Study On The Protective Effects On Drug Hepatic Injury And Synergistic Anti-inflammative Effect On Compatibility With Aconitum Brachypodum Diels Of Verattilla Bailonii Franch

Verattilla bailonii Franch belongs to the genus of Veratrilla baillonii?Family Gentianaceae?.Its root has been used as a traditional medicine of China and it used for the Hyperactivity cough,diarrhea,burns and so on.Its chemical compositions are mainly ridoid and xanthogenones,which possess hepatoprotective,anti-inflammatory,analgesic effects and so on.Tripterygium wilfordii Hook F.belongs to the genus of Tripterygium,which has been used for dampness,Huoxuetongluo,swelling,analgesia and insecticide detoxification.But it is limited by its hepatotoxicity in clinic.Aconitum brachypodum Die1 is often used by Yunnan natives to treat pain and inflammation-related diseases.In our laboratory,we revealed that Verattilla bailonii Franch can protect from aconitum-induced toxicity.Gene chip analysis showed that there existed synergistic effect between Aconitum brachypodum Die1 and Verattilla bailonii Franch.The present study will focus on two experiments.One is to investigate the hepatoprotection of Verattilla bailonii Franch on Tripterygium-induced toxicity.Another is to study the synergistic anti-inflammatory effect between Aconitum brachypodum Diel and Verattilla bailonii Franch.1.The water extract of Veratrilla baillonii Franch?WVBF?attenuates tripterygium wilfordii glycosides?TG?-induced hepatoxicity in Vivo and in Vitro¹Drug induced hepatic injury model was prepared using TG?270 mg/kg?administration for 4 days,then WVBF?25,50,100 mg/kg?was given for another 7days.Clinical course and body weights as well as morphological changes in target organs were examined.The serum biochemistry was assessed to delineate the pathophysiological link among TG exposure.Hepatic expression of MicroRNA-122was measured in order to understand the underlying mechanism.The effect of 20?M triptolide?TP?and six monomers on the survival rate of WVBF on HepG2 cells was assessed by MTT assay.ROS level was measured by fluorescence probe DCFH-DA,and Q-PCR?Realtime Polymerase Chain Reaction?was used to measure the expression of mitochondrion DNA?mtDNA?and metabolic enzymes CYP3A4/1A2levels.Mitochondrial membrane potential???m loss?was analyzed using flowcytometry assay.The TG-induced hepatotoxicity was demonstrated by the decreased body weight,increased liver to body weight ratio,altered hepatic histology,increased plasma levels of hepatic enzymes and hepatic expression of MicroRNA-122?miR-122?in comparison with the treatment of water in both male and female mice.Pre-treatment with WVBF for 7 days attenuated all aforementioned TG-induced toxic effects including oxidative stress in the liver.The results showed that the optimum concentration of 20?M TP was the best and gnetipoicrosdie was the main active component in WVBF.Treatment with gentiopicroside for 24 h could protect the HepG2 hepatocytes from TP–induced toxic effects such as decrease of mtDNA,CYP3A4/1A2 and??m loss.WVBF can reduce the TP-induced acute toxic symptom,secondly,the serum chemistry index indicate that 25 mg/kg,50 mg/kg,100 mg/kg dose of WVBF reduce the liver disorder induced by TP to some extent.2.Efficacy of different compatibilities of Aconitum brachypodum Diels?CFA?and WVBFLPS-induced RAW264.7 cell was used to explore the synergistic anti-inflammatory effects between CFA and WVBF.Isobologram analytical method and interaction index for analysis were used to determine which is the best compatibility of the proportion of CFA and WVBF.The results indicated that CFA?WVBF and the CFA of WVBF in compatibility with the ratio of 1:2,1:5 and 1:10 all significantly inhibited LPS-induced NO production in RAW264.7 cell.However,only 1:10 group of interaction index was less than 1,showing synergistic anti-inflammmtory effects.The combined use of CFA and WVBF could obviously inhibition iNOS,TNF-?,COX-2,IL-6 mRNA expression in RAW264.7,reduce the ROS level and calcium concentration,and its therapeutic effect in suppressing iNOS,TNF-?mRNA expression,ROS releasing and calcium concentration was better than separately used.Furthermore,the curative effect was the best when the compatibility of CFA and WVBF reached 1:10.The reduced NO production,iNOS mRNA expression,the ROS level and calcium concentration may be the drug treatment of synergistic anti-inflammatory mechanism.Conclusions:?1?WVBF can reduce TG-induced liver dysfunction in mice by down-regulation of MicroRNA-122 expression,serum biochemical indicators?ALT,AST,ALP,LDH?and MDA increase as well as increase TG-induced decrease of GSH and SOD contents.?2?The main active component of gnetipoicrosdie in WVBF can attenuate TP-induced mitochondrial DNA loss,mitochondrial membrane potential loss,and reducing intracellular ROS release.?3?1:10 of the mixture of CFA and WVBF is the best ratio,and synergistic anti-inflammatory effect is achieved by inhibiting LPS-induced iNOS gene expression in RAW264.7 cells,increased intracellular ROS release,and increased Ca²⁺concentration.