mat H group).After 12 hours of culture,the changes of intracellular calcium level were detected by Fura-3/AM calcium fluorescence detection reagent and fluorescence enzyme labeling instrument.Meanwhile,the effects of Matrine on calcium homeostasis in normal L02 cells were studied by transient stimulation experiments with Matrine and calcium channel inhibitor,thapsigargin(Tg)and 2-aminoethoxydiphenyl borate(2-APB),and the interaction between Matrine and SERCA was verified by computer molecular docking.In addition,the regulation of Matrine on SERCA was verified by detecting SERCA activity.Results 1.(1)In high-fat diet-induced NAFLD mice,Matrine could effectively improve the fatty lesions of liver,reduce the weight of mice and the levels of TC and TG in serum,and inhibit the expression of lipid synthesis-related proteins in liver.(2)In methionine-deficient diet-induced NASH mice,Matrine could effectively improve the fatty and inflammatory lesions of liver,reduce the levels of ALT,AST and proinflammatory factors in serum,and inhibit the expression of ERS-related proteins in liver.2.In palmitic acid-induced hepatic L02 cells,low and medium doses of Matrine could significantly reduce lipid accumulation and lipid synthesis-related protein expression in hepatocytes,reduce ROS level,alleviate mitochondrial damage and inhibit ER stress response and apoptosis in hepatic L02 cells,while high dose of Matrine had no significant effect.3.(1)In long-term incubation experiments,Matrine at low and medium dose inhibited the increase of cytoplasmic calcium in PA-induced L02 hepatocytes.In transient stimulation experiments,Matrine increased cytoplasmic calcium in L02 hepatocytes in a concentration-dependent manner.(2)Matrine could partially inhibit the elevation of cytosolic calcium ions by classical SERCA inhibitors(Tg).Molecular docking and activity experiments showed that Matrine has a inhibiting effect on SERCA.Low and medium doses of Matrine could exert competitive inhibition,indirectly restore the SERCA activity,improve the calcium balance of endoplasmic reticulum and inhibit the endoplasmic reticulum stress response,but high dose of Matrine mainly played the role of inhibiting SERCA activity,and had no significant improvement on the endoplasmic reticulum stress response.Conclusions 1.Matrine can inhibit liver fatty lesions,inflammatory response and ERS response,thereby improving the status of NAFLD in mice.2.Matrine can improve ERS response by competitive inhibition of SERCA to restore calcium balance,and play a protective role in NAFLD.3.This study preliminarily clarified the pharmacodynamics and mechanism of Matrine in improving NAFLD,which provides a new perspective for explaining the mechanism of Matrine regulating ERS response and lays a theoretical foundation for expanding the clinical application of Matrine.