Protective Effect And Its Mchanism Of Xiao-Chai-Hu Decoction On The Liver Injury Liver Induced By Lead In Mice

Lead is a toxic heavy metal that caused health risks worldwide.Although the use of lead is decreasing,however,a large amount of lead is still discharged into the nature through the air and water,seriously affecting the health of humans and animals.Lead can enter human and animal bodies through the respiratory and digestive systems and accumulate in various tissues and organs of the body.Liver is the organ with the largest accumulation of lead among all organs in the body,and it is also the key organ for detoxification and metabolism.So protecting the liver is very important in treating lead poisoning.Xiao-Chai-Hu decoction(XCHD)which recorded in “Shang Han Lun” is one of the famous Chinese herbal prescriptions.It consists of bupleurum,scutellaria baicalensis,Codonopsis pilosula,Pinellia ternata,Glycyrrhiza uralensis(processed),Zingiber officinale(cut)and Ziziphus jujubas and has a good therapeutic effect on liver diseases.At present,there has been no report on the effect of XCHD on alleviating liver injury caused by lead.In this research,acute and chronic lead poisoning model of ICR mice were used for investigating the effect of XCHD on liver injury induced by lead,and screening the effective fraction of XCHD in protecting the liver injury by the same way.The main components of n-butanol in XCHD were determined by LC-MS spectrometry.At the same time,RNA-seq transcriptome sequencing was carried out on the liver tissue under the action of effective fraction,and the mechanism of the protective lead induced liver injury of XCHD was discussed from the change of m RNA level.Firstly,the liver index,serum biochemical indexes and pathological sections were used to evaluate the protective effect of XCHD on the liver injury model of mice.The results showed that after the treatment of lead acetate,the liver index and the content of AST and ALT in serum increased significantly,the liver tissue section showed that the cell space was enlarged,the hepatic sinuses were dilated obviously,the hepatic cells were arranged disorderly,accompanied by obvious inflammatory cell infiltration.However,the liver indext of mice treated with lead acetate + XCHD had no significant change compared with the control group,and the serum AST and ALT indexes were significantly decreased compared with the model group.The liver tissue section showed that the liver cells were arranged neatly,the hepatic sinusoids were slightly dilated,and the tissue damage was relatively small.The results showed that XCHD had obvious protective effect on liver injury by lead in mice,and the n-butanol fraction was the effective fraction of XCHD.On this basis,the main ingredients of n-butanol fraction of XCHD was analyzed by LC-MS.,and nine compounds were identified,including diacetoxy-6-gingerdiol from Zingiber officinale,chrysin-6-C-α-L-arabinopyranosyl-8-C-β-D-glucopyranoside,baicalin,wogonin-5-O-glucoside,oroxylin A 7-O-glucuronide,wogonoside from Scutellaria baicalensis,tangshenoside I from c Codonopsis pilosula,glycyrrhizic acid and isoquercetin from Glycyrrhiza.Subsequently,the liver tissue was sequenced by RNA-seq,and the results showed that the transcriptional status of 3429 genes were changed by lead,among which 1684 genes were significantly up-regulated and 1745 genes were significantly down-regulated.Compared with the lead acetate model group,143 genes with abnormal expression were improved in the lead acetate + effective fraction group,among which71 genes were significantly up-regulated and 72 genes were significantly down-regulate.16 key genes were found in n-butanol fraction of XCHD to improve liver injury by key driving analysis,including: Tubb2 a,Stip1,Cyp4a12 a,Cyp2c50,Ugt1a1,Cyp3a11,Cyp4a12 b,Ahsa1,Cyp2c54,Tubb4 b,Esr1,Hsp90aa1,Tuba1 a,Tuba1c,and Hsph1.these genes were significantly enriched in five key metabolic pathways: oxidative phosphorylation,thermogenesis,Steroid hormone biosynthesis,retinol metabolism and bile secretion pathway.These results suggest that the mechanism of n-butanol fraction of XCHD against liver injury may lie in the regulation of a variety of hepatic metabolic processes.Then,q PCR was used to verify the transcriptome results by detecting the changes of Cirbp,Hsp90aa1,Esr1,Cyp4a12 b and Ugt1a1 genes in liver tissue at m RNA level,and the results showed that the trend of changes was consistent with the transcriptome results.Finally,the changes of Cirbp,Esr1 and Ugt1a1 proteins were detected by Western-blot.The results showed that it can significantly up-regulate the protein levels of Cirbp and Ugt1a1 in liver,and down-regulate the protein level of Esr1 in liver,which further confirmed and discussed the mechanism of protecting liver injury by improving metabolism and regulating endocrine process.