NF-κB、MCP-1在糖尿病大鼠肾组织的表达及丹红注射液和二甲双胍的干预作用
发布时间：2018-08-28 18:31
【摘要】：目的：糖尿病肾病（diabetic nephropathy，DN）是常见的以蛋白尿为临床特征的糖尿病微血管并发症之一。糖尿病可通过多种途径损害肾脏，并会累及肾脏所有结构，最终导致结节性和弥漫性肾小球硬化。DN的发病机理较为复杂，目前传统的认识主要有遗传因素、高血糖、晚期糖基化终末产物（advanced glycation end products，AGEs）形成、多元醇旁路活性增加、血流动力学异常、氧化应激、血脂异常等。此外，炎性反应在DN的发病中也起着重要作用，核因子-kappaB（nuclear factor-kappaB，NF-κB）是DN炎性反应过程中的重要介质之一，激活后调控许多细胞因子的表达，单核细胞趋化蛋白-1(monocyte chemoattractant protein-1，MCP-1)是单核/巨噬细胞特异性的趋化因子，趋化单核/巨噬细胞在肾组织的浸润。MCP-1的基因启动子和增强子序列存在NF-κB的结合位点。丹红注射液是由传统中药丹参、红花科学配方经现代工艺提取精炼而成的复方注射液，其中丹参味苦性寒，为君药，红花性温味辛，为臣药，二甲双胍可通过抑制高糖所诱发的炎性反应、降低氧化应激、改善微循环等途径发挥治疗DN的作用。 本研究通过制造1型糖尿病大鼠模型的方法，即腹腔注射途径给予大剂量链脲佐菌素（streptozotocin，STZ），并应用丹红注射液和二甲双胍进行干预，通过HE染色及免疫组织化学、分子生物学等方法观察实验动物肾组织病理形态的改变及NF-κB、MCP-1的表达变化来探讨NF-κB、MCP-1在DN发生发展中的作用及丹红注射液和二甲双胍的肾脏保护作用机制。 方法：从48只健康雄性SD大鼠中随机抽取10只作为正常对照组（A组），剩余作为造模组。造模组大鼠一次性腹腔注射大剂量链脲佐菌素（55mg/kg）。72小时后，采尾静脉血测定血糖连续3天大于16.7mmol/L，确定1型糖尿病造模成功。将造模成功大鼠随机分为糖尿病对照组（B组）、糖尿病丹红注射液（2ml/kg/d）干预组（C组）、糖尿病二甲双胍（300mg/kg/d）干预组（D组）。药物干预共持续8周。各组大鼠自由进食饮水，实验过程中不予任何降糖药物。分别于造模前及干预后第8周末测定大鼠体重；末次给药后，用代谢笼收集24小时尿液，测定24小时尿量，放射免疫法检测24小时尿白蛋白定量；腹主动脉采血测定血糖（bloodglucose，BG）、低密度脂蛋白胆固醇（low-density lipoprotein cholesterol，LDL-C）、总胆固醇（total cholesterol，TC）、甘油三酯（triglyceride，TG）、血肌酐（serum creatinine，Scr）、尿素氮（blood urea nitrogen，BUN）水平。迅速分离两侧肾脏除去包膜并称重，计算肾脏肥大指数（kidney hypertrophy index）：左、右肾重均值/体重。左侧肾组织制备光镜切片，应用HE染色观察肾组织病理形态改变，应用免疫组化的方法检测肾组织NF-κB蛋白的表达，右侧肾组织应用实时荧光定量PCR法检测NF-κBmRNA、MCP-1mRNA的表达。实验结果采用SPSS16.0统计学软件分析处理，符合正态、方差齐性的数据多组比较采用方差分析，组间比较采用LSD检验，以P＜0.05为差异有统计学意义。 结果： 1实验第8周末，各组大鼠血糖、体重的比较 B组、C组、D组三组的体重较A组明显下降（P均＜0.01），B组、C组、D组三组之间比较无明显差异（P均＞0.05）。B组、C组和D组血糖较A组显著升高，D组血糖较B组下降（P均＜0.01），B组、C两组之间无明显差异（P＞0.05）。 2实验第8周末，各组大鼠生化指标的比较 B组、C组和D组的TC、TG和LDL-C较A组明显升高，差异有统计学意义（P均＜0.01），C组、D组的TC、TG和LDL-C明显低于B组，差异有统计学意义（P均＜0.01）。B组、C组和D组Scr、BUN明显高于A组，差异有统计学意义（P均＜0.01），C组、D组Scr较B组明显下降，差异有统计学意义（P均＜0.01），C组、D组BUN较B组下降，差异有统计学意义（P＜0.05，P＜0.01）。 3实验第8周末，各组大鼠24小时尿白蛋白定量的比较 B组、C组和D组24小时尿白蛋白定量较A组明显升高，差异有统计学意义（P均＜0.01），C组、D组24小时尿白蛋白定量较B组显著下降（P均＜0.01）。 4实验第8周末，各组大鼠肾脏肥大指数的比较 B组、C组和D组肾脏肥大指数明显高于A组，差异有统计学意义（P均＜0.01），C组、D组与B组比较，肾脏肥大指数降低，差异有统计学意义（P＜0.05，P＜0.01）。 5HE染色结果的比较 B组大鼠肾小球肥大增生，肾小囊囊腔缩小，部分肾小管细胞出现空泡样变性。C组、D组病变程度较B组改善。 6肾组织NF-κB蛋白表达水平 B组、C组、D组大鼠肾组织NF-κB蛋白表达较A组显著增加（P均＜0.01），C组、D组表达较B组明显减少（P均＜0.01）。 7肾组织NF-κBmRNA、MCP-1mRNA的表达水平 与A组比较，B组、C组、D组大鼠肾组织NF-κBmRNA、MCP-1mRNA表达明显增加（P均＜0.01），与B组比较，C组、D组NF-κBmRNA的表达明显减少（P均＜0.01），，MCP-1mRNA的表达也有所减少（P均＜0.05）。 结论： 1与正常大鼠比较，糖尿病大鼠肾组织NF-κB、MCP-1表达水平明显升高，提示糖尿病肾病的发生和发展可能与NF-κB、MCP-1介导的炎性反应密切相关。 2丹红注射液和二甲双胍除可能通过改善血脂、减少尿白蛋白防治糖尿病肾病外，还可能抑制炎性因子NF-κB、MCP-1的表达发挥其肾脏保护作用。
[Abstract]:Objective: Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus characterized by proteinuria. Diabetes can damage the kidney through various ways and involve all the structures of the kidney, eventually leading to nodular and diffuse glomerulosclerosis. The main genetic factors are hyperglycemia, advanced glycation end products (AGEs), increased polyol bypass activity, hemodynamic abnormalities, oxidative stress, dyslipidemia and so on. In addition, inflammatory reaction plays an important role in the pathogenesis of DN. Nuclear factor-kappa B (NF-kappa B) is an inflammatory factor in DN. Monocyte chemoattractant protein-1 (MCP-1) is a monocyte/macrophage specific chemokine, which chemoattracts the infiltration of monocyte/macrophage into kidney tissue. The promoter and enhancer of MCP-1 gene contain NF-kappa B. Danhong injection is a compound injection made from traditional Chinese medicine Salvia miltiorrhiza and safflower scientific formula extracted and refined by modern technology. Salvia miltiorrhiza has a bitter cold taste, is a monarch medicine, safflower Wenweixin is a subject medicine, metformin can inhibit inflammation induced by high glucose, reduce oxidative stress and improve microcirculation. The action of DN.
In this study, a rat model of type 1 diabetes mellitus was established by intraperitoneal injection of high-dose streptozotocin (STZ) and intervention with Danhong injection and metformin. The pathological changes of renal tissue and NF-kappa B, M in experimental animals were observed by HE staining, immunohistochemistry and molecular biology. To explore the role of NF-kappa B and MCP-1 in the development of DN and the renal protective mechanism of Danhong injection and metformin.
METHODS: Ten healthy male SD rats were randomly selected as normal control group (group A) and the rest as model group. After one-off intraperitoneal injection of high-dose streptozotocin (55mg/kg) for 72 hours, the blood glucose of tail venous blood was measured for 3 days more than 16.7mmol/L to determine the success of type 1 diabetes modeling. The rats were divided into diabetic control group (group B), diabetic Danhong injection (2ml/kg/d) intervention group (group C) and diabetic metformin (300mg/kg/d) intervention group (group D). After administration, 24-hour urine was collected by metabolic cage, 24-hour urine volume was measured, 24-hour urinary albumin was measured by radioimmunoassay, blood glucose (BG), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglyceride (TG), and abdominal aorta blood glucose (BG) were measured. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured. The kidneys on both sides were separated quickly and the capsules were removed and weighed. The kidney hypertrophy index (KHI) was calculated: the mean weight of left and right kidneys / body weight. Methods The expression of NF-kappa B protein in kidney tissue was detected, and the expression of NF-kappa B mRNA and MCP-1 mRNA in right kidney tissue were detected by real-time fluorescence quantitative PCR. The experimental results were analyzed by SPSS16.0 statistical software, which accorded with normal, and the data of homogeneity of variance were analyzed by variance analysis. Statistical significance.
Result:
1 the blood glucose and body weight of rats in each group at the end of experiment eighth.
The body weight of group B, group C and group D was significantly lower than that of group A (P < 0.01). There was no significant difference among group B, group C and group D (P > 0.05). The blood glucose of group B, group C and group D was significantly higher than that of group A, and group D was significantly lower than that of group B (P < 0.01). There was no significant difference between group B and group C (P > 0.05).
2 at the end of experiment eighth, the biochemical indexes of rats in each group were compared.
TC, TG and LDL-C in group B, C and D were significantly higher than those in group A (P < 0.01). TC, TG and LDL-C in group C and D were significantly lower than those in group B (P < 0.01). Scr and BUN in group B, C and D were significantly higher than those in group A (P < 0.01). Scr in group C and D was significantly lower than that in group B (P < 0.01). Significance (P < 0.01), C group and D group BUN decreased compared with B group, the difference was statistically significant (P < 0.05, P < 0.01).
3 at the end of experiment eighth, the 24 hour urine albumin quantitation of rats in each group was compared.
The 24-hour urinary albumin levels in group B, group C and group D were significantly higher than those in group A (P < 0.01). The 24-hour urinary albumin levels in group C and group D were significantly lower than those in group B (P < 0.01).
4 Comparison of kidney hypertrophy index in each group at the end of experiment eighth.
The renal hypertrophy index of group B, group C and group D was significantly higher than that of group A (P < 0.01). The renal hypertrophy index of group C and group D was lower than that of group B (P < 0.05, P < 0.01).
Comparison of 5HE staining results
In group B, glomerular hypertrophy and cystic cavity narrowing were observed, and some tubular cells showed vacuolar degeneration.
6 expression level of NF- kappa B protein in renal tissue
The expression of NF-kappa B protein in group B, group C and group D was significantly higher than that in group A (P < 0.01). The expression of NF-kappa B protein in group C and group D was significantly lower than that in group B (P < 0.01).
7 the expression level of NF- kappa BmRNA and MCP-1mRNA in renal tissue
Compared with group A, the expression of NF-kappa B mRNA and MCP-1 mRNA in group B, group C and group D increased significantly (P < 0.01). Compared with group B, the expression of NF-kappa B mRNA in group C and group D decreased significantly (P < 0.01), and the expression of MCP-1 mRNA also decreased (P < 0.05).
Conclusion:
Compared with normal rats, the expression levels of NF-kappa B and MCP-1 in kidney tissue of diabetic rats were significantly higher, suggesting that the occurrence and development of diabetic nephropathy may be closely related to the inflammatory response mediated by NF-kappa B and MCP-1.
Danhong injection and metformin may not only prevent and treat diabetic nephropathy by improving blood lipid and reducing urinary albumin, but also inhibit the expression of inflammatory factors NF-kappa B and MCP-1.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692
本文编号：2210241
[Abstract]:Objective: Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus characterized by proteinuria. Diabetes can damage the kidney through various ways and involve all the structures of the kidney, eventually leading to nodular and diffuse glomerulosclerosis. The main genetic factors are hyperglycemia, advanced glycation end products (AGEs), increased polyol bypass activity, hemodynamic abnormalities, oxidative stress, dyslipidemia and so on. In addition, inflammatory reaction plays an important role in the pathogenesis of DN. Nuclear factor-kappa B (NF-kappa B) is an inflammatory factor in DN. Monocyte chemoattractant protein-1 (MCP-1) is a monocyte/macrophage specific chemokine, which chemoattracts the infiltration of monocyte/macrophage into kidney tissue. The promoter and enhancer of MCP-1 gene contain NF-kappa B. Danhong injection is a compound injection made from traditional Chinese medicine Salvia miltiorrhiza and safflower scientific formula extracted and refined by modern technology. Salvia miltiorrhiza has a bitter cold taste, is a monarch medicine, safflower Wenweixin is a subject medicine, metformin can inhibit inflammation induced by high glucose, reduce oxidative stress and improve microcirculation. The action of DN.
In this study, a rat model of type 1 diabetes mellitus was established by intraperitoneal injection of high-dose streptozotocin (STZ) and intervention with Danhong injection and metformin. The pathological changes of renal tissue and NF-kappa B, M in experimental animals were observed by HE staining, immunohistochemistry and molecular biology. To explore the role of NF-kappa B and MCP-1 in the development of DN and the renal protective mechanism of Danhong injection and metformin.
METHODS: Ten healthy male SD rats were randomly selected as normal control group (group A) and the rest as model group. After one-off intraperitoneal injection of high-dose streptozotocin (55mg/kg) for 72 hours, the blood glucose of tail venous blood was measured for 3 days more than 16.7mmol/L to determine the success of type 1 diabetes modeling. The rats were divided into diabetic control group (group B), diabetic Danhong injection (2ml/kg/d) intervention group (group C) and diabetic metformin (300mg/kg/d) intervention group (group D). After administration, 24-hour urine was collected by metabolic cage, 24-hour urine volume was measured, 24-hour urinary albumin was measured by radioimmunoassay, blood glucose (BG), low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), triglyceride (TG), and abdominal aorta blood glucose (BG) were measured. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were measured. The kidneys on both sides were separated quickly and the capsules were removed and weighed. The kidney hypertrophy index (KHI) was calculated: the mean weight of left and right kidneys / body weight. Methods The expression of NF-kappa B protein in kidney tissue was detected, and the expression of NF-kappa B mRNA and MCP-1 mRNA in right kidney tissue were detected by real-time fluorescence quantitative PCR. The experimental results were analyzed by SPSS16.0 statistical software, which accorded with normal, and the data of homogeneity of variance were analyzed by variance analysis. Statistical significance.
Result:
1 the blood glucose and body weight of rats in each group at the end of experiment eighth.
The body weight of group B, group C and group D was significantly lower than that of group A (P < 0.01). There was no significant difference among group B, group C and group D (P > 0.05). The blood glucose of group B, group C and group D was significantly higher than that of group A, and group D was significantly lower than that of group B (P < 0.01). There was no significant difference between group B and group C (P > 0.05).
2 at the end of experiment eighth, the biochemical indexes of rats in each group were compared.
TC, TG and LDL-C in group B, C and D were significantly higher than those in group A (P < 0.01). TC, TG and LDL-C in group C and D were significantly lower than those in group B (P < 0.01). Scr and BUN in group B, C and D were significantly higher than those in group A (P < 0.01). Scr in group C and D was significantly lower than that in group B (P < 0.01). Significance (P < 0.01), C group and D group BUN decreased compared with B group, the difference was statistically significant (P < 0.05, P < 0.01).
3 at the end of experiment eighth, the 24 hour urine albumin quantitation of rats in each group was compared.
The 24-hour urinary albumin levels in group B, group C and group D were significantly higher than those in group A (P < 0.01). The 24-hour urinary albumin levels in group C and group D were significantly lower than those in group B (P < 0.01).
4 Comparison of kidney hypertrophy index in each group at the end of experiment eighth.
The renal hypertrophy index of group B, group C and group D was significantly higher than that of group A (P < 0.01). The renal hypertrophy index of group C and group D was lower than that of group B (P < 0.05, P < 0.01).
Comparison of 5HE staining results
In group B, glomerular hypertrophy and cystic cavity narrowing were observed, and some tubular cells showed vacuolar degeneration.
6 expression level of NF- kappa B protein in renal tissue
The expression of NF-kappa B protein in group B, group C and group D was significantly higher than that in group A (P < 0.01). The expression of NF-kappa B protein in group C and group D was significantly lower than that in group B (P < 0.01).
7 the expression level of NF- kappa BmRNA and MCP-1mRNA in renal tissue
Compared with group A, the expression of NF-kappa B mRNA and MCP-1 mRNA in group B, group C and group D increased significantly (P < 0.01). Compared with group B, the expression of NF-kappa B mRNA in group C and group D decreased significantly (P < 0.01), and the expression of MCP-1 mRNA also decreased (P < 0.05).
Conclusion:
Compared with normal rats, the expression levels of NF-kappa B and MCP-1 in kidney tissue of diabetic rats were significantly higher, suggesting that the occurrence and development of diabetic nephropathy may be closely related to the inflammatory response mediated by NF-kappa B and MCP-1.
Danhong injection and metformin may not only prevent and treat diabetic nephropathy by improving blood lipid and reducing urinary albumin, but also inhibit the expression of inflammatory factors NF-kappa B and MCP-1.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692
【引证文献】
相关期刊论文 前1条
1 任松;李贵森;;丹红注射液对尿蛋白和肾功能影响的Meta分析[J];中国中西医结合肾病杂志;2015年09期
相关硕士学位论文 前1条
1 陈秀芬;基于文献挖掘的中药治疗糖尿病用药筛选及作用机制研究[D];北京中医药大学;2016年
本文编号：2210241
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