Research Autophagy Mechanism On Emodin Combined With KGF And HIF-1αin Hypoxic Cell Stress Damage

Object:It was protected that the hypoxia environment damage of intestinal mucosa to improve the structure and function of intestinal mucosa barrier by the combined strategy of gene therapy and traditional Chinese medicine extract. The research aim to use rat small intestinal crypt epithelial cells(IEC-6) for the vitro model, establishing new treatments by using emodin combined hypoxiainducible factor 1α(HIF-1α) and keratinocyte growth factor(KGF) in order to prove the protection of hypoxia stress injury of intestinal epithelial cells by autophagy mechanism.Methods: MTT technique was used to observe the optimal concentration.IEC-6 were infected by recombinant adenovirus that contained GFP gene,in order to detect the best multiplicity of infection(MOI) from adenovirus to IEC-6.The cell models of hypoxia on IEC-6 damage was divided into 4 groups:cells without any drugs;cells with emodin;cells with adenovirus contain HIF-1α and KGF;cells with emodin combined adenovirus containing HIF-1α and KGF.To detect cell survival rate and cell cycle distribution with MTT and flow cytometer at 0 hours、6 houers and 24 hours under hypoxia environment(the oxygen content is5%). At 6 houers and 24 hours under hypoxia environment(the oxygen content is 5%),by using transmission electron microscope(TEM),Real time-PCR and Western-blot to observe autophagsome、detect the Beclin-1 and LC3 DNA expression and identify the Beclin-1 and LC3 protein expression.The expression of HIF-1αand KGF were tested through Real time-PCR、immunofluorescent assay( IFA) and Western-blot.Results:Emodin can obviously promote the growth of IEC-6 cell when the concentration of 5μmol/L.The best MOI was 100 pfu/cell,according to detect the efficiency and survival rate of infection from adenovirus with GFP gene to IEC-6 by fluorescence and MTT assay. IEC-6 cells were infected by adenovirus with HIF-1α gene and KGF gene by the optimal MOI. As a result,under hypoxia environment(the oxygen content is 5%),the group of emodin combined adenovirus containing HIF-1α and KGF has better protective effect on IEC-6 cells by MTT;the group that contain HIF-1α and KGF could obviously block cells in G1 phase(P<0.01) while the group of emodin could obviously promote cells into the split phase(P<0.01),however, the group of emodin combined adenovirus contain HIF-1α and KGFalso promoted cells into the S phase(P<0.01),therefore,the role of promoting cells into the split phase by emodin is greater than the effects of blocking by the group of adenovirus with HIF-1α gene and KGF gene,what’s more the group of emodin combined adenovirus contain HIF-1α and KGF has effect on promoting cell growth. Autophagosome can be observed by electron Microscope,which caused by undergoing hypoxia in 6 hours and 24 hours.As compared with normoxic control( NC) group,the expression quantity of the autophagy related genes-Beclin-1 and LC3 was arised in hypoxic condition at 6 and 24 hours( P<0.01) by Real-time PCR. Especially the expression quantity of the autophagy related genes-Beclin-1 and LC3 in hypoxic condition at 6 hours was more than that at 24 hours( P<0.05),however,the genes expression in the group of emodin combined adenovirus containing HIF-1α and KGF was the most of all the groups( P<0.05).Western-blot testified that Beclin-1 and LC3 protein expressed in the hypoxic condition at 6and 24 hours and the proteins expression in the group of emodin combined adenovirus containing HIF-1α and KGF was the most of all the groups( P<0.05).Stable expression of HIF-1α and KGF was confirmed in IEC-6 cells under hypoxic condition at 0、6 and 24 hours by Real time-PCR and immunofluorescence.The HIF-1α protein was stable expression by Western-blot and there were no significant difference in the normoxic control(NC)group and the hypoxia groups(P>0.05).Conclusion: That emodin combined cytokines(HIF- 1α and KGF) was found to confer protection on cells in hypoxic intestinal stress damage.That emodin combined cytokines(HIF- 1α and KGF) by autophagy regulating the protection in hypoxic intestinal stress damag.The exogenous HIF-1αand KGF genes could expressed in IEC-6 cells in both normoxia and Hypoxia groups which contain the two genes. That emodin combined adenovirus contain HIF-1α and KGF jointly plays a role in promoting the occurrence of autophagy to protect the IEC-6 cells under hypoxia environment.