枸杞籽油对小鼠睾丸支持细胞衰老模型的抗炎作用

doi:10.6040/j.issn.1671-7554.0.2020.1081

摘要/Abstract

摘要： 目的探讨枸杞籽油对小鼠睾丸支持细胞(TM4)衰老模型的抗炎作用及机制。方法以不同浓度(50、100、150、200 mmol/L)的D-半乳糖(D-Gal)以不同时间(24、36、48 h)体外作用TM4细胞,通过CCK-8法和β-半乳糖苷酶染色筛选最佳时间浓度的D-Gal建立TM4衰老模型,Western blotting 检测衰老因子P16^INK4A、P21^Waf1/Cip1、γH2A.X以及半乳糖苷酶蛋白的表达。以不同浓度(1%、3%、5%、10%)枸杞籽油含药血清提前干预TM4细胞衰老模型,并用CCK-8筛选枸杞籽油含药血清最佳浓度。Western blotting检测细胞炎症因子白细胞介素-1(IL-1β)、肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)与血红素加氧酶(HO-1)的表达以及p-P65/P65的相对表达量;细胞免疫荧光(IF)定位NF-κB表达。结果 D-Gal建立TM4衰老模型结果显示,与正常细胞比较,200 mmol/L浓度的D-Gal干预48 h对细胞增殖有抑制作用,其抑制率为(14.890±0.892)%,差异有统计学意义(F=12.820,P=0.004;t=17.230,P<0.001),同时β-半乳糖苷酶染色结果显示,与正常细胞比较,细胞蓝染阳性率上升(t=5.243,P<0.001)。P16^INK4A(t=2.364, P=0.033)、P21^Waf1/Cip1(t=2.908, P=0.011)、γH2A.X(t=2.511, P=0.025)蛋白表达以及半乳糖苷酶(t=2.299, P=0.037)表达在衰老模型组升高。枸杞籽油抗TM4衰老的结果显示,3%浓度的枸杞籽油含药血清干预衰老TM4细胞后活性上升(t=5.399, P<0.001)。与正常细胞比较,TM4细胞衰老相关蛋白P16^INK4A(t=7.211, P<0.001)、P21^Waf1/Cip1(t=8.248, P<0.001)、γH2A.X(t=4.145, P<0.001)以及半乳糖苷酶(t=7.288, P<0.001)表达下调;与模型组相比,3%浓度的枸杞籽油含药血清干预后炎症因子TNF-α(t=4.992, P<0.001)、IL-1β(t=2.921, P=0.011)、IL-6(t=4.985, P<0.001)的表达下调,HO-1 (t=6.047, P<0.001)与IL-10(t=2.315, P=0.036)表达上调;衰老TM4细胞p-P65/P65相对表达量在3%浓度的枸杞籽油含药血清干预后下调(t=24.630, P<0.001)。结论 3%枸杞籽油含药血清干预,对衰老TM4细胞有明显的抗炎作用,其机制可能是下调核因子κB进而抑制下游炎症因子IL-1β、TNF-α、IL-6表达,并启动抑炎因子HO-1、IL-10的表达。

关键词: 枸杞籽油, TM4细胞, 炎症因子, NF-κB通路

Abstract: Objective To investigate the effects of Lycium barbarum seed oil(LBSO)on anti-inflammation in the sub-acutely aging Sertoli cells of mice(TM4 cells)induced by D-Galactose(D-Gal)and to explore the mechanism. Methods TM4 cells were treated with different concentrations(50, 100, 150 and 200 mmol/L)of D-Gal for different periods(24 h, 36 h, 48 h)in vitro. The optimal intervention time and concentration of D-Gal were determined with CCK-8 and β-galactosidase staining to establish the TM4 aging model. The expressions of aging-related factors P16^INK4A, P21^Waf1/Cip1, H2A.X, and β-galactosidase were detected with Western blotting. The TM4 aging models were intervened with different concentrations(1%, 3%, 5%, 10%)of LBSO containing serum in advance, and the optimal concentration was determined with CCK-8. The expressions of interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), interleukin-10(IL-10), heme oxygenase-1(HO-1)and the relative expression of p-P65/P65 were detected with Western blotting. The expression of NF-κB was located with immunofluorescence(IF). Results The inhibition rate of TM4 cells was(14.890±0.892)% when cells were treated with D-gal at 200 mmol/L for 48 h(F=12.820, P=0.0040; t=17.230, P<0.001). β-galactosidase staining indicated when compared with control group, the stained cells had increased positive rate ［(63.000±9.388)% vs(11.390±2.959)%, t=5.243, t=5.243, P<0.001)］. The expressions of P16^INK4A(t=2.364, P=0.033), P21^Waf1/Cip1(t=2.908, P=0.011), γH2A.X(t=2.511, P=0.025)and β-galactosidase(t=2.299, P=0.037)significantly increased in the aging model. CCK-8 detection showed a significant increasing trend in cell activity when 3% LBSO containing serum interfered with aging model ［(1.941±0.021)vs(1.784±0.019), t=5.399, P<0.001］, while the expressions of P16^INK4A(t=7.211, P<0.001), P21^Waf1/Cip1(t=8.248, P<0.001), γH2A.X(t=4.145, P<0.001)and β-galactosidase(t=7.288, P<0.001)downregulated. Compared to the model group, after 3% LBSO intervention, TM4 cells showed decreased expressions of TNF-α(t=4.992, P<0.001), IL-1β(t=2.921, P=0.011), IL-6(t=4.985, P<0.001), and p-P65/P65(t=24.630, P<0.001), but increased expressions of HO-1(t=6.047, P<0.001)and IL-10(t=2.315, P=0.036). Conclusion LBSO containing serum at concentration of 3% has an obvious anti-inflammatory effect on aging TM4 cells. The possible mechanism is down-regulation of NF-κB inhibits the expressions of IL-1β, TNF-α and IL-6, but activates the expressions of HO-1 and IL-10.

Key words: Lycium barbarum seed oil, TM4 cells, Inflammatory factors, NF-κB pathway

中图分类号:

R277.5

杨张杰,王宇欣,陈冬梅,赵帅,虎娜,马良宏,马会明. 枸杞籽油对小鼠睾丸支持细胞衰老模型的抗炎作用[J]. 山东大学学报 (医学版), 2020, 58(12): 15-22.

YANG Zhangjie, WANG Yuxin, CHEN Dongmei, ZHAO Shuai, HU Na, MA Lianghong, MA Huiming. Effects of Lycium barbarum seed oil(LBSO)on anti-inflammation in the aging Sertoli cells of mice(TM4 cells)[J]. Journal of Shandong University (Health Sciences), 2020, 58(12): 15-22.

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