Development And Application Of Screening Method Of Hepatoprotective Composition In Traditional Chinese Medicines With Hepatocyte Mitochondria As Target

BackgroundLiver is not only the most important metabolism organ,but also one of the most vulnerable organs.Liver injury has become one of the important diseases that threaten human health.Mitochondria are the cell"engines"and play a fundamental role in energy metabolism,free radicals production,apoptotic regulation and lipid metabolism.Research results have shown that the damage of structure or functions of hepatocellular mitochondria will lead to liver injury,whilst many traditional Chinese medicines?TCMs?can regulate mitochondria to protect the injured liver,and its medicinal substances are still unclear.Therefore,it is an important way to reveal the substance basis of hepatoprotective action of TCMs and find lead compounds by using mitochondria as targets to screen active ingredients of traditional Chinese medicines.However,the current method of screening liver active ingredients of TCMs using mitochondria as targets is limited,and it is necessary to prepare a monomer compound and then carry out pharmacological experiments.Therefore,it is necessary to find an analysis method that can rapidly and effectively screening the hepatoprotective active ingredients from a complex system of TCMs without any separation and purification.ObjectDevelopment of an analysis method for rapid and effective fishing hepatoprotective ingredients using hepatocyte mitochondria as a target,and its application in TCMs to find hepatoprotective compounds,and to provide a strong basis for revealing the material basis that regulate the mitochondria and exert hepatoprotective effects.MethodFresh liver tissue of SPF-grade SD male rats was quickly excised and homogenized,immediately followed by differential Centrifugation to obtain hepatic mitochondria?HM?.The structural intactness,purity and biological functions of the prepared HM fractions were examined through transmission electron microscopy,a commercial Mitochondrial Fraction Purity Marker Enzyme Assay Kit?SDH/AP?and fluorescence quenching,respectively.A new mitochondria-targeted screening methods of hepatoprotective constituents that had the capability of?recognition-separation-identification?were separately developed by combining affinity-ultrafiltration with Liquid Chromatography/Mass Spectrometry?LC/MS?.Afterward,the feasibility of the screening methods was evaluated using positive drugs?silybin,daidzein?,negative drugs?amoxicillin,glucuronolides?and mixed standard solution?comprised of positive and negative drugs?.The effects of important conditions?concentration of mitochondria,concentration of screened sample and incubation time?of the screening methods to screening results was investigated using screened TCMs extracts?Peucedani Radix and Notopterygii Rhizoma et Radix?.The two Chinese herbal medicines?Peucedani Radix and Notopterygii Rhizoma et Radix?were screened by the developed screening method,and the chemical structures of screened bioactive constituents were assigned using LC/MS and standards.To verify the direct effect of the screened compounds on mitochondria,the Membrane permeability transition pore?mPTP?opening,membrane potential???m?and ATPase activity of isolated hepatic mitochondria were examined through Ultraviolet Spectrophotometry,Fluorescence Spectrophotometry,and ATPase Assay Kits,respectively.The hepatoprotective effect of screened compounds was investigated in vitro on an acute liver injury model induced by CCl₄ in L02 cells,through the survival rate,alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,malondialdehyde?MDA?and superoxide dismutase?SOD?.In addition,further verification of the hepatoprotective activity of the compounds in vivo was implemented in a mouse model of CCl₄-induced injury,by examining the hepatic index,liver morphology,content of ALT and AST on serum,MDA,glutathione?GSH?and SOD in liver tissue,and mitochondria-involved indexes(mPTP,??m,Na⁺-K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,and MDA).ResultsIntegrity,purity and biological functions evaluation of HM.The representative electron micrographs of the isolated HM reveal that the mitochondrial cristae and membrane were intact and continuous,which suggests the high structural integrity of the HM.The detected results show that the mitochondria-marker was at least 20 times than the lysosome-marker in the purified mitochondria,suggesting a high level of purity.In addition,the satisfactory results in which the fluorescence intensity of the HM fraction was remarkably augmented after CCCP treatment,suggesting the collapse of??m of the HM,indicate that the enriched HM still possessed their biological functions.Development of the screening system and evaluation of its feasibility.The screening method could efficiently recognize bioactive constituents binding to mitochondria?positive drugs?from mixtures.The recognized bioactive constituents could be isolated,whilst inactive impurities were directly eliminated by ultrafiltration technique.Finally,the structural characteristics of corresponding bioactive constituents were conveniently identified by LC/MS,and this verified the feasibility of the method.Investigation of important influential factor of screening method.Three conditions could significantly affect the screening results?numbers and species of screened bioactive compounds?of the two methods.In a general,the primarily condtions was as follows:mitochondrial concentration was 1.00 g/L,sample concentration was 12.68g/L and incubation time was 90 min.Screening the different samples,however,these influential factors should be separately optimized to obtain optimal screening results.Study on the screening of hepatoprotectie constituents of TCMs.Totally 19bioactive constituents that could bind with mitochondria were discovered from 2 TCMs.Fourteen kinds of active compounds were assigned by LC/MS and reference products,and all the 14 active compounds were found as new mitochondrial ligands.Validation of screening results using isolated liver mitochondria were found that 7 bioactive constituents screened could significantly inhibit the mPTP opening,reduce mitochondrial membrane potential,increase ATPase activity on isolated liver mitochondria,and further protect mitochondria.Screening of hepatoprotective of compounds by in vitro and in vivo.It was found that 5 screened compounds?praeruptorin A,praeruptorin B,praeruptorin D,praeruptorin E and isoimperatorin?could significant increase in cell viability and superoxide dismutase?SOD?,and the notable decrease in alanine transaminase?ALT?,aspartate transaminase?AST?and malondialdehyde?MDA?,and play a role in liver protection in vitro.In addition,2 screened compounds?praeruptorin B and isoimperatorin?could obviously reduce liver index,improve liver morphological lesions,reduce serum ALT and AST levels,decrease the liver MDA content,the significant elevation SOD and glutathione?GSH?,and effectively improve mitochondrial damage(decrease mPTP openness,recovery??m,increase mitochondrial Na+-K+-ATPase,Ca2+-Mg2+-ATPase activity and decrease MDA content,which effectively improve the liver injury.ConclusionIn summary,we had developed a HM-targeted screening methods with the ability of?recognition-separation-identification.This provide a new approach for the rapid and effective screening of hepatoprotective ingredients in the complex system of TCMs.Total 19 HM-targeted molecules were successfully captured from Peucedani Radix and Notopterygii Rhizoma et Radix,and 7 of them can be directly affected by mitochondria to affect the mitochondrial mPTP openness,??m and ATPase activity.These components may be the main material basis for the direct action of traditional Chinese medicine on liver mitochondria.Furthermore,the hepatoprotective abilities of seven screened compounds were exposed in vitro and in vivo.The results obtained will help to reveal the medicinal substances that have been screened to regulate the mitochondria and exert hepatoprotective effects.