Effect And Mechanisms Of Curcumin On The Apoptotic Of HUC-MSC

Spinal cord injury(SCI)is a serious disaster,it leads to damage to nerve function,which significantly reduces the quality of patients’ life,and imposes a serious burden on their families and society.The spontaneous recovery ability after SCI is poor,there’s still no effective treatment to it.Stem cell transplantation has brought new hopes to the treatment of SCI,it has become a popular direction in the SCI researches in recent years,and has shown some therapeutic effects.At the same time,previous studies also found that transplanted stem cells just survived for a short period at the transplant site,which made the transplanted stem cells unable to continue to play a biological role and seriously limited the treatment effect.Human umbilical cord mesenchymal stem cells(hUC-MSC)are derived from the umbilical cord of fetal.It is abundant in source and easy to obtain,also don’t have obvious damage to donors.hUC-MSC is easy to be isolated and cultured.These characteristics make it have great potential in tissue engineering application and have become a hot topic in recent years.However,hUC-MSC will rapidly lose after being transplanted to the injury site,which limits its therapeutic efficacy.This may be caused by the pathological environment of ischemia,hypoxia,hemorrhage and secondary inflammation post-injury,which leads to apoptosis of hUC-MSC.Perhaps the treatment effect of hUC-MSC transplantation therapy can be improved by increasing the proportion and time of hUC-MSC survival.Curcumin(cur)is a compound extracted from powdered dry Curcuma Longa rhizome.Its powerful effects of antioxidant,anti-inflammatory and anti-fibrotic have been widely confirmed.We have found that curcumin can inhibit the expression of inflammatory factors and reactive gliosis after spinal cord injury,and promote the improvement of hindlimb motor function in rats after spinal cord injury.Recent studies have reported that curcumin can inhibit rat kidney and cardiomyocyte apoptosis,indicating that curcumin also has an anti-apoptotic effect.It is necessary to be studied furtherly about whether curcumin can also inhibit the apoptosis of hUC-MSC.Based on the above theory,we assume that curcumin can inhibit the occurrence of apoptosis in hUC-MSC.In this study,we induce human umbilical cord mesenchymal stem cells apoptosis by TNF-α and treat with curcumin.to find the effect and mechanisms of curcumin on the apoptotic of hUC-MSC。It may provide a preliminary laboratory basis for the treatment of spinal cord injury and other diseases by curcumin combined with hUC-MSC transplantation.Part one: the Effect of curcumin on the apoptotic of hUC-MSCObjectiveTo investigate the effect of curcumin(cur)on apoptosis of human umbilical cord mesenchymal stem cells(hUC-MSC)intervened by TNF-α.MethodshUC-MSC cultured in vitro were given 5 ng/mL TNF-α to induce apoptosis,and treated with different concentrations of cur.LDH release assay CCK-8 and flow cytometry was used to assess the anti-apoptosis effect of cur.Western blot(WB)was used to detect the expression of tumor necrosis factor receptor(TNFR)downstream apoptosis-related MAPK signaling pathway(ERK,JNK,P38)protein.Results1.LDH release assay showed that the TNF-α concentration ≥5 ng/mL resulted in significant differences in apoptosis compared with the control group2.When the concentrations were between 0.5 μmol/L and 4 μmol/L,cur inhibited the hUC-MSC apoptosis induced with TNF-α by concentration dependent,Flow cytometry also confirmed that 4 μmol/L cur significantly inhibited the apoptosis of hUC-MSCs(P<0.05).3.WB analysis indicated that Cur activated ERK,there was no statistic difference in the expression of JNK and P38 with or without cur.ConclusionCurcumin can inhibit the apoptosis of hUC-MSC induced by TNF-αThe effect of curcumin inhibits the apoptosis of hUC-MSC induced with TNF-α may result from it activated ERK。Part two: The mechanisms of curcumin inhibiting the apoptosis of human umbilical cord mesenchymal stem cells TNF-α-inducedObjectiveFurther,investigating the mechanisms of curcumin inhibits TNF-α-induced human umbilical cord mesenchymal stem cells apoptosisMethodshUC-MSC cultured in vitro were given 5 ng/mL TNF-α to induce apoptosis and treated with 4 μmol/L cur.U0126 was used to inhibit the activation of ERK.Western blot(WB)was used to detect the expression of ERK and its downstream anti-apoptosis related protein Bcl2 and Bad.Flow cytometry was used to assess the anti-apoptosis effect of cur when ERK is effectively inhibited.Results1.U0126 can inhibit the activation of ERK effectively.2.The pro-survival protein Bcl2 expression and Bad phosphorylation of hUC-MSC increase after ERK are activated and decrease when ERK is inhibited.3.Flow cytometry confirmed that curcumin no longer inhibited TNF-α-induced hUC-MSC apoptosis after ERK was inhibited.ConclusionCurcumin can inhibit TNF-α-induced apoptosis of hUC-MSC by activating ERK and promotes pro-survival protein Bad phosphorylation and Bcl2 expression.