| Objective: Diabetic nephropathy(DN) is one of the microvascularcomplications of diabetes mellitus, which is very common and characteristicof albuminuria in clinical practice. Diabetes mellitus can damage kidney inmore than one way and all structures will be impaired, resulting in nodularand diffuse glomerular sclerosis in the end. The pathogenesis of DN iscomplicated. Now the traditional understanding includes genetic factor,hyperglycemia, formation of advanced glycation end products(AGEs),increased polyol bypass activities, hemodynamic disorder, oxidative stress anddyslipidemia. In addition, inflammation is also considered to contribute tomorbidity of DN. Nuclear factor-kappaB(NF-κB) is one of the importantmediators in the process of inflammation of DN and regulates many cytokinesexpression. Monocyte chemoattractant protein-1(MCP-1) is a specificchemokine of monocyte/macrophage. MCP-1can also induce infiltration ofmonocyte/macrophage in the renal tissue. MCP-1gene promoter and enhancerof sequence have the NF-κB binding sites. Danhong injection, a kind ofcompound injection, extracted and refined by modern technology, is made upof two kinds of traditional Chinese medicine, salvia miltiorrhiza and safflower.Salvia miltiorrhiza, cold in property, bitter in flavor, is the sovereign drug.Safflower, warm in property, acrid in flavor, is the minister drug. Metforminplays a role in the treatment of DN through inhibiting inflammatory responseinduced by hyperglycemia, reducing oxidative stress and improvingmicrocirculation. In this study, type1diabetic rat model was established byintroperitoneal injection of high-dose streptozotocin(STZ). Danhong injectionand metformin were applied to diabetic rats for intervention. The change ofrenal pathological morphology and the expression of NF-κB and MCP-1in renal tissue was detected with methods of HE, immunohistochemistry andmolecular biology to explore the effect of NF-κB and MCP-1in the genesisand development of DN and the renal protective mechanism of Danhonginjection and metformin.Methods: All the SD rats were randomly divided into10normal rats(group A) and36experimental rats. Experimental rats were injected withstreptozotocin(55mg/kg) intraperitoneally once. After72hours, the caudalvenous blood glucose was measured. If the blood glucose levels were higherthan16.7mmol/L in the subsequent three days, we thought that type1diabeticmodels were established successfully. Then we divided diabetic rats into threegroups: including diabetic control group(group B), diabetic group withtreatment of Danhong injection (2ml/kg/d)(group C) and diabetic group withtreatment of metformin (300mg/kg/d)(group D). The whole experiment lastedeight weeks. During the experiment, all rats could eat and drink freely, but thehypoglycemic agents were not given to them. Body weight was measured atthe beginning of the experiment and at the end of eight weeks. After the lastdrug administration,24-hour urine volumes were measured.24-hour urinealbumin was measured by radioimmunoassay. Blood glucose (BG),low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC),triglyceride (TG), serum creatinine (Scr), blood urea nitrogen (BUN) weredetected. Separate bilateral kidneys rapidly, remove envelopes, weigh andcalculate kidney hypertrophy index: mean weight of left and right kidney/body weight. The left renal specimens were examined by HE to observe themorphous of renal tissue. The expression of NF-κB in renal tissue wasdetected by immunohistochemistry. Real-time fluorescence quantitative PCRwas used to detect the expression of NF-κBmRNA and MCP-1mRNA in rightrenal tissue. The experiment data were dealt with SPSS16.0. If the data wereconsistent with normal distribution and homogeneity of variance, analysis ofvariance was used to compare muitiple-group data and LSD test was adoptedto compare inter-group data. Results：1At the end of eight weeks, comparison of body weight and blood glucose inevery group: the body weight of group B, group C and group D weresignificantly lighter than that of group A(all P＜0.01).There was no statisticaldifference in body weight among group B, group C and group D(all P＞0.05).The blood glucose of group B, group C and group D were significantly higherthan that of group A(all P＜0.01), but the blood glucose of group D decreasedcompared with group B(P＜0.01). There was no statistical difference in bloodglucose between group B and group C(P＞0.05).2At the end of eight weeks, comparison of biochemical indexes in everygroup: TC, TG and LDL-C of group B, group C and group D weresignificantly higher than that of group A(all P＜0.01). All the above indexesdecreased compared with group B(all P＜0.01). Scr and BUN of group B,group C and group D were higher than that of group A, the difference wasstatistically significant(all P＜0.01). Compared with group B, Scr of group Cand group D dramatically decreased(all P＜0.01). BUN of group C and groupD were lower than that of group B(P＜0.05，P＜0.01).3At the end of eight weeks, comparison of24-hour urine albumin in everygroup: compared with group A,24-hour urine albumin of group B, group Cand group D were markedly higher(all P＜0.01).24-hour urine albumin ofgroup C and group D were lower than that of group B(all P＜0.01).4At the end of eight weeks, comparison of kidney hypertrophy index in everygroup: compared with group A, kidney hypertrophy index of group B, groupC and group D was statistically higher(all P＜0.01), while the index decreasedin group C and group D compared with group B(P＜0.05，P＜0.01).5HE staining in every group at the end of eight weeks: in group B, rats hadcharacteristics of glomerular hypertrophy and proliferation and the lumen ofthe renal capsule was shrunk. There was vacuolar degeneration in some renaltubular cells. The lesion of group C and group D improved compared withgroup B.6Expression of NF-κB protein in every group at the end of eight weeks: theexpression of NF-κB protein in renal tissue in group B, group C and group D significantly increased compared with group A(all P＜0.01). The index ofgroup C and group D decreased compared with group B(all P＜0.01).7Expression of NF-κBmRNA and MCP-1mRNA in renal tissue in everygroup at the end of eight weeks: the expression of NF-κBmRNA andMCP-1mRNA in renal tissue in group B, group C and group D was highercompared with group A(all P＜0.01). Expression of NF-κBmRNA in group Cand group D decreased compared with group B(all P＜0.01). Expression ofMCP-1mRNA in group C and group D was also lower than that of group B(all P＜0.05).Conclusions:1Compared with normal rats, the expression of NF-κB and MCP-1in therenal tissue were higher. The occurrence and development of diabeticnephropathy might have a close relationship with inflammatory responsemediated by NF-κB and MCP-1.2Except reducing the levels of blood lipid and urine albumin, Danhonginjection and metformin might have renal protection by reducing theexpression NF-κB and MCP-1. |
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