| Objective: To investigate the inhibiting effects of plumbagin on hepatocytes and to explore the protective role of MaFGF in the injury of plumbagin on hepatocytes of SD rats. To study the toxicity mechanism of plumbagin and the protective mechanism of MaFGF,and also to provide experimental evidence and the theoretical basis for clinical application.Methods: stripping the capsule and cutting the liver of SD rats about 1 day,The liver tissues of 1mm3 could be obtained.then,These tissues were digested in two steps by 0.25%trypsin for about 3 minutes and 0.1%collagenase for about 10 minutes.Hepatocytes we obtained were cultured with DMEM-HG in well plates and culture bottles. Hepatocytes were identified by PAS: First, The amylase was used to act on hepatocytes as the control group,In the experiment group, take several of microsopic field of vision and count the number of negative and positive cells. Results revealed that the number of positive cells accounted for 95%. and could meet needs for other subsequent studies. Positive cells were seeded in culture plates of 96 wells: (1)Different concentrations of MaFGF were added into culture media for 24h, The viability and proliferation were determined with MTT assay. ( 2 ) After 72h culture,Plumbagin with different concentration was added, and MaFGF with different concentration was added further as the experimental group after 24h of the Plumbagin addition. MTT assays were performed to test the cell viability after 24h treated by MaFGF. (3)The injury model was induced by Plumbagin. And the protective effect of MaFGF on the injured hepatocytes was observed. The statistical analysis was executed by SPSS 13.0 software, using chi-square test, analysis of variance for statistical, statistically significant level was"a=0.05".Results: ( 1 ) The cultured cells had the typical multi-angular morphological features of epithelium, After hepatocytes was detected by PAS. Microscopically, The cytoplasm is filled with granular pink,and with no staining in the nucleus.These were positive cells. Treated with amylase,the negative cells exhibited colorless in the cytoplasm. (2)Proliferation activity in hepatocytes:No significant difference was noted between low-concentration (3.12μg/L) MaFGF group and the control group(P>0.05),while medium and high doses MaFGF groups showed significant differences as against the control group(P<0.01), but no significant difference was observed between the MaFGF of medium and high concentration groups(.P>0.05)(.3)The inhibitory effect of the plumbagin on hepatocytes increased with the increase of concentration.(4)The inhibition rate decreases with increasing of concentration of MaFGF when treated with Plumbagin and different concentration MaFGF, But the IC50 increased with MaFGF concentration increasing. (5)The biochemical and enzyme indexes were significantly different between the Plumbagin group and control group (P <0.05 or P <0.01); Compared with Plumbagin group, the SOD activity was significandy higher, but the contents of NO,MDA,LDH,GPT,GOT were significantly lower (P <0.05 or P <0.01)in Plumbagin+MaFGF groups. The activity and contents of the biochemical and enzyme in Plumbagin+MaFGF groups were not declined to normal levels, comparing with control group, and there was still significant difference.Conclusion:①thought PAS identified,isolated successfully hepatocytes from SD rats by trypsin +collagenase digestion and tissue plant methods.②It showed that MaFGF could promote the proliferation on hepatocytes at proper concentrations. But their activity did not perform concentration-effect relationship;③The result indicated the success of the foundation of the model injured by Plumbagin:Plumbagin could inhibit hepatocytes proliferation in vitro correlated with concentration dependent manner.④MaFGF had some protective effects on the hepatocyte injured with plumbagin and the effects was concentration dependent.
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