骨髓间充质干细胞可减轻脂多糖所致肾损伤大鼠的细胞焦亡

Bone marrow mesenchymal stem cells attenuate pyroptosis lipopolysaccharide-induced renal injury rats

目的:探讨骨髓间充质干细胞(BMSC)移植对肾损伤大鼠体内细胞焦亡的影响及其机制。方法:体外分离培养4~5周龄清洁级雌性SD大鼠的BMSC,取第3代细胞用于后续实验。采用成组设计,随机将15只6周龄清洁级雌性SD大鼠分为对照组、肾损伤模型组和BMSC组,每组5只。采用经尾静脉注射脂多糖(LPS)1 mg/kg构建肾损伤模型;对照组以相同途径给予等量生理盐水。BMSC组于制模后2 h经尾静脉注射BMSC 0.5 mL(含BMSC 2×10^(6)个);肾损伤模型组和对照组给予等量生理盐水。于术后3 d取各组大鼠腹主动脉血,采用苦味酸法检测血肌酐(SCr)水平,采用二乙酰一肟法检测血尿素氮(BUN)水平,采用酶联免疫吸附试验(ELISA)检测血胱抑素C(Cys C)和白细胞介素(IL-1β、IL-18)水平。处死大鼠取肾组织,采用实时荧光定量聚合酶链反应(qRT-PCR)检测大鼠肾组织NOD样受体蛋白3(NLRP3)、天冬氨酸特异性半胱氨酸蛋白酶1(caspase-1)的mRNA表达;采用蛋白质免疫印迹试验(Western blotting)检测大鼠肾组织NLRP3、caspase-1的蛋白表达。结果:体外培养显示,骨髓细胞悬液培养24 h后出现大量圆形贴壁细胞和悬浮细胞,培养4~5 d后有大量长梭形细胞聚集性贴壁生长,仍可见明显的杂质细胞,经胰酶消化并传代至第3代以长梭形贴壁细胞为主,基本纯化为BMSC。体内实验显示,与对照组比较,肾损伤模型组大鼠SCr、BUN、Cys C、IL-1β、IL-18水平均明显升高〔SCr(μmol/L):85.22±2.29比21.80±0.59,BUN(mmol/L):11.50±0.64比5.86±0.83,Cys C(mg/L):0.13±0.01比0.11±0.02,IL-1β(ng/L):31.49±1.42比4.74±0.49,IL-18(ng/L):29.01±1.95比1.52±0.03,均P<0.05〕,NLRP3及caspase-1的mRNA和蛋白表达水平也均明显升高〔NLRP3 mRNA(2-ΔΔCt):3.635±0.296比1.000±0.002,caspase-1 mRNA(2-ΔΔCt):4.020±0.228比1.001±0.003;NLRP3蛋白(NLRP3/β-actin):1.560±0.868比0.902±0.036,caspase-1蛋白(caspase-1/β-actin):1.392±0.097比0.895±0.046,均P<0.05〕。与肾损伤模型组比较,BMSC组大鼠SCr、BUN、IL-1β、IL-18水平均明显下降〔SCr(μmol/L):51.64±3.84比85.22±2.29,BUN(mmol/L):9.90±0.46比11.50±0.64,IL-1β(ng/L):24.20±1.45比31.49±1.42,IL-18(ng/L):12.97±1.25比29.01±1.95,均P<0.05〕,NLRP3及caspase-1的mRNA和蛋白表达水平也均明显下降〔NLRP3 mRNA(2-ΔΔCt):1.488±0.136比3.635±0.296,caspase-1 mRNA(2-ΔΔCt):1.643±0.143比4.020±0.228;NLRP3蛋白(NLRP3/β-actin):1.227±0.053比1.560±0.868,caspase-1蛋白(caspase-1/β-actin):1.159±0.107比1.392±0.097,均P<0.05〕。结论:在体内,BMSC可以减轻LPS所致肾损伤大鼠的细胞焦亡。

Objective To investigate the effect and mechanism of bone marrow mesenchymal stem cell(BMSC)on pyroptosis of rats with kidney injury.Methods Bone marrow of 4-5 week-old female Sprague-Dawley(SD)rats was isolated in vitro and BMSC was obtained.The third generations of BMSC were used to further experiments.Fifteen 6 week-old SD rats were cluster-randomized divided into control group,kidney injury group and BMSC group(5 rats in each group).Rats in kidney injury group were injected with lipopolysaccharide(LPS)1 mg/kg via tail vein;the control group was given the same amount of normal saline.BMSC group was injected with 0.5 mL BMSC(including 2×10^(6) BMSC)via tail vein after modeling;the kidney injury group received the same amount of normal saline.On day 3 after these injections,serum creatinine(SCr)was detected by picric acid method,and blood urea nitrogen(BUN)was detected by diacetyl monoxime.The levels of cystatin C(Cys C),interleukins(IL-1βand IL-18)in blood were detected by enzyme-linked immunosorbent assay(ELISA).The rats were then sacrificed and their kidneys were removed for subsequent detection.The mRNA expression levels of NOD-like receptor protein 3(NLRP3)and cysteinyl aspartate-specific protease-1(caspase-1)of kidney were detected by quantificational real-time quantitative polymerase chain reaction(qRT-PCR).The protein expression levels of NLRP3 and caspase-1 of kidney were detected by Western blotting.Results In vitro,after bone marrow cell suspension was cultured for 24 hours,a large number of round adherent cells and suspended cells appeared in each culture flask.After 4-5 days of culture,a large number of long spindle cells adhered to the wall,and there were still obvious impurity cells.After trypsin digestion and passage to the third generation,the long spindle adherent cells grew mainly in the culture flask and were basically purified as BMSC.In vivo,compared with the control group,the levels of SCr,BUN,Cys C,IL-1βand IL-18 in kidney injury group were increased[SCr(μmol/L):85.22±2.29 vs.21.80±0.59,BUN(mmol/L):11.50±0.64 vs.5.86±0.83,Cys C(mg/L):0.13±0.01 vs.0.11±0.02,IL-1β(ng/L):31.49±1.42 vs.4.74±0.49,IL-18(ng/L):29.01±1.95 vs.1.52±0.03,all P<0.05].The mRNA and protein expression levels of NLRP3,caspase-1 were significantly increased[NLRP3 mRNA(2-ΔΔCt):3.635±0.296 vs.1.000±0.002,caspase-1 mRNA(2-ΔΔCt):4.020±0.228 vs.1.001±0.003;NLRP3 protein(NLRP3/β-actin):1.560±0.868 vs.0.902±0.036,caspase-1 protein(caspase-1/β-actin):1.392±0.097 vs.0.895±0.046,all P<0.05].Compared with kidney injury group,the levels of SCr,BUN,IL-1βand IL-18 in BMSC group were significantly decreased[SCr(μmol/L):51.64±3.84 vs.85.22±2.29,BUN(mmol/L):9.90±0.46 vs.11.50±0.64,IL-1β(ng/L):24.20±1.45 vs.31.49±1.42,IL-18(ng/L):12.97±1.25 vs.29.01±1.95,all P<0.05].The mRNA and protein expression levels of NLRP3,caspase-1 were significantly decreased[NLRP3 mRNA(2-ΔΔCt):1.488±0.136 vs.3.635±0.296,caspase-1 mRNA(2-ΔΔCt):1.643±0.143 vs.4.020±0.228;NLRP3 protein(NLRP3/β-actin):1.227±0.053 vs.1.560±0.868,caspase-1 protein(caspase-1/β-actin):1.159±0.107 vs.1.392±0.097,all P<0.05].Conclusion In vivo,BMSC may attenuate pyroptosis in LPS-induced kidney injury rats.