| Objective Endemic arsenism caused by coal-burning is a special type of endemic arsenism only distributed in Guizhou and Shanxi province in China.In addition to the typical skin lesions,the patients also have serious liver damage and liver cirrhosis is one of the main reasons for this type of arsenic poisoning death.Due to the pathogenic mechanism of arsenic poisoning has not been fully elucidated,the mechanism of liver damage becomes the research focus.Early research found that the occurrence and development of liver injury caused by arsenic related to oxidative stress and apoptosis,and p53 which had functions of controlling cell cycle progression and apoptosis had complex cross-talk with the mechanism of arsenic poisoning.However,the effect of p53-mediated apoptosis on arsenic induced liver injury from coal-burning remains unclear.To further clarify the mechanism of arsenic induced liver injury from coal-burning,in our study,based on the population resource of arsenic poisoning caused by coal-burning in Guizhou province,combined with in vivo animal experiment and in vitro experiment,the regulation of p53 expression and the effects of p53-mediated apoptotic pathway on arsenic induced liver injury were investigated by gene transcription,protein expression and posttranslational modification detection.In addition,the intervention effects and mechanism of Ginkgo biloba extract on arsenic induced liver injury were explored to provide new ideas and directions for the prevention and therapy strategies of arsenism.Methods Investigation spot and objects:In the endemic arsenism area with coal-burning-pollution(Jiaole village of Xinren county,Guizhou province),according to the standard of diagnosis for endemic arsenism(WS/T 211-2001)207 cases which exposed to arsenic were selected as the observation objects and then were divided into non-patient group(46 cases)and patient group(161 cases).According to diagnostic criteria of occupational toxic hepatopathy(GBZ 59-2010),the patient group was divided into non-obvious hepatopathy group(49 cases),mild hepatopathy group(65 cases)and moderate to severe hepatopathy group(47 cases).64 residents were selected as the control group in Daguoduo village without arsenic pollution about 13km away from the endemic arsenism area Establishment of arsenic poisoning model and Ginkgo biloba extract intervention model:Establishment of arsenic poisoning model:24 Wistar rats were divided randomly into four groups:normal control group,low-dose arsenic poisoning group,medium-dose and high-dose arsenic poisoning group.Arsenic poisoning rats were fed with feed containing different proportions of corn flour dried by high arsenic coal(arsenic contents were 25,50 and 100 mg/kg respectively)for 3 months.Normal control group was fed with standard food without arsenic.Establishment of Ginkgo biloba extract intervention model:24 Wistar rats were divided randomly into four group:normal control group,high-dose arsenic poisoning group,Ginkgo biloba extract intervention group and pure Ginkgo biloba extract group.Normal control group was fed with standard food for 4.5 months.After exposed to arsenic for 3 months,the high-dose arsenic poisoning group was fed with standard food for 1.5 months and the Ginkgo biloba extract intervention group was administered intragastrically with 25mg/kg.bw Ginkgo biloba extract,6 d/w,for 1.5 months.After fed with standard food for 3 months,the pure Ginkgo biloba extract group was administered intragastrically with 25mg/kg.bw Ginkgo biloba extract,6 d/w,for 1.5 months.Establishment of arsenic poisoning cellular model and intervention cellular model:Establishment of arsenic poisoning cellular model:L-02 cells were maintained in medium containing sodium arsenite(0,1,5,25,50 and 100 μmol/L)for 24 h.Establishment of intervention cellular model:L-02 cells were pretreated with PFTa(p53 inhibitor),CGK733(ATM/ATR inhibitor)and Ginkgo biloba extract,and then were treated with sodium arsenite(50 μmol/L)for 24 h.Observation indexes and methods:The arsenic contents of urine,hair and liver were detected by HG-ICP-OES.The liver function indexes of ALT,ChE,ALB,TBA and STB were measured.The pathological changes of liver were detected by HE staining.In addition,the apoptosis of hepatocytes was detected by TUNEL.The expressions of p53,Bax,Bcl-2,Fas and DR5 mRNA of peripheral blood and hepatocytes were performed by RT-qPCR.Finally,the expressions of p-p53(ser 20),p53,p-Chkl(Ser345),Chkl,Bax,Bcl-2,Fas and DR5 protein were detected by western blotting.Results The results of population research:The arsenic contents of urine and hair increased with the aggravation of hepatopathy and the arsenic contents of each patient group were significantly higher than the control group and non-patient group.Serum TBA of mild and moderate to severe hepatopathy group were significantly higher than the control group,non-patient group and non-obvious hepatopathy group.Serum ALB,ChE of each patient group were significantly lower than the control group and non-patient group.Results of RT-qPCR revealed that compared with the control group,non-patient group and non-obvious hepatopathy group,Bax and DR5 mRNA in peripheral blood of mild and moderate to severe hepatopathy group were significantly higher and p53 mRNA was significantly lower.There was no significantly difference of Bcl-2 and Fas mRNA in each group.The results of in vivo animal experiments and Ginkgo biloba extract intervention experiments:The arsenic contents of urine,hair and liver increased with the increase of arsenic dose.Compared with the control group,serum TBA of medium-dose and high-dose arsenic poisoning group were significantly higher,ALB of high-dose arsenic poisoning group and ChE of low-dose,medium-dose and high-dose arsenic poisoning group were significantly lower.Histopathological examination showed that arsenic poisoning rats appeared different degree of hepatocytes swelling,spotty necrosis or piecemeal necrosis,fibrous connective tissue proliferation and inflammatory cells infiltration.The general situation,growth inhibition,arsenic contents,liver function indexes and pathological changes of rats showed that arsenic poisoning model was established successfully.The apoptotic rate of hepatocytes increased in a dose-dependent manner induced by arsenic.Results of RT-qPCR and western blotting revealed that,with the increase of arsenic dose,p53 mRNA of peripheral blood and hepatic tissue while p-Chkl,p-p53,p53 protein of hepatic tissue increased,which accompanied by the increase of Bax,DR5 mRNA and Bax,.DR5,Fas protein.Bcl-2 mRNA and protein were increased firstly and then decreased and only Bcl2 mRNA of hepatic tissue in high-dose arsenic poisoning group was significantly lower than the normal control group.Ginkgo biloba extract can relieve toxic effects of rats induced by arsenic and improve liver function.After treated with Ginkgo biloba extract,the apoptotic rate of hepatocytes decreased and p53 mRNA,p-p53,p53 protein of hepatic tissue decreased,which accompanied by the decrease of Bax,DR5 mRNA and Bax,DR5,Fas protein.The results of in vitro experiments and intervention experiments:Results of RT-qPCR revealed that,with the increase of arsenic dose,p53 mRNA of L-02 cells decreased and p53 mRNA of 25,50 and 100 μmol/L NaAsO2 group were significantly lower than the control group.Western blotting showed that p-Chkl,p-p53,p53 protein were increased firstly and then decreased.Compared with the control group,p-Chkl and p-p53 protein of 25,50 μmol/L NaAsO2 group and p53 protein of 1,5,25 and 50 μmol/L NaAsO2 group were significantly higher.Bax,Bcl-2 and DR5 mRNA increased in a dose-dependent manner and Bax protein of 25,50,100 μmol/L NaAsO2 group and DR5 protein of 25,50 μmol/L NaAsO2 group were significantly higher than the control group.There was no significantly difference of Fas mRNA and Bcl-2、Fas protein in each group.p53 inhibitor PFTaand ATM/ATR inhibitor CGK733 inhibited the expressions of Bax,Bcl-2,DR5 mRNA and Bax,DR5 protein induced by arsenic in L-02 cells.In contrary,the inhibitors increased the mRNA expression of p53.Pretreatment with Ginkgo biloba extract also inhibited the expressions of Bax,Bcl-2,DR5 mRNA and Bax,DR5 protein induced by arsenic in L-02 cells,while increased the mRNA expression of p53.Conclusion Arsenic may influence the activation of ATR-Chkl kinases which then participate in the phosphorylation of p53.Phosphorylated p53 accumulate and induce the expression of Bax,DR5 mRNA and protein which may result in apoptosis of hepatocytes through intrinsic mitochondrial and extrinsic death receptor pathway.p53-mediated apoptotic pathway may participate in the occurrence and development of arsenic liver injury causeed by coal-burning.p53 inhibitor PFTα and ATM/ATR inhibitor CGK733 may down-regulate the overexpressions of Bax,DR5 mRNA and protein induced by arsenic.The results provide evidence to the hypothesis that arsenic can induce p53-mediated apoptosis through ATR-Chkl kinases and point out new ideas and directions for the prevention and therapy of arsenism.Ginkgo biloba extract may partially abrogate arsenic induced hepatocyte apoptosis by inhibiting the activation of ATR-Chkl-p53 pathway which may be one of the mechanisms associated with Ginkgo biloba extract relieving liver damage and improving liver function. |
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