猪肺炎支原体乳酸脱氢酶在诱导猪支气管上皮细胞凋亡中的作用

Effects of LDH in Mesomycoplasma(Mycoplasma)hyopneumoniae on Apoptosis of Porcine Bronchial Epithelial Cells

猪肺炎支原体感染会引起宿主上皮细胞的损伤及凋亡。上皮屏障的破坏往往导致细菌和病毒的继发感染,给养猪业造成严重的经济损失,但具体的致病机制及毒力因子尚未完全阐明。前期研究发现猪肺炎支原体乳酸脱氢酶(lactate dehydrogenase, LDH)不仅参与猪肺炎支原体的代谢还与猪肺炎支原体的毒力相关。因此,本研究通过原核表达及亲和层析获得猪肺炎支原体乳酸脱氢酶重组蛋白(recombinant LDH, rLDH),之后分别用不同浓度的rLDH与猪支气管上皮细胞(porcine bronchial epithelial cell, PBEC)孵育,经显微镜观察rLDH对细胞形态的影响,通过CCK-8法检测rLDH对细胞活力的影响。选取对细胞形态及活力影响不显著的50和100μg·mL^(-1) rLDH分别与PBEC孵育,通过流式细胞仪检测细胞凋亡率,反转录荧光定量PCR(reverse transcription-quantitative polymerase chain reaction, RT-qPCR)法和免疫印记检测凋亡相关基因caspase 3、caspase 8、caspase 9、Bax、BCL-2的转录水平和蛋白水平。结果显示:猪肺炎支原体rLDH呈剂量依赖性地诱导PBEC凋亡,其作用与猪肺炎支原体菌株一致。用rLDH及Mhp 168孵育后,PBEC的促凋亡因子Bax、caspase 3和caspase 9的mRNA水平显著上调,凋亡抑制因子BCL-2的mRNA水平显著下调,caspase 8的mRNA水平未发生显著变化。促凋亡因子Bax、caspase 3的蛋白表达水平显著上调,凋亡抑制因子BCL-2的蛋白表达水平显著下调。结果表明,猪肺炎支原体可能通过LDH诱导PBEC内源性细胞凋亡,凋亡相关因子caspase 3、caspase 9、Bax和BCL-2在其中发挥重要作用。

Mesomycoplasma(Mycoplasma)hyopneumoniae(Mhp)infection can cause damage and apoptosis of host epithelial cells,and the breakdown of the epithelial barrier often leads to the secondary infection of other bacteria and viruses,causing serious economic losses to the swine industry.However,the pathogenicity and virulence determinants of Mhp are not yet completely elucidated.Previous studies have found that Mhp lactate dehydrogenase(LDH)not only involved in the metabolism of Mhp,but also related to its virulence.Therefore,in this study,recombinant LDH(rLDH)was obtained through prokaryotic expression and affinity chromatography,and different concentrations of rLDH were used to interact with porcine bronchial epithelial cell(PBEC).The effect of rLDH on cellular morphology was observed by microscope,and the effect of rLDH on cellular viability was detected by CCK-8 method.The rLDH concentrations(50 and 100μg·mL^(-1))were selected for further analysis by that there was no significant effect on cellular morphology and viability when it incubated with PBEC,and the apoptosis rate of cells was detected by flow cytometry.The transcriptional and expression levels of apoptosis-related genes caspase3,caspase 8,caspase 9,Bax and BCL-2 were detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and Western blot.The results showed that rLDH induced apoptosis of PBEC in a dose-dependent manner,the function of that was similar with Mhp.The mRNA levels of caspase 3,caspase 9,and Bax of PBEC after incubation with rLDH and Mhp 168 were significantly up-regulated and the mRNA level of BCL-2 was significantly down-regulated,while the mRNA of caspase 8 was not changed significantly.The protein expression levels of caspase 3 and Bax were significantly up-regulated and the protein expression level of BCL-2 was significantly down-regulated.The results showed that Mhp could induce the endogenous apoptosis of PBEC through LDH,in which the apoptosis-related factors caspase 3,caspase 9,Bax and BCL-2 playing important roles.