基于沉默信息调节因子1/p65通路探讨竹叶黄酮对过氧化氢诱导的奶牛乳腺上皮细胞焦亡的保护作用机制

Protective Mechanism of Bamboo Leaf Flavonoids on Hydrogen Peroxide-Induced Pyroptosis of Bovine Mammary Epithelial Cells Was Discussed Based on Silent Information Regulator 1/p65 Pathway

本试验旨在通过过氧化氢(H_(2)O_(2))诱导的奶牛乳腺上皮细胞(bMECs)焦亡模型探讨竹叶黄酮(BLF)预处理缓解bMECs焦亡的作用机制。试验以bMECs为研究对象,设对照组、BLF组、H_(2)O_(2)组、BLF+H_(2)O_(2)组、BAY 11-7082+H_(2)O_(2)组、BLF+BAY 11-7082+H_(2)O_(2)组、EX 527+H_(2)O_(2)组和BLF+EX 527+H_(2)O_(2)组,BLF作用浓度为80μg/mL,H_(2)O_(2)作用浓度为800μmol/L,使用BAY 11-7082和EX 527干预p65和沉默信息调节因子1(SIRT1)的表达。利用PCR和Western Blot技术检测细胞内焦亡相关基因和蛋白的表达水平,利用流式细胞术检测焦亡细胞数量,并利用免疫荧光技术检测细胞内凋亡相关斑点样蛋白(ASC)斑点的荧光强度,以观测SIRT1/p65通路在BLF缓解H_(2)O_(2)诱导的bMECs焦亡中发挥的作用。结果显示:1)H_(2)O_(2)诱导的bMECs焦亡中p 65的mRNA和蛋白表达水平显著升高(P<0.05),BLF处理后p 65的mRNA和蛋白表达水平则显著降低(P<0.05);BLF与BAY 11-7082对p65表达及核转位的抑制效果显著(P<0.05),可有效缓解bMECs焦亡,同时二者也显著降低了bMECs焦亡相关基因和蛋白的表达水平(P<0.05),并增强了ASC斑点的荧光强度。2)H_(2)O_(2)诱导的bMECs焦亡中SIRT1的mRNA和蛋白表达水平显著降低(P<0.05),BLF处理后SIRT1的mRNA和蛋白表达水平则显著升高(P<0.05);EX 527对抑制SIRT1、促进p65的表达及核转位的效果显著(P<0.05);EX 527也显著升高了bMECs焦亡相关基因和蛋白的表达水平(P<0.05),并增强了ASC斑点的荧光强度。综上所述,80μg/mL的BLF通过抑制p65的表达和核转位,缓解H_(2)O_(2)诱导的bMECs焦亡和NLRP3炎症小体激活;SIRT1的表达在H_(2)O_(2)诱导的bMECs焦亡中被下调,BLF缓解H_(2)O_(2)诱导的bMECs焦亡是通过上调SIRT1的表达来抑制p65的表达来实现的。

This study aimed to explore the mechanism by which bamboo leaf flavonoids(BLF)pretreatment alleviates pyroptosis in bovine mammary epithelial cells(bMECs)induced by hydrogen peroxide(H_(2)O_(2)).bMECs were used as the research subjects,and control group,BLF group,H_(2)O_(2) group,BLF+H_(2)O_(2) group,BAY 11-7082+H_(2)O_(2) group,BLF+BAY 11-7082+H_(2)O_(2) co-treatment group,EX 527+H_(2)O_(2) group and BLF+EX 527+H_(2)O_(2) co-treatment group were set up in this experiment.BLF was administered at a concentration of 80μg/mL,and H_(2)O_(2) was used at 800μmol/L.BAY 11-7082 and EX 527 were used to inhibit the expression of p65 and silent information regulator 1(SIRT1),respectively.Gene and protein expression levels in the cells were detected using PCR and Western Blot.The number of pyroptotic cells was determined by flow cytometry.The fluorescence intensity of apoptosis-associated speck-like protein(ASC)spots was detected by immunofluorescence to observe the role of the SIRT1/p65 pathway in BLF-mediated alleviation of H_(2)O_(2)-induced bMECs pyroptosis.The results showed as follows:1)p 65 mRNA and protein expression levels were significantly increased in H_(2)O_(2)-induced bMECs pyroptosis(P<0.05),and significantly decreased following BLF treatment(P<0.05).BLF and BAY 11-7082 significantly inhibited p65 expression and nuclear translocation(P<0.05),effectively alleviating bMECs pyroptosis.The expression levels of pyroptosis-related genes and proteins in bMECs were significantly reduced by BLF and BAY 11-7082(P<0.05),as well as the fluorescence intensity of ASC spots was reduced.2)SIRT1 mRNA and protein expression levels were significantly decreased in H_(2)O_(2)-induced bMEC pyroptosis(P<0.05),and significantly increased following BLF treatment(P<0.05).EX 527 significantly inhibited SIRT1 expression,while promoted p65 expression and nuclear translocation(P<0.05).EX 527 also significantly increased the expression levels of pyroptosis-related genes and proteins in bMECs(P<0.05),and enhanced ASC spots fluorescence intensity.In summary,80μg/mL BLF alleviates H_(2)O_(2)-induced bMECs pyroptosis and NLRP3 inflammasome activation by inhibiting p65 expression and nuclear translocation.SIRT1 expression is downregulated in H_(2)O_(2)-induced bMEC pyroptosis,and BLF alleviates bMECs pyroptosis by upregulating SIRT1 expression,thereby inhibiting p65 expression and exerting a protective effect on the cells.