Effect Of Different Kinds Of Calculus Bovis Combined With Realgar On Arsenic Induced Hepatotoxicity

Realgar is a sulfide mineral,mainly containing arsenic tetrasulfide(As4S4),which has the functions of detoxification,insect killing,dampness,phlegm removal and malaria control.It has been used in China for thousands of years.Because of the toxicity of soluble arsenic in Realgar and its preparation,therefore,the safe use and supervision of Realgar is very important.At present,the safety and scientificity of clinical application of Realgar have been questioned,and the mechanism of its toxic components and toxic effects has not been fully clarified.It has been proved that the toxic components of Realgar may be related to arsenites(As(Ⅲ))in the soluble arsenic.As(Ⅲ)has strong binding force with mercaptol.When it gathers in cells,it can improve ROS level by inhibiting glutathione(GSH)and mercaptoenzyme activity,thus directly affecting the metabolism,redox process,inflammatory immunity,proliferation and apoptosis of cells.The cytotoxic effect of arsenic mainly shows oxidative damage,apoptosis,and eventually leads to target organ damage.In human body,four kinds of arsenic As(Ⅲ),As(Ⅴ),MMA and DMA can be detected.The transport of arsenic mainly depends on multidrug resistance associated protein(MRP2).Liver is the main place for arsenic metabolism and transport,so the hepatotoxicity of Realgar should be paid more attention.Natural Calculus Bovis(NCB,CB)is a commonly used precious traditional Chinese medicine,which can be used to clear away heat,detoxify and calm the nerves.Artificial Calculus Bovis(ACB)and cultured Calculus Bovis sativus(CBS)are widely used in the market because of their scarce sources and high price.CBS is often considered to be an ideal substitute for CB.At present,it is widely used in the long-term treatment of chronic liver disease,which can prevent the accumulation of phlegm and blood stasis in the liver.According to our research on the compatibility of ancient Niuhuang Realgar formula,we found that the ancients often used them together.The compatibility theory of bezoar Realgar pair is still mainly recorded in ancient books of traditional Chinese medicine,and modern research is rare.Our previous study found that Calculus Bovis contains many active components,mainly bile acids can activate nuclear receptor farnesoid X receptor(FXR),and then activate MRP2,which may reduce the accumulation of arsenic in cells and reduce the damage through this pathway.And bile acids,bilirubin,taurine and other active ingredients of Calculus Bovis may also be combined with Realgar through anti-inflammatory,anti-oxidation,regulation of intestinal flora structure and other aspects to achieve the purpose of enhancing efficiency and reducing toxicity.Objective:Comprehensive study of different kinds of Calculus Bovis and Realgar compatibility to affect the absorption,metabolism and transport of arsenic in the liver and the mechanism of chronic arsenic poisoning through intestinal flora,so as to provide the basis for the theoretical research of different Calculus Bovis and Realgar compatibility prescriptions,and provide solutions and scientific theoretical support to ensure the safety of Calculus Bovis and Realgar clinical medication.Methods:(1)In animal experiment,ICR mice were used as the research object,and different doses of Realgar were given by gavage for 7 days and 28 days respectively to establish the liver toxicity injury model;ACB,CBS,FXR agonist obacholic acid(OCA)combined with Realgar were given by gavage for 28 days,serum and liver tissue were collected at the end of the administration cycle.The levels of ALT,AST and ALP in serum were detected;HE,Sirius red and Masson staining were used to evaluate the degree of pathological damage of liver tissue;DHE and DCFH-DA staining were used to observe the production of ROS;Meanwhile,the contents of TSH,GSH,MDA and the activities of SOD,GPX,GST and other antioxidant enzymes in liver tissue were detected;The secretion of cytokines was detected by ELISA;The expression of Pro-apoptotic proteins Caspase-3,-8,-9 in liver tissue was observed by immunofluorescence microscope,and the expression of various cytokines and apoptotic proteins in liver was detected by protein chip.(2)The serum and liver of mice fed by gavage for 7 days and 28 days were taken,the effects of Realgar on the metabolism of 35 kinds of metal elements in serum and liver were detected by ICP-MS.at the same time,As(Ⅲ),As(Ⅴ),MMA and DMA were detected by HPLC-ICP-MS.the effects of Calculus Bovis on the metabolism and distribution of As(Ⅲ),As(Ⅴ),MMA and DMA in liver and serum of mice were analyzed.(3)In vitro experiments,L02 human normal hepatocytes were used as the research objects.CCK-8 and SRB methods were used to screen the toxic dose of Realgar soluble arsenic and verify the protective effects of three kinds of Calculus Bovis and FXR agonists;The ROS was detected by DHE and DCFH-DA staining;The antioxidant enzymes and lipid peroxidation indexes MDA,such as TSH,GSH,SOD,etc.were detected in liver tissues.The secretion of inflammatory factors in L02 cells was detected by ELISA.The expression of Caspase-3,8,9 in L02 cells was observed by immunofluorescence microscope.In order to further verify the protective effect of FXR/MRP2 on Calculus Bovis,FXR and MRP2 inhibitors were used to detect the protein expression of FXR and MRP2 in vivo and in vitro by immunofluorescence double staining and Western blotting,and the content of arsenic in L02 cells was detected by ICP-MS;the expression of several apoptotic proteins was detected by protein chip.(4)Bile acid targeted metabonomics based on UPLC-MS/MS was used to study the effects of different sources of Calculus Bovis and Realgar on liver bile acid profiles of normal mice after 28 days of administration,and to analyze the effects of bile acid homeostasis in mice.16sr DNA sequencing was used to study the changes of intestinal microflora in mice treated with different Realgar for 28 days,and the changes of intestinal microflora in mice treated with ACB and CBS combined with Realgar for 28 days,to screen the different flora affected by Calculus Bovis and Realgar compatibility compatibility,and analyze the correlation between the different bacteria and arsenic metabolism and bile acid level,so as to explore the candidate intestinal bacteria related to hepatotoxicity affected by different kinds of Calculus Bovis,the correlation between different bacteria and liver arsenic and bile acid was analyzed.Result:(1)After administration for 7 days and 28 days,Realgar could reduce body weight,increase liver to body ratio,and cause liver toxicity injury in mice.After administration for 7 days,Realgar could increase ALT and AST levels,but it had no significant effect on serum liver function indexes after administration for 28 days.After 28 days of administration,HE,Sirius red and Masson staining showed that Realgar could lead to liver pathological changes in a dose-dependent manner,leading to mild fibrosis.ACB,CBS and OCA can improve liver injury,portal inflammatory cell infiltration,hepatocyte degeneration and necrosis,and reduce collagen deposition in varying degrees;Meanwhile,ACB,CBS and OCA can increase the content of TSH and GSH,increase the activity of SOD and other antioxidant enzymes,reduce the level of MDA,reduce the secretion of inflammatory factors,and inhibit the activity of,pro apoptotic protein caspase-3-8,-9.The expression of inflammatory factors and apoptotic proteins in liver was decreased by protein chip(2)ICP-MS was used to detect the metabolism of metal elements in serum and liver of mice fed with Realgar for 7 days and 28 days.It was found that Realgar could significantly affect the content of metal elements in serum and liver,such as V,Mo,Zn,Cu,Se and other trace elements.The content of As(Ⅲ),As(Ⅴ),DMA,DMA in serum and liver of mice fed with Realgar for 7 days and 28 days were significantly increased in a dose-dependent manner.ACB,CBS and OCA can improve the disorder of metal metabolism,reduce the content of various valence arsenic in liver and serum,but do not affect the methylation of arsenic.(3)CCK-8 and SRB were used to screen the damage dose of androgen soluble arsenic to L02 cells was lppm,and the protective effects of three kinds of taurine and FX agonists were verified.The three kinds of rhubarb and FXR agonists can improve the TSH and GSH content of L02 cells,increase the activity of SOD and other antioxidant enzymes,reduce MDA level,moderately reduce the secretion of cytokines,reduce the activity of Caspase-3,-8 and-9,and reduce the content of arsenic in the cells.Many apoptotic protein expression is reduced by protein chip detection.The inhibition of FXR/MRP2 can counteract the protective effect of CBS,increase the accumulation of arsenic in cells,promote oxidative stress and improve the expression of apoptosis related proteins in L02 cells.(4)16Sr RNA high-throughput sequencing showed that the diversity and richness of intestinal flora in mice induced by Realgar decreased significantly.CBS improved the diversity of intestinal flora in mice induced by Realgar,and ACB had no significant effect.In OTU taxonomic level,CBS and ACB could affect several different strains.CBS and ACB can reverse the effect of Realgar,but OCA has no obvious effect on this kind of flora.Realgar three dose groups can increase the content of bile acids in mice liver,ACB,CBS,OCA can improve the bile acid structure.At the same time,the correlation analysis showed that all kinds of cholic acids were associated with Campilobacterota,Actinobacteriota,unclassified_k__norank_d__Bacteria.It is inversely proportional to bacteria and directly proportional to Bacteria,Firmicutes and patescibacteria.At the genus level,cholic acids were also correlated with different bacterial populations.Under the interference of two kinds of Calculus Bovis and OCA,the correlation between intestinal microflora and liver cholic acid was significantly interfered.At the class and genus level,the correlation logarithm decreased.Only LCA and TDCA were correlated with a variety of different microflora.Conclusion:(1)Realgar administration for 7 days and 28 days can induce oxidative stress imbalance in mice liver and L02 cells,increase the secretion of inflammatory factors,and eventually lead to hepatocyte apoptosis,liver injury and mild fibrosis.Different kinds of Calculus Bovis combined with Realgar for 28 days can reduce the imbalance of liver oxidative stress,reduce the secretion of inflammatory factors,reduce the damage of hepatocyte apoptosis and mild fibrosis.(2)Different kinds of Calculus Bovis combined with Realgar can significantly improve the metabolic disorder of Mo,V,Zn,Cu,Se and other trace elements in serum and liver of mice caused by Realgar,and inhibit the content of arsenic in liver,serum and L02 cells.(3)Different kinds of Calculus Bovis can activate the FXR/MRP2 pathway and promote arsenic efflux in varying degrees,while inhibiting the FXR/MRP pathway can reverse the effect of Calculus Bovis.(4)Different doses of Realgar can reduce the diversity and richness of intestinal flora in mice,and disturb the structure of intestinal flora.Different kinds of Calculus Bovis can also affect the diversity and richness of intestinal flora in mice,and can regulate the composition of intestinal flora and improve the disorder of intestinal flora in mice.In vitro CBS is better than ACB.(5)CBS may be better than ACB in reducing arsenic accumulation in hepatocytes,anti apoptosis and regulating intestinal flora structure.