白毛藤乙醇提取物抑制HaCaT细胞增殖及炎性因子表达机制OA

Objective To investigate the effects of ethanol extract of Solanum lyratum(SLT)on the proliferation and inflammatory factor expression in human immortalized keratinocytes(HaCaT cells),and to elucidate the underlying molecular mechanisms.Methods HaCaT cells were used to establish a psoriasis-like cell model via stimulation with 20 ng/mL TNF-α for 24 h.Experimental groups included the control group,TNF-α group,and SLT+TNF-α group.The cytotoxicity and optimal concentrations of SLT were determined using the CCK-8 assay,and concentrations of 200,400,and 800 μg/mL were selected for subsequent experiments.Cell proliferation was assessed by CCK-8,while cell cycle distribution(G1 and S phases)was analyzed by flow cytometry.The mRNA levels of inflammatory cytokines interleukin(IL)-6,IL-1 β,and tumor necrosis factor α(TNF-α)were quantified by PCR.Western Blot was employed to detect the expression of proliferation-related protein Cornulin(CRNN),cell cycle regulator CyclinD1,and phosphorylation levels of protein kinase B(Akt).Additionally,immunofluorescence and Western Blot were used to evaluate the activation status of the nuclear factor-kappa B(NF-κB)pathway,including nuclear and cytoplasmic levels of NF-κB p65,I kappa B alpha(IκBα),and phosphorylated IκBα.Results Compared with the control group,the proliferation ability of cells in the TNF-α group was enhanced(P＜0.05),the proportion of HaCaT cells in the G1 phase decreased and the proportion in the S phase increased(P＜0.01),and the expression levels of CyclinD1 and p-Akt proteins increased(P＜0.01).The mRNA expression levels of IL-6,IL-1β and TNF-α and the expression of NF-κB P65 in the nucleus increased(P＜0.01,P＜0.05),the expressions of NF-κB P65 and IκBα in the cytoplasm decreased(P＜0.01),and the expression of p-IκBα increased(P＜0.01).Compared with the TNF-α group,the low-dose SLT group exhibited reduced cell proliferation ability(P＜0.05),while the medium-and high-dose SLT groups showed significantly reduced cell proliferation ability(P＜0.01).All SLT dose groups had significantly increased apoptosis rates(P＜0.01),increased proportion of HaCaT cells in G1 phase and decreased proportion in S phase(P＜0.01),decreased protein expression of CRNN and CyclinD1(P＜0.01).The medium-and high-dose SLT groups showed decreased p-Akt expression(P＜0.01).The medium-dose SLT group showed decreased mRNA expression of IL-6,IL-1β,and TNF-α(P＜0.01).The low-and medium-dose SLT groups showed decreased nuclear NF-κB P65 expression(P＜0.01),increased cytoplasmic NF-κB P65 and IκBα expression(P＜0.01),and decreased p-IκBα expression(P＜0.01).Conclusions SLT inhibits TNF-α-promoted HaCaT cell proliferation in a concentration-dependent manner.The mechanism may involve inhibition of cell cycle progression,reduction in the expression of the CRNN and CyclinD1,and suppression of Akt signaling pathway activation.Furthermore,SLT can downregulate TNF-α-induced expression of inflammatory factors IL-6,IL-1β,and TNF-α by interfering with the activation of the NF-κB signaling pathway.