图1 6组大鼠肾组织病理形态学观察结果(HE染色)
Published:15 June 2024,
Received:28 December 2023,
Revised:11 April 2024
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To investigate the effect of dioscin on renal injury in septic rats and its possible mechanism.
The septic rat model was induced by using cecal ligation and puncture. Sixty model rats were randomly divided into model group (0.5% sodium carboxymethyl cellulose solution), dioscin low-dose, medium-dose and high-dose groups (30, 60, 120 mg/kg) and dexamethasone group (positive control, 10 mg/kg), with 12 rats per group; another 12 rats were selected as the sham operation group (0.5% sodium carboxymethyl cellulose solution). After 15 minutes of modeling, rats in each group were injected with medicine/0.5% sodium carboxymethyl cellulose solution via the tail vein. Twenty-four hours after administration, the levels of creatinine (Cr), blood urea nitrogen (BUN), neutrophil gelatinase associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), interleukin 6 (IL-6), IL-1β and tumor necrosis factor-α (TNF-α) in serum and malondialdehyde (MDA) in renal tissue, superoxide dismutase (SOD) activity and the protein expressions of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NOD-like receptor protein 3 (NLRP3) were detected; renal histomorphology was observed.
Compared with model group, pathological injury of renal tissue was improved significantly in dioscin low-dose, medium-dose and high-dose groups; the levels of Cr, BUN, NGAL, KIM-1, IL-6, IL-1β and TNF-α in serum, MDA level and protein expression of NLRP3 in renal tissue were decreased significantly (P<0.05); SOD activity in renal tissue, protein expressions of Nrf2 and HO-1 were increased significantly (P<0.05), in a dose-dependent manner (P<0.05). The pathological damage of renal tissue in the dioscin high-dose group was similar to dexamethasone group, and there was no statistically significant difference in the levels of the above indicators (P>0.05).
Dioscin can activate the Nrf2/HO-1 signaling pathway to inhibit NLRP3 inflammasome, and realize the inhibition of inflammatory factors and oxidative stress, so as to protect the kidney injury in sepsis.
脓毒症是感染引发的全身性炎症综合征,会造成多器官功能衰竭,影响患者生命安全,已成为当今临床重症患者的重要死亡原因之一。脓毒症患者肾损伤发生率高达50%,该类患者预后差、死亡率高,且目前尚无有效治疗方法[
薯蓣皂苷为薯蓣根提取物,具有抗肿瘤、抗感染、抗炎、抗病毒等多种药理作用。据报道,薯蓣皂苷可减轻脂多糖诱导的急性肺损伤后的炎症反应和氧化应激[
核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)/血红素加氧酶1(heme oxygenase-1,HO-1)信号通路具有调节细胞氧化-抗氧化、炎症反应等多种生物活性,是机体内调节氧化应激的关键途径[
HCC300型全自动生化分析仪、Sense型酶标仪均购自上海堃择实业有限公司;B302型光学显微镜购自上海普赫光电科技有限公司;QIAxpert型分光光度计、KST-6000型凝胶成像分析系统均购自上海金鹏分析仪器有限公司。
薯蓣皂苷原料药(批号YS-U-0701,纯度98%)购自美国Sigma公司;地塞米松注射液(阳性对照药物,批号20220116,规格5 mg/支)购自郑州卓峰制药有限公司;羧甲基纤维素钠(sodium carboxymethyl cellulose,CMC-Na)溶液、BCA蛋白检测试剂盒(批号分别为MK-2823、MK-30116)均购自上海麦克林生化科技股份有限公司;中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase associated lipocalin,NGAL)、肾损伤分子1(kidney injury molecule-1,KIM-1)、白细胞介素6(interleukin-6,IL-6)、IL-1β、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)酶联免疫吸附检测(ELISA)试剂盒以及兔源Nrf2、HO-1、NLRP3、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)一抗和辣根过氧化物酶标记的羊抗兔IgG二抗(批号分别为Y223258、Y234370、Y119623、Y255439、Y236738、Y233432、Y141443、Y137568、Y263823、Y243789)均购自上海翌圣生物科技股份有限公司;苏木精-伊红(HE)染色试剂盒、丙二醛(malondialdehyde,MDA)检测试剂盒、超氧化物歧化酶(superoxide dismutase,SOD)检测试剂盒和RIPA蛋白裂解液(批号分别为D983478、D234482、D498347、D239844)均购自上海帛龙生物科技有限公司。
本研究所用动物为SPF级雄性SD大鼠,共81只,9~10周龄,体重348~366 g,购自成都达硕实验动物有限公司[生产许可证号:SCXK(川)2021-004]。购入后,大鼠饲养于我院动物实验房内,环境温度(25±0.5) ℃,相对湿度(50±5)%,12 h光/12 h暗循环。本研究通过了我院伦理委员会批准(审批号:JCLS2023第41号)。
参照相关文献[
造模15 min后,薯蓣皂苷低、中、高剂量组大鼠分别尾静脉注射30、60、120 mg/kg薯蓣皂苷(用0.5%CMC-Na溶液稀释成质量浓度分别为3、6、12 mg/mL的溶液,现配现用)[
给药后24 h,对大鼠心脏穿刺取血,室温静置2 h,以3 000 r/min离心10 min,收集上层血清,置于-20 ℃冰箱中保存,备用;取血后,脱颈椎处死大鼠,快速取出肾组织,将左肾用10%甲醛固定,右肾保存于-80 ℃冰箱中备用。
取出血清,以全自动生化分析仪检测血清中肌酐(creatinine,Cr)、尿素氮(blood urea nitrogen,BUN)水平,采用ELISA法以酶标仪检测血清中NGAL、KIM-1、IL-6、IL-1β、TNF-α水平。
取固定于10%甲醛中的左肾组织适量,常规石蜡包埋、切片(厚6 µm),行HE染色、二甲苯透明、封片后,在光学显微镜下观察肾组织病理形态学变化并拍照。
取冻存的右肾组织约30 mg,解冻后制备组织匀浆,分别采用硫代巴比妥酸比色法、黄嘌呤氧化酶法以分光光度计检测肾组织中MDA水平和SOD活性。
采用Western blot法检测。取冻存的右肾组织约50 mg,解冻后制备匀浆,以蛋白裂解液裂解20 min提取组织中总蛋白,采用BCA法定量蛋白浓度,然后电泳分离蛋白样品并转膜;室温下将膜封闭2 h后,在4 ℃下与相应的一抗[Nrf2(稀释比例1∶790)、HO-1(稀释比例1∶790)、NLRP3(稀释比例1∶830)、GAPDH(稀释比例1∶850)]一起孵育24 h;室温下用二抗(稀释比例1∶1 700)孵育1 h后,以电化学发光试剂显影,使用Image J软件对蛋白表达进行定量分析。以目的蛋白与内参蛋白(GAPDH)条带灰度值的比值表示目的蛋白的表达量。
使用SPSS 25.0软件对数据进行统计分析。计量资料以x±s表示,多组间比较采用单因素方差分析,组间两两比较采用SNK-q检验。检验水准α=0.05。
与假手术组比较,模型组大鼠血清中Cr、BUN、NGAL、KIM-1水平均显著升高(P<0.05);与模型组比较,各给药组大鼠血清中上述指标水平均显著降低(P<0.05),且薯蓣皂苷的作用具有剂量依赖性(P<0.05);薯蓣皂苷高剂量组大鼠上述指标水平与地塞米松组比较,差异均无统计学意义(P>0.05)。结果见
组别 | Cr/(μmol/L) | BUN/(mmol/L) | NGAL/(ng/L) | KIM-1/(ng/L) |
---|---|---|---|---|
假手术组 | 38.25±4.06 | 6.45±1.14 | 26.16±3.15 | 19.46±2.05 |
模型组 | 80.85±8.25a | 18.95±3.38a | 144.25±9.27a | 92.45±7.30a |
薯蓣皂苷低剂量组 | 64.56±7.02b | 13.64±2.44b | 96.56±9.15b | 64.31±6.62b |
薯蓣皂苷中剂量组 | 55.48±6.22bc | 10.86±2.06bc | 60.19±7.23bc | 47.60±4.13bc |
薯蓣皂苷高剂量组 | 44.62±4.73bcd | 7.80±1.52bcd | 37.51±4.10bcd | 29.01±3.58bcd |
地塞米松组 | 43.18±4.52bcd | 7.73±1.57bcd | 35.38±4.37bcd | 27.58±3.17bcd |
a:与假手术组比较,P<0.05;b:与模型组比较,P<0.05;c:与薯蓣皂苷低剂量组比较,P<0.05;d:与薯蓣皂苷中剂量组比较,P<0.05。
与假手术组比较,模型组大鼠血清中IL-6、IL-1β、TNF-α水平均显著升高(P<0.05);与模型组比较,各给药组大鼠血清中IL-6、IL-1β、TNF-α水平均显著降低(P<0.05),且薯蓣皂苷的作用具有剂量依赖性(P<0.05);薯蓣皂苷高剂量组大鼠上述指标水平与地塞米松组比较,差异均无统计学意义(P>0.05)。结果见
组别 | IL-6 | IL-1β | TNF-α |
---|---|---|---|
假手术组 | 7.12±0.82 | 42.16±3.15 | 51.35±6.77 |
模型组 | 16.46±2.13a | 203.16±14.50a | 141.36±11.18a |
薯蓣皂苷低剂量组 | 13.04±2.01b | 148.45±9.17b | 119.48±13.05b |
薯蓣皂苷中剂量组 | 10.35±1.76bc | 110.48±9.02bc | 110.95±13.59bc |
薯蓣皂苷高剂量组 | 8.03±1.13bcd | 76.15±7.35bcd | 86.15±9.67bcd |
地塞米松组 | 8.15±1.16bcd | 73.46±6.92bcd | 82.48±8.35bcd |
a:与假手术组比较,P<0.05;b:与模型组比较,P<0.05;c:与薯蓣皂苷低剂量组比较,P<0.05;d:与薯蓣皂苷中剂量组比较,P<0.05。
假手术组大鼠肾组织未见明显病理变化;模型组大鼠肾组织可见系膜增生、肾小管萎缩,肾小管间质被大量炎症细胞浸润;薯蓣皂苷低剂量组大鼠肾组织可见系膜增生、大量炎症细胞浸润肾小管;薯蓣皂苷中剂量组大鼠肾组织炎症浸润情况缓解;薯蓣皂苷高剂量组和地塞米松组大鼠肾组织可观察到正常的肾小管。结果见
图1 6组大鼠肾组织病理形态学观察结果(HE染色)
黑色箭头:炎症细胞浸润。
与假手术组比较,模型组大鼠肾组织中MDA水平显著升高(P<0.05),SOD活性显著降低(P<0.05);与模型组比较,各给药组大鼠肾组织中MDA水平均显著降低(P<0.05),SOD活性均显著升高(P<0.05),且薯蓣皂苷的作用具有剂量依赖性(P<0.05);薯蓣皂苷高剂量组大鼠肾组织中上述指标水平与地塞米松组比较,差异均无统计学意义(P>0.05)。结果见
组别 | MDA/(nmol/mg) | SOD/(U/mg) |
---|---|---|
假手术组 | 5.46±0.60 | 116.15±8.08 |
模型组 | 20.16±3.18a | 43.48±5.60a |
薯蓣皂苷低剂量组 | 15.23±3.25b | 64.02±5.96b |
薯蓣皂苷中剂量组 | 11.60±2.17bc | 82.89±7.25bc |
薯蓣皂苷高剂量组 | 7.73±1.02bcd | 102.38±9.03bcd |
地塞米松组 | 7.86±1.26bcd | 105.15±8.52bcd |
a:与假手术组比较,P<0.05;b:与模型组比较,P<0.05;c:与薯蓣皂苷低剂量组比较,P<0.05;d:与薯蓣皂苷中剂量组比较,P<0.05。
与假手术组比较,模型组大鼠肾组织中Nrf2、HO-1蛋白表达量显著降低(P<0.05),NLRP3蛋白表达量显著升高(P<0.05);与模型组比较,各给药组大鼠肾组织中Nrf2、HO-1蛋白表达量均显著升高(P<0.05),NLRP3蛋白表达量均显著降低(P<0.05),且薯蓣皂苷的作用具有剂量依赖性(P<0.05);薯蓣皂苷高剂量组大鼠上述蛋白表达量与地塞米松组比较,差异均无统计学意义(P>0.05)。结果见
图2 6组大鼠肾组织中Nrf2、HO-1、NLRP3蛋白表达的电泳图
组别 | Nrf2/GAPDH | HO-1/GAPDH | NLRP3/GAPDH |
---|---|---|---|
假手术组 | 1.95±0.15 | 2.76±0.38 | 0.41±0.06 |
模型组 | 0.23±0.03a | 0.09±0.01a | 1.68±0.20a |
薯蓣皂苷低剂量组 | 0.67±0.08b | 0.72±0.05b | 1.30±0.18b |
薯蓣皂苷中剂量组 | 1.02±0.10bc | 1.13±0.08bc | 0.98±0.13bc |
薯蓣皂苷高剂量组 | 1.37±0.11bcd | 1.76±0.12bcd | 0.52±0.08bcd |
地塞米松组 | 1.43±0.13bcd | 1.69±0.17bcd | 0.49±0.09bcd |
a:与假手术组比较,P<0.05;b:与模型组比较,P<0.05;c:与薯蓣皂苷低剂量组比较,P<0.05;d:与薯蓣皂苷中剂量组比较,P<0.05。
肾脏对于维持机体中水、酸碱平衡和排泄代谢等具有重要作用。当出现肾损伤后,肾小球滤过功能丧失,Cr、BUN在体内积累,危害机体健康,而脓毒症为常见的肾损伤诱因。NGAL可维持肾组织正常功能,炎症出现时其水平会升高,其在肾损伤检测中的灵敏性和特异性都很强[
脓毒症通过多种途径影响肾损伤,而炎症介质过度释放与脓毒症肾损伤的发生发展关系密切。IL-6、IL-1β、TNF-α作为标志性炎症介质,在发生脓毒症肾损伤后其水平会升高[
Nrf2是调节细胞对抗外来异物和氧化损伤的关键转录因子,参与修复炎症、对抗氧化应激等多种生理过程。正常情况下,Nrf2与抗氧化反应元件相互作用,调节机体氧化还原平衡;当细胞处于应激状态时,Nrf2入核启动抗氧化基因HO-1的表达,从而实现对机体的保护[
综上所述,薯蓣皂苷可能是通过激活Nrf2/HO-1信号通路抑制NLRP3炎性小体,实现对炎症因子表达和氧化应激的抑制,从而改善脓毒症肾损伤。但是本研究只初步探究了薯蓣皂苷对脓毒症肾损伤的影响及可能机制,未设置Nrf2/HO-1信号通路抑制剂/激动剂来进行机制验证,接下来本课题组将进一步开展相关研究。
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