NF-κB、MCP-1在糖尿病大鼠腎組織的表達(dá)及丹紅注射液和二甲雙胍的干預(yù)作用
發(fā)布時間：2018-08-28 18:31
【摘要】：目的：糖尿病腎�。╠iabetic nephropathy，DN）是常見的以蛋白尿?yàn)榕R床特征的糖尿病微血管并發(fā)癥之一。糖尿病可通過多種途徑損害腎臟，并會累及腎臟所有結(jié)構(gòu)，最終導(dǎo)致結(jié)節(jié)性和彌漫性腎小球硬化。DN的發(fā)病機(jī)理較為復(fù)雜，目前傳統(tǒng)的認(rèn)識主要有遺傳因素、高血糖、晚期糖基化終末產(chǎn)物（advanced glycation end products，AGEs）形成、多元醇旁路活性增加、血流動力學(xué)異常、氧化應(yīng)激、血脂異常等。此外，炎性反應(yīng)在DN的發(fā)病中也起著重要作用，核因子-kappaB（nuclear factor-kappaB，NF-κB）是DN炎性反應(yīng)過程中的重要介質(zhì)之一，激活后調(diào)控許多細(xì)胞因子的表達(dá)，單核細(xì)胞趨化蛋白-1(monocyte chemoattractant protein-1，MCP-1)是單核/巨噬細(xì)胞特異性的趨化因子，趨化單核/巨噬細(xì)胞在腎組織的浸潤。MCP-1的基因啟動子和增強(qiáng)子序列存在NF-κB的結(jié)合位點(diǎn)。丹紅注射液是由傳統(tǒng)中藥丹參、紅花科學(xué)配方經(jīng)現(xiàn)代工藝提取精煉而成的復(fù)方注射液，其中丹參味苦性寒，為君藥，紅花性溫味辛，為臣藥，二甲雙胍可通過抑制高糖所誘發(fā)的炎性反應(yīng)、降低氧化應(yīng)激、改善微循環(huán)等途徑發(fā)揮治療DN的作用。 本研究通過制造1型糖尿病大鼠模型的方法，即腹腔注射途徑給予大劑量鏈脲佐菌素（streptozotocin，STZ），并應(yīng)用丹紅注射液和二甲雙胍進(jìn)行干預(yù)，通過HE染色及免疫組織化學(xué)、分子生物學(xué)等方法觀察實(shí)驗(yàn)動物腎組織病理形態(tài)的改變及NF-κB、MCP-1的表達(dá)變化來探討NF-κB、MCP-1在DN發(fā)生發(fā)展中的作用及丹紅注射液和二甲雙胍的腎臟保護(hù)作用機(jī)制。 方法：從48只健康雄性SD大鼠中隨機(jī)抽取10只作為正常對照組（A組），剩余作為造模組。造模組大鼠一次性腹腔注射大劑量鏈脲佐菌素（55mg/kg）。72小時后，采尾靜脈血測定血糖連續(xù)3天大于16.7mmol/L，確定1型糖尿病造模成功。將造模成功大鼠隨機(jī)分為糖尿病對照組（B組）、糖尿病丹紅注射液（2ml/kg/d）干預(yù)組（C組）、糖尿病二甲雙胍（300mg/kg/d）干預(yù)組（D組）。藥物干預(yù)共持續(xù)8周。各組大鼠自由進(jìn)食飲水，實(shí)驗(yàn)過程中不予任何降糖藥物。分別于造模前及干預(yù)后第8周末測定大鼠體重；末次給藥后，用代謝籠收集24小時尿液，測定24小時尿量，放射免疫法檢測24小時尿白蛋白定量；腹主動脈采血測定血糖（bloodglucose，BG）、低密度脂蛋白膽固醇（low-density lipoprotein cholesterol，LDL-C）、總膽固醇（total cholesterol，TC）、甘油三酯（triglyceride，TG）、血肌酐（serum creatinine，Scr）、尿素氮（blood urea nitrogen，BUN）水平。迅速分離兩側(cè)腎臟除去包膜并稱重，計算腎臟肥大指數(shù)（kidney hypertrophy index）：左、右腎重均值/體重。左側(cè)腎組織制備光鏡切片，應(yīng)用HE染色觀察腎組織病理形態(tài)改變，應(yīng)用免疫組化的方法檢測腎組織NF-κB蛋白的表達(dá)，右側(cè)腎組織應(yīng)用實(shí)時熒光定量PCR法檢測NF-κBmRNA、MCP-1mRNA的表達(dá)。實(shí)驗(yàn)結(jié)果采用SPSS16.0統(tǒng)計學(xué)軟件分析處理，符合正態(tài)、方差齊性的數(shù)據(jù)多組比較采用方差分析，組間比較采用LSD檢驗(yàn)，以P＜0.05為差異有統(tǒng)計學(xué)意義。 結(jié)果： 1實(shí)驗(yàn)第8周末，各組大鼠血糖、體重的比較 B組、C組、D組三組的體重較A組明顯下降（P均＜0.01），B組、C組、D組三組之間比較無明顯差異（P均＞0.05）。B組、C組和D組血糖較A組顯著升高，D組血糖較B組下降（P均＜0.01），B組、C兩組之間無明顯差異（P＞0.05）。 2實(shí)驗(yàn)第8周末，各組大鼠生化指標(biāo)的比較 B組、C組和D組的TC、TG和LDL-C較A組明顯升高，差異有統(tǒng)計學(xué)意義（P均＜0.01），C組、D組的TC、TG和LDL-C明顯低于B組，差異有統(tǒng)計學(xué)意義（P均＜0.01）。B組、C組和D組Scr、BUN明顯高于A組，差異有統(tǒng)計學(xué)意義（P均＜0.01），C組、D組Scr較B組明顯下降，差異有統(tǒng)計學(xué)意義（P均＜0.01），C組、D組BUN較B組下降，差異有統(tǒng)計學(xué)意義（P＜0.05，P＜0.01）。 3實(shí)驗(yàn)第8周末，各組大鼠24小時尿白蛋白定量的比較 B組、C組和D組24小時尿白蛋白定量較A組明顯升高，差異有統(tǒng)計學(xué)意義（P均＜0.01），C組、D組24小時尿白蛋白定量較B組顯著下降（P均＜0.01）。 4實(shí)驗(yàn)第8周末，各組大鼠腎臟肥大指數(shù)的比較 B組、C組和D組腎臟肥大指數(shù)明顯高于A組，差異有統(tǒng)計學(xué)意義（P均＜0.01），C組、D組與B組比較，腎臟肥大指數(shù)降低，差異有統(tǒng)計學(xué)意義（P＜0.05，P＜0.01）。 5HE染色結(jié)果的比較 B組大鼠腎小球肥大增生，腎小囊囊腔縮小，部分腎小管細(xì)胞出現(xiàn)空泡樣變性。C組、D組病變程度較B組改善。 6腎組織NF-κB蛋白表達(dá)水平 B組、C組、D組大鼠腎組織NF-κB蛋白表達(dá)較A組顯著增加（P均＜0.01），C組、D組表達(dá)較B組明顯減少（P均＜0.01）。 7腎組織NF-κBmRNA、MCP-1mRNA的表達(dá)水平 與A組比較，B組、C組、D組大鼠腎組織NF-κBmRNA、MCP-1mRNA表達(dá)明顯增加（P均＜0.01），與B組比較，C組、D組NF-κBmRNA的表達(dá)明顯減少（P均＜0.01），，MCP-1mRNA的表達(dá)也有所減少（P均＜0.05）。 結(jié)論： 1與正常大鼠比較，糖尿病大鼠腎組織NF-κB、MCP-1表達(dá)水平明顯升高，提示糖尿病腎病的發(fā)生和發(fā)展可能與NF-κB、MCP-1介導(dǎo)的炎性反應(yīng)密切相關(guān)。 2丹紅注射液和二甲雙胍除可能通過改善血脂、減少尿白蛋白防治糖尿病腎病外，還可能抑制炎性因子NF-κB、MCP-1的表達(dá)發(fā)揮其腎臟保護(hù)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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R587.2;R692
本文編號：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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R587.2;R692
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 任松;李貴森;;丹紅注射液對尿蛋白和腎功能影響的Meta分析[J];中國中西醫(yī)結(jié)合腎病雜志;2015年09期
相關(guān)碩士學(xué)位論文 前1條
1 陳秀芬;基于文獻(xiàn)挖掘的中藥治療糖尿病用藥篩選及作用機(jī)制研究[D];北京中醫(yī)藥大學(xué);2016年
本文編號：2210241
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