目的基于核因子激活的B细胞的NOD-样受体热蛋白结构域相关蛋白3(NOD-like receptor pyrin domain-containing protein 3,NLRP3)/白细胞介素-18(interleukin-18,IL-18)/κ-轻链(nuclear factor-activated B cellκ-light chain,NF-κB)...目的基于核因子激活的B细胞的NOD-样受体热蛋白结构域相关蛋白3(NOD-like receptor pyrin domain-containing protein 3,NLRP3)/白细胞介素-18(interleukin-18,IL-18)/κ-轻链(nuclear factor-activated B cellκ-light chain,NF-κB)信号通路探讨化痰安神汤抑制神经元细胞凋亡的作用机制。方法体外培养HT22小鼠海马神经元细胞,C57小鼠灌胃化痰安神方后制备含药小鼠血清,CCK-8法筛选化痰安神方含药血清最佳干预浓度。采用不同浓度含药血清处理HT22细胞后,予脂多糖(lipopolysaccharide,LPS)诱导其发生凋亡,CCK-8法L-乳酸脱氢酶(L-lactate dehydrogenase,L-LDH)生化检测法和Annexin V/PI染色法用于检测细胞损伤与凋亡情况,Western blotting法用于测定通路相关蛋白P-NF-κB/NF-κB、凋亡相关的斑点样蛋白(apoptosis-associated speck-like protein containing,ASC)、NLRP3、Caspase-1的表达,酶联免疫吸附测定法用于测定IL-18的表达。过表达NLRP3后重复试验,进行单基因功能验证。结果化痰安神方含药血清可改善LPS诱导的细胞损伤和凋亡,降低细胞L-LDH浓度(P<0.05);显著下调NLRP3/IL-18/NF-κB信号通路相关蛋白的转录表达(P<0.05);显著降低细胞内IL-18浓度;同时过表达NLRP3可逆转化痰安神方含药血清对HT22细胞凋亡抑制作用(P<0.05)。结论化痰安神方能够通过抑制NLRP3/IL-18/NF-κB信号通路改善神经元细胞凋亡。展开更多
目的:通过盲肠穿刺结扎法建立脓毒症大鼠模型,观察四逆汤是否可以改善脓毒症SD大鼠肾损伤,探讨四逆汤是否通过抑制NLRP3-Caspase-1-IL-1β/IL-18信号通路改善脓毒症SD大鼠肾损伤。方法:100只雌性大鼠,按照大鼠体重进行初步分类,采用轮...目的:通过盲肠穿刺结扎法建立脓毒症大鼠模型,观察四逆汤是否可以改善脓毒症SD大鼠肾损伤,探讨四逆汤是否通过抑制NLRP3-Caspase-1-IL-1β/IL-18信号通路改善脓毒症SD大鼠肾损伤。方法:100只雌性大鼠,按照大鼠体重进行初步分类,采用轮次分配法,确保每组大鼠体重分布均匀。平均分成3大组,每组30只老鼠,从中随机选取1组作为空白组,剩下两组使用CLP法进行造模,分别是实验组、对照组两个大组。随后又分别按大鼠体重采用轮次分类分为8小时组、16小时组和24小时组三个小组,每个小组10只;其中死亡10只,继续造模补充;实验组采用四逆汤灌胃、对照组和空白组采用生理盐水灌胃。分别在8 h、16 h、24 h时间点,取各组大鼠肾组织进行HE染色,计算肾损伤评分,WB检测肾组织中NLRP3、Caspase-1蛋白表达,ELISA法检测血清IL-1β、IL-18表达情况。结果:1) 肾组织HE染色后高倍镜下对照组8小时开始可见肾脏正常形态被破坏,肾小球体积明显增大,肾小囊腔扩大,系膜细胞肿胀、间质炎性细胞浸润;16小时较8小时更加严重,并且间质有明显出血及大量炎症细胞浸润;24小时肾小球缺血皱缩,失去正常形态;球–囊间隙扩大,炎性细胞渗出增多,肾小管上皮细胞肿胀。实验组大鼠8小时及16小时的肾脏病理改变较对照组明显减轻。肾损伤评分中,肾损伤随着时间的延长而逐渐加重(P P P P > 0.05)。3) ELISA检测血清IL-1β、IL-18后发现,总趋势是从8小时开始明显上升(P P P > 0.05)。这与WB检查结果相似。结论:四逆汤可能通过介导NLRP3-Caspase-1-IL-1β/IL-18信号通路改善脓毒症急性肾损伤SD大鼠肾损伤。Objective: Sepsis rat model was established by cecal puncture and ligation to observe whether Sini Decoction could improve kidney injury of septic SD rats, and to explore whether Sini Decoction could improve kidney injury of septic SD rats by inhibiting NLRp3-caspase-1-IL-1β/IL-18 signaling pathway. Methods: 100 female rats, initially classified according to the weight of the rats, using a round-robin allocation method to ensure an even distribution of weights in each group. Divided into 3 large groups on average, each group of 30 rats, from which 1 group was randomly selected as the blank, and the remaining two groups were used for modeling with the CLP method. A total of 100 female rats were initially classified according to their body weights. The round-robin allocation method was used to ensure a uniform distribution of body weights among each group. The rats were evenly divided into three major groups, with 30 rats in each group. One group was randomly selected as the blank group, and the remaining two groups were used to establish the model using the CLP method, namely the experimental group and the control group. Subsequently, according to the body weights of the rats, each of these two groups was further divided into three subgroups (8-hour group, 16-hour group, and 24-hour group) using the round-robin classification method, with 10 rats in each subgroup. Ten rats died during the process, and new rats were used to continue the modeling to make up for the loss. The experimental group was administered Sini Decoction by gavage, while the control group and the blank group were administered normal saline by gavage. At the time points of 8 h, 16 h, and 24 h, the renal tissues of the rats in each group were collected for HE staining, and the renal injury score was calculated. Western blotting (WB) was used to detect the protein expressions of NLRP3 and Caspase-1 in the renal tissues, and the enzyme-linked immunosorbent assay (ELISA) was used to detect the expressions of serum IL-1β and IL-18. Results: 1) After HE staining of the renal tissues, under a high-power microscope, in the control group, the normal morphology of the kidneys was found to be damaged starting from 8 hours, with a significant increase in the volume of the glomeruli, expansion of the renal capsule cavity, swelling of the mesangial cells, and infiltration of interstitial inflammatory cells. The situation at 16 hours was more severe than that at 8 hours, with obvious interstitial bleeding and a large number of inflammatory cell infiltrations. At 24 hours, the glomeruli were ischemic and shrunk, losing their normal morphology;the glomerular-capsular space was enlarged, the exudation of inflammatory cells increased, and the tubular epithelial cells were swollen. The renal pathological changes in the experimental group at 8 hours and 16 hours were significantly milder than those in the control group. In the renal injury score, the renal injury gradually worsened over time (P P P P > 0.05). 3) Serum IL-1β and IL-18 were detected by ELISA, and they began to increase at 8 h (P P P > 0.05). This is similar to the WB results. Conclusion: Sini Decoction may improve renal function in SD rats with sepsis acute kidney injury by mediating NLRP3-Caspase-1-IL-1β/IL-18 signaling pathway.展开更多
文摘目的:通过盲肠穿刺结扎法建立脓毒症大鼠模型,观察四逆汤是否可以改善脓毒症SD大鼠肾损伤,探讨四逆汤是否通过抑制NLRP3-Caspase-1-IL-1β/IL-18信号通路改善脓毒症SD大鼠肾损伤。方法:100只雌性大鼠,按照大鼠体重进行初步分类,采用轮次分配法,确保每组大鼠体重分布均匀。平均分成3大组,每组30只老鼠,从中随机选取1组作为空白组,剩下两组使用CLP法进行造模,分别是实验组、对照组两个大组。随后又分别按大鼠体重采用轮次分类分为8小时组、16小时组和24小时组三个小组,每个小组10只;其中死亡10只,继续造模补充;实验组采用四逆汤灌胃、对照组和空白组采用生理盐水灌胃。分别在8 h、16 h、24 h时间点,取各组大鼠肾组织进行HE染色,计算肾损伤评分,WB检测肾组织中NLRP3、Caspase-1蛋白表达,ELISA法检测血清IL-1β、IL-18表达情况。结果:1) 肾组织HE染色后高倍镜下对照组8小时开始可见肾脏正常形态被破坏,肾小球体积明显增大,肾小囊腔扩大,系膜细胞肿胀、间质炎性细胞浸润;16小时较8小时更加严重,并且间质有明显出血及大量炎症细胞浸润;24小时肾小球缺血皱缩,失去正常形态;球–囊间隙扩大,炎性细胞渗出增多,肾小管上皮细胞肿胀。实验组大鼠8小时及16小时的肾脏病理改变较对照组明显减轻。肾损伤评分中,肾损伤随着时间的延长而逐渐加重(P P P P > 0.05)。3) ELISA检测血清IL-1β、IL-18后发现,总趋势是从8小时开始明显上升(P P P > 0.05)。这与WB检查结果相似。结论:四逆汤可能通过介导NLRP3-Caspase-1-IL-1β/IL-18信号通路改善脓毒症急性肾损伤SD大鼠肾损伤。Objective: Sepsis rat model was established by cecal puncture and ligation to observe whether Sini Decoction could improve kidney injury of septic SD rats, and to explore whether Sini Decoction could improve kidney injury of septic SD rats by inhibiting NLRp3-caspase-1-IL-1β/IL-18 signaling pathway. Methods: 100 female rats, initially classified according to the weight of the rats, using a round-robin allocation method to ensure an even distribution of weights in each group. Divided into 3 large groups on average, each group of 30 rats, from which 1 group was randomly selected as the blank, and the remaining two groups were used for modeling with the CLP method. A total of 100 female rats were initially classified according to their body weights. The round-robin allocation method was used to ensure a uniform distribution of body weights among each group. The rats were evenly divided into three major groups, with 30 rats in each group. One group was randomly selected as the blank group, and the remaining two groups were used to establish the model using the CLP method, namely the experimental group and the control group. Subsequently, according to the body weights of the rats, each of these two groups was further divided into three subgroups (8-hour group, 16-hour group, and 24-hour group) using the round-robin classification method, with 10 rats in each subgroup. Ten rats died during the process, and new rats were used to continue the modeling to make up for the loss. The experimental group was administered Sini Decoction by gavage, while the control group and the blank group were administered normal saline by gavage. At the time points of 8 h, 16 h, and 24 h, the renal tissues of the rats in each group were collected for HE staining, and the renal injury score was calculated. Western blotting (WB) was used to detect the protein expressions of NLRP3 and Caspase-1 in the renal tissues, and the enzyme-linked immunosorbent assay (ELISA) was used to detect the expressions of serum IL-1β and IL-18. Results: 1) After HE staining of the renal tissues, under a high-power microscope, in the control group, the normal morphology of the kidneys was found to be damaged starting from 8 hours, with a significant increase in the volume of the glomeruli, expansion of the renal capsule cavity, swelling of the mesangial cells, and infiltration of interstitial inflammatory cells. The situation at 16 hours was more severe than that at 8 hours, with obvious interstitial bleeding and a large number of inflammatory cell infiltrations. At 24 hours, the glomeruli were ischemic and shrunk, losing their normal morphology;the glomerular-capsular space was enlarged, the exudation of inflammatory cells increased, and the tubular epithelial cells were swollen. The renal pathological changes in the experimental group at 8 hours and 16 hours were significantly milder than those in the control group. In the renal injury score, the renal injury gradually worsened over time (P P P P > 0.05). 3) Serum IL-1β and IL-18 were detected by ELISA, and they began to increase at 8 h (P P P > 0.05). This is similar to the WB results. Conclusion: Sini Decoction may improve renal function in SD rats with sepsis acute kidney injury by mediating NLRP3-Caspase-1-IL-1β/IL-18 signaling pathway.
