健脾益肠散对TNF-α诱导的树突状细胞自噬相关蛋白及基因表达的影响OA

Objective To observe the effect of Jianpi Yichang San(JPYCS)on the expression of autophagy-related proteins and genes in tumor necrosis factor-α(TNF-α)-induced dendritic cells(DCs).Methods Dendritic cells were divided into the following groups:normal group(treated with DC-specific culture medium),model group(treated with TNF-α),negative control group(TNF-α+blank serum),positive drug group(TNF-α+sulfasalazine serum),test drug group(TNF-α+JPYCS serum),inhibitor group[TNF-α+3-methyladenine(3-MA,5 mmol·L-1)],activator group[TNF-α+rapamycin(RAPA,1 μmol·L-1)],inhibitor+test drug group(TNF-α+3-MA 5 mmoL·L-1+JPYCS serum),and activator+test drug group(TNF-α+RAPA 1moL·L-1+JPYCS serum).Cells in the normal group were treated with specific medium for 24 hours.For other groups,an inflammatory model was established by induction with 10 ng·mL-1 TNF-α;then,serum groups were treated with 10％corresponding medicated serum for 24 hours,while the inhibitor and activator groups were treated with 3-MA or RAPA,respectively,for 24 hrous.Cell proliferation was assessed using the Cell Counting Kit-8(CCK-8)assay.Positive expression of autophagy-related myosin-like BCL2 interacting protein and microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ)was detected by immunofluorescence.Protein expression of Beclin-1,LC3-Ⅱ,Ubiquitin-binding protein62(p62/SQSTM1),autophagy-related protein 5(Atg5),and autophagy-related protein 7(Atg7)in DCs was determined by Western Blot.mRNA expression of p62/SQSTM1,Atg5,and Atg7 was measured by real-time quantitative polymerase chain reaction(Real-time PCR).Results Compared with the normal group,the model group showed significantly decreased cell proliferation capacity(P<0.01).Protein expression of Beclin-1,LC3-Ⅱ,Atg5,and Atg7,as well as mRNA expression of Atg5 and Atg7,were significantly reduced(P<0.01)in the model group,while protein and gene expression of p62 were significantly increased(P<0.01).The test drug group significantly enhanced cell proliferation capacity(P<0.01),upregulated the expression of Beclin-1,LC3-Ⅱ,Atg5,and Atg7(P<0.01),and downregulated the expression of p62(P<0.01).The inhibitor+test drug group reversed the abnormal expression of the aforementioned indicators under 3-MA intervention(P<0.01).The activator+test drug group exerted a synergistic effect with RAPA,further upregulating the expression of these indicators(P<0.01).Conclusion Jianpi Yichang San enhances the expression of autophagy-related proteins and genes in TNF-α-induced dendritic cells,promoting autophagy.This may be one of its mechanisms for regulating intestinal immune tolerance and alleviating UC inflammation.