三溴丙酮酸通过调控有氧糖酵解–细胞焦亡抑制低氧诱导的大鼠PASMCs增殖

3-BP Inhibits Hypoxia-Induced Proliferation of RPASMCs by Regulating Aerobic Glycolysis-Pyroptosis

该文探讨了三溴丙酮酸(3-bromopyruvate,3-BP)对低氧诱导的大鼠肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)的作用及机制。取生长状态良好的PASMCs,在其细胞密度为60%~70%时,细胞饥饿处理24 h。随机将细胞分成4组:正常对照组(Normal组)、正常+3-BP组(Normal+3-BP组)、低氧造模组(Hypoxia组)、低氧+3-BP组(Hypoxia+3-BP组)。通过CCK-8检测3-BP抑制低氧诱导PASMCs增殖的最适浓度;EDU检测各组细胞增殖情况;Western blot检测各组细胞己糖激酶2(hexokinase 2,HK2)、丙酮酸脱氢酶(pyruvate dehydrogenase,PDH)、M2型丙酮酸激酶(pyruvate kinase isozyme type 2,PKM2)、血清核苷酸结合寡聚结构域样受体蛋白3(nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domain-containing 3,NLRP3)、GSDMD蛋白N-端片段(gasdermin D,GSDMD-N)、半胱氨酸蛋白酶-1(cysteinyl aspartate specific proteinase-1,Caspase-1)、白细胞介素-1β(interleukin-1 beta,IL-1β)、白细胞介素-18(interleukin-18,IL-18)增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的蛋白表达变化;乳酸试剂盒检测各组细胞乳酸含量;免疫荧光法检测各组细胞HK2、NLRP3基因表达情况;ELISA法检测各组细胞IL-1β、IL-18蛋白含量。结果显示,3-BP抑制低氧诱导的PAMSCs增殖的有效浓度为40μmol/L。与Normal组相比,Hypoxia组PAMSCs增殖显著增多(P<0.0001),PCNA蛋白表达水平明显上升(P<0.05),HK2、PKM2蛋白表达水平显著上升(P<0.01),PDH蛋白表达水平明显下降(P<0.0001),NLRP3蛋白表达水平上升(P<0.01)、GSDMD-N蛋白表达水平上升(P<0.001)、P20蛋白表达水平上升(P<0.05)、IL-1β蛋白表达水平上升(P<0.0001)、IL-18蛋白表达水平上升(P<0.05);乳酸含量明显增多(P<0.0001);免疫荧光显示HK2阳性表达细胞数显著增加(P<0.001)、NLRP3阳性表达细胞数显著增加(P<0.0001);各组肺组织IL-1β含量增多(P<0.001)、IL-18含量增多(P<0.01)。与Hypoxia组相比,Hypoxia+3-BP组PAMSCs增殖显著减少(P<0.001),PCNA蛋白表达水平下降(P<0.01),HK2蛋白表达水平明显下降(P<0.05)、PKM2蛋白表达水平明显下降(P<0.001),PDH蛋白表达水平上升(P<0.01),NLRP3、GSDMD-N、IL-18蛋白表达水平下降(P<0.05),P20、IL-1β蛋白表达水平下降(P<0.01);乳酸含量明显降低(P<0.001);免疫荧光显示HK2阳性表达细胞数显著减少(P<0.001)、NLRP3阳性表达细胞数显著减少(P<0.0001);各组肺组织IL-1β、IL-18蛋白含量降低(P<0.05)。总之,3-BP通过调控有氧糖酵解–细胞焦亡抑制低氧诱导的大鼠PASMCs增殖。

This article investigates the effect and mechanism of 3-BP(3-bromopyruvate)on hypoxiainduced PASMCs(pulmonary artery smooth muscle cells)in rats.PASMCs in good growth condition were taken.When the cell density was 60%-70%,the cells were starved for 24 h.The cells were randomly divided into four groups:normal control group(Normal group),normal+3-BP group(Normal+3-BP group),hypoxia model group(Hypoxia group),hypoxia+3-BP group(Hypoxia+3-BP group).CCK8 assay was used to detect the optimal concentration of 3-BP to inhibit hypoxia-induced PASMCs proliferation.Edu was used to detect cell proliferation in each group.The protein expressions of HK2,PDH,PKM2,NLRP3,GSDMD-N,caspase-1,P20,IL-1β,IL-18 and PCNA in each group were detected by Western blot.Lactic acid content of cells was detected by lactic acid kit.Immunofluorescence method was used to detect the protein expression of HK2 and NLRP3 in each group.The protein levels of IL-1β and IL-18 in each group were detected by Elisa.The results showed that the effective concentration of 3-BP to inhibit the proliferation of hypoxia-induced PAMSCs was 40μmol/L.Compared with the Normal group,the proliferation of PAMSCs in the Hypoxia group was significantly increased(P<0.0001);the expression of PCNA protein was significantly increased(P<0.05);the expression of HK2 and PKM2 protein was significantly increased(P<0.01);the expression of PDH protein was significantly decreased(P<0.0001);the expression of NLRP3 protein was significantly increased(P<0.01);the expression of GSDMD-N protein was significantly increased(P<0.001);the expression of P20 protein was significantly increased(P<0.05);the expression of IL-1β protein was significantly increased(P<0.0001);the expression of IL-18 protein was significantly increased(P<0.05).The lactic acid content increased significantly(P<0.0001).Immunofluorescence showed that the number of cells with positive expression of HK2 was significantly increased(P<0.001),and the number of cells with positive expression of NLRP3 was significantly increased(P<0.0001).The content of IL-1β and IL-18 in lung tissues increased(P<0.001),the content of IL-18 in lung tissues increased(P<0.01).Compared with the Hypoxia group,the proliferation of PAMSCs in the Hypoxia+3-BP group was significantly decreased(P<0.001);the protein expression of PCNA was decreased(P<0.01);the protein expression of HK2 was decreased(P<0.05);the protein expression of PKM2 was decreased(P<0.001)and the protein expression of PDH was increased(P<0.01).The protein expression of NLRP3,GSDMD-N and IL-18 decreased(P<0.05),and the expression of P20 and IL-1β proteins decreased(P<0.01).The lactate content was significantly decreased(P<0.001).The lactic acid content was significantly decreased(P<0.001).Immunofluorescence showed that the number of cells with positive expression of HK2 was significantly decreased(P<0.001),and the number of cells with positive expression of NLRP3 was significantly decreased(P<0.0001).The protein contents of IL-1β and IL-18 in lung tissues of each group decreased(P<0.05).In conclusion,3-BP inhibits hypoxia-induced proliferation of rat PASMCs by regulating aerobic glycolysis-pyroptosis.