Study On Hepatototoxicity Mechanism Of Cyclovirobuxine D

Aim:To observe the hepatotoxicity of Cyclovirobuxine D(CVB-D)and investigate the mechanisms.Methods:1.MTT method was used to determine the effect of CVB-D at different concentrations on the cell ability of L-02for different time.morphological changes were observed after L-O2was treated with CVB-D；The content of LDH、SOD、MDA in supernatant, GSH and Na+,K+-ATPase、Ca2+,Mg2+-ATPase、Ca2+-ATPase in L-O2were detected；FCM were used to observe the cell cycle and detect L-O2cell apoptosis by annexinV/PI staining;We observed cell morphology by electron microscope and cell nucleus morphology by Hoechst33258staining;Besides,we used laser scanning confocal fluorescence microscope to test the effects of CVB-D on mitochondrial membrane potential and intracellular Ca2+of L-O2cell.2. The SD rats were randomized into4groups,control group and CVB-D(3、6、12mg·kg-1)groups, the drugs were given by intragastric administration and the control group was given equivalence solvent.After CVB-D was ig given for eight weeks,the coefficient of liver and pathological changes in hepatic tissue were observed.3.SOD、MDA in serum and homogenate and Na+,K+-ATPase、Ca2+,Mg2+-ATPase、Ca2+-ATPase and GSH in homogenate were also detected by kits.Besides,we tested the differential gene expression between the CVB-D12mg·kg-1group and control group using gene chip technology. And then the expression of Na+,K+-ATPase、 Ca2+,Mg2+-ATPase、Ca2+-ATPase、Bax and Bcl-2mRNA were detected by RT-PCR.Results:1.CVB-D inhibited L-O2cells viability with time-and dose-dependent manner: L-O2cells morphology became round、contraction and off the wall after deal with CVB-D and the LDH release increased with dose-dependent manner(P<0.05,P<0.01):AnnexinV/PI staining confirmed the apoptosis was induced by CVB-D in L-O2cell and with significant necrosis company with significant chromatin condensation,CVB-D(10μM、20μM)can increase the phase of G2/M and reduce the phase of S;There are no significant change about SOD、MDA、GSH,but the ability of Na+,K+-ATPase、Ca2+,Mg2+-ATPase、Ca2+-ATPase were significantly decreased. CVB-D increased intracellular Ca2+and decreased mitochondrial membrane potential of L-O2cell.2.The coefficient of female rats’ liver were increased significantly;the content of ALT increased in CVB-D3mg·kg-1group(P<0.01),AST and ALP were increased in CVB-D6mg·kg-1group(P<0.05,P<0.01),ALT、AST、TBIL and ALP were increased in CVB-D12mg·kg-1group(P<0.05, P<0.01).Histopathological observation showed that CVB-D can cause toxic injury in rat liver;Gene chip results showed that CVB-D may affect the MAPK signal pathway, FGF、Ras、IL-1、TNF、P53were up-regulated in the pathway.In apoptosis pathway CytC、 Apaf-1、Bax、AIF were up-regulated,CDK1、CycA、APC/C、PTTG were down-regulated in cell cycle pathway,P21、CyclinD、Cdc2、BAI-1、p53R2were down-regulated in P53pathway. 3.CVB-D can not affect the ccontent of SOD、MDA in serum and SOD、MDA、GSH in homogenate, but the ATPase activity was reduced; RT-PCR results showed that CVB-D could down-regulate the expression of Ca2+-ATPase, Ca2+,Mg2+-ATPase, Na+,K+-ATPase,and Bcl-2mRNA in liver, but the expression of Bax was obversed.Conclusion:1.CVB-D can inhibit the activity of L-O2cells significantly, the mechanism may relate to inhibit the Na+,K+-ATPase、Ca2+,Mg2+-ATPase and Ca2+-ATPase activity, causing intracellular calcium overload, induced apoptosis and necrosis, but not the oxidative damage.2.Oral administration of large dose of CVB-D for a long time have significant toxic effects in rats, microarray gene showed that CVB-D can affect the metabolic pathway、cell apoptosis、calcium binding channels, the genes changed may induce the Hepatotoxicity.