Effects Of Human Umbilical Cord Mesenchymal Stem Cells Transfected With HGF On Hepatic Stellate Cells Via NF-κB Signaling Pathway

Objective:In this experiment,HGF-transfected hUCMSCs and HSCs-T6 were indirectly co-cultured to explore whether hUCMSCs could decrease HSCs-T6 activity,promote its apoptosis,inhibit its activation,and reduce the expression of Collagen I and Collagen III by inhibiting the activation of NF-κB signaling pathway,which would provide a new idea to alleviate or even reverse liver fibrosis.Methods:HSCs-T6 and HGF-transfected hUCMSCs were cultured by Transwell indirect co-culture plates.HGF-transfected hUCMSCs were cultured on the upper layer,and HSCs-T6 were cultured on the lower layer.The cell number ratio was 1:1.The experiment was divided into four groups:Control group（HSCs-T6）,HGF group（hUCMSCs transfected by lentivirus inserted with HGF gene fragment co-cultured with HSCs-T6）,hUCMSC group（hUCMSCs co-cultured with HSCs-T6）,and LV5-NC group（hUCMSCs transfected by lentivirus inserted without HGF gene fragment cocultured with HSCs-T6）.After culturing for 24 h,48h and 72 h,the morphology of HSCs-T6 in each group was observed under an inverted microscope,the cell viability was determined by CCK-8assay and Hoechst 33342 staining was used to observe the apoptosis.What’s more,the protein expressions of Bcl-2,Bax,α-SMA,Collagen Ⅰ and Collagen Ⅲ in each group of HSCs-T6 were detected by immunocytochemistry and the protein expressions of Bcl-2,Bax,α-SMA,Collagen Ⅰ,Collagen Ⅲ,P65,P50 and IκBα were detected by Western Blot,the mRNA expressions of CollagenⅠ,Collagen Ⅲ,P65,P50 and IκBα were detected by RT-qPCR.Results:1.Microscope observation showed that after co-culturing for 48 h and 72 h,HSCs-T6 in HGF,hUCMSC and LV5-NC group were sparsely arranged compared with control group and it was the most significant in HGF group.2.The results of CCK-8 assay showed that after co-culturing for 48 h and 72 h,the OD values of HGF,hUCMSC and LV5-NC group were significantly decreased compared with control group（P<0.05）.In addition,after co-culturing for 72 h,the OD values of HGF,hUCMSC and LV5-NC group were significantly decreased compared with control group（P<0.05）and they were significantly decreased in HGF group compared with hUCMSC and LV5-NC group（P<0.05）.whereas there were no significant differences between hUCMSC and LV5-NC group.3.Hoechst 33342 staining results showed that compared with Control group,nuclear pyknosis and nuclear disintegration occurred in HGF group,hUCMSC group and LV5-NC group after 48 and 72h co-culture.Compared with hUCMSC group and LV5-NC group,HGF group had more obvious nuclear pyknosis and fragmentation after 72 h co-culture.4.Immunocytochemistry results showed that the protein expressions of Bcl-2,α-SMA,Collagen Ⅰ and Collagen Ⅲ in HGF,hUCMSC and LV5-NC group were significantly decreased compared with control group（P<0.05）while Bax was significantly increased（P<0.05）.After coculturing for 72 h,the above indicators in HGF group were significantly decreased compared with hUCMSC and LV5-NC group（P<0.05）,while the expression of Bax was significantly increased（P<0.05）.In addition,there were no significant differences between hUCMSC and LV5-NC group.5.Western Blot results showed that when co-cultured for 48 h and 72 h,the protein expressions of Bcl-2,α-SMA,Collagen Ⅰ,Collagen Ⅲ,P65 and P50 in HGF,hUCMSC and LV5-NC group were significantly decreased compared with control group（P<0.05）,while Bax and IκBα were increased（P<0.05）.Moreover,after co-culturing for 72 h,the protein expressions of Bcl-2,α-SMA,Collagen Ⅰ,Collagen Ⅲ,P65 and P50 in HGF group were significantly decreased compared with hUCMSC and LV5-NC group（P<0.05）while Bax and IκBα were significantly increased（P<0.05）.In addition,there were no significant differences between hUCMSC and LV5-NC group.6.RT-qPCR results showed that the mRNA expressions of Collagen Ⅰ,Collagen Ⅲ,P65 and P50 in HGF,hUCMSC and LV5-NC group were significantly decreased compared with control group when co-cultured for72h（P<0.05）whereas IκBα was increased（P<0.05）.Moreover,in HGF group,the mRNA expression of Collagen Ⅰ,Collagen Ⅲ,P65 and P50 were significantly decreased compared with hUCMSC and LV5-NC group（P<0.05）while IκBα was significantly increased（P<0.05）.In addition,there were no significant differences between hUCMSC and LV5-NC group.Conclusion:HGF-transfected hUCMSCs may inhibit the activation of NF-κB signaling pathway,decrease the activity of HSCs-T6,promote its apoptosis,inhibit the activation of HSCs-T6,and decrease the production of Collagen I and Collagen III.