摘要
运用荧光定量PCR和 Western blot方法检测健康、酮病奶牛乳腺组织中肉碱棕榈酰转移酶1A(carnitine palmitoyltransferase 1a,CPT1A)及储存操作 Ca2+通道(store-operated Ca2+entry,SOCE)通路相关分子Orai1、Orai3、STIM1和STIM2的表达水平;在体外乳腺细胞培养过程中,细胞处理分为对照组、BHBA组、CGA组、CGA+BHBA 组.使用 20 mg/L 绿原酸(chlorogenic acid,CGA)刺 激细胞 12 h 后用 1.2 mmol/L BHBA 刺 激细胞24 h,运用荧光定量PCR和 Western blot方法检测不同处理细胞中CPT1A及SOCE通路相关分子Orai1、Orai3、STIM1和STIM2的表达水平.结果显示,与健康奶牛相比,酮病奶牛乳腺组织中CPT1A的转录水平和表达水平均极显著降低(P＜0.01),而Orai1、Orai3、STIM1和STIM2的转录水平和表达水平均显著升高(P＜0.05).与对照组相比,BH-BA刺激降低了 CPT1A的转录水平和表达水平(P＜0.01),升高了 Orai1、Orai3、STIM1和STIM2的转录水平和表达水平(P＜0.05);与对照组相比,CGA组CPT1A、Orai1、Orai3、STIM1和STIM2的转录水平和表达水平无明显变化(P＞0.05);与BHBA组相比,使用CGA和BHBA共同刺激使CPT1A的转录水平和表达水平升高(P＜0.05),Orai1、Orai3、STIM1和STIM2的转录水平和表达水平降低P＜0.05).结果表明,绿原酸增加酮病奶牛乳腺细胞的CPT1A的表达并降低SOCE的表达.
Abstract
This experiment is to explore whether chlorogenic acid(CGA)affects the oxidation and SOCE expression of mammary cells in ketosis dairy cows.First,the expression levels of CPT1A and SOCE pathway-related molecules Orai1,Orai3,STIM1 and STIM2 in mammary tissue of healthy and ketosis dairy cows were detected by fluorescent quantitative PCR and western blot.The four groups used for experiment were the control group,BHBA group,CGA group,and CGA+BHBA group.The cells were stimulated with 20 mg/L CGA for 12 h and then stimulated with 1.2 mmol/L BHBA for 24 h.The expression of the molecules Orai1,Orai3,STIM1 and STIM2 linked to the CPT1A and SOCE pathways in various treated cells was assessed using fluorescent quantita-tive PCR and western blot.Compared with healthy cows,the transcript level and expression level of CPT1A in the mammary gland tissue of ketosis cows were significantly decreased(P＜0.01),while the transcript levels and expression levels of Orai1,Orai3,STIM1 and STIM2 were significantly increased(P＜0.05).CPT1 A transcription and expression levels were reduced by BHBA stimulation in comparison to the control group(P＜0.01),and increased Orai1,Orai3,STIM1 and Transcript level and expression level of STIM2(P＜0.05).The transcript levels and ex-pression levels of CPT1 A,Orai1,Orai3,STIM1 and STIM2 in the CGA group did not differ signif-icantly from those in the control group(P＞0.05).The expression level increased(P＜0.05),and the transcript level and expression level of Orai1,Orai3,STIM1 and STIM2 decreased(P＜0.05).The results showed that chlorogenic acid could increase the expression of CPT1A and decrease the expression of SOCE in mammary cells of cows in ketosis.
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