【摘要】 目的：探讨糖尿病大鼠肾组织肿瘤坏死因子α(TNF-α)、核转录因子-κB(NF-κB)对肾组织细胞凋亡的诱导以及雷帕霉素干预的作用。方法：选择2型糖尿病大鼠(GK大鼠)20只，均分为糖尿病模型组(DM组)，雷帕霉素治疗组(DMR组，建立糖尿病模型后给予雷帕霉素治疗);另选择10只Wistar雄性大鼠作为正常对照组。分别在4周、8周后检测各组肾组织细胞凋亡情况，NF-κB，TNF-α的表达水平以及血脂、血糖水平。结果：模型制作后 4周、8周，与正常对照组和DMR组比较，DM组大鼠肾组织细胞凋亡数[RCA， 4周(0.217±0.031)、(0.272±0.031)比(0.545±0.031)、8周(0.358±0.031)、(0.350±0.031)比(0.811±0.031)]均明显增多，NF-κBp65[OD： 4周(0.160±0.027)、(0.131±0.027)比(0.411±0.027)、8周(0.232±0.027)、(0.275±0.027)比(0.634±0.027)]、TNF-α[OD： 4周(0.242±0.027)、(0.275±0.027)比(0.617±0.027)、8周(0.385±0.027)、(0.342±0.027)比(0.912±0.027)]表达均明显升高(P均<0.01)。相关分析显示，肾组织NF-κBp65与TNF-α，RCA与TNF-α，NF-κBp65均呈正相关关系(r=0.956,0.953,0.886，P均<0.01)。结论：糖尿病病大鼠的肾细胞凋亡显著增加，雷帕霉素干预组肾细胞凋亡显著减少，NF-κB、TNF-α表达显著增加，其保护作用可能是NF-κB、TNF-α阻滞细胞的凋亡。
Abstract：Objective：To investigate the apoptosis of renal cells induced by tumor necrosis factor alpha (TNF-α) and nuclear factor-κB (NF-κB) in diabetic rats and intervention of rapamycin.Methods：A total of 20 rats (Goto-Kakizaki rats) with type 2 diabetes mellitus (T2DM) were randomly and equally divided into DM model group (DM group) and rapamycin treatment group (DMR group， received rapamycin treatment after DM model was established); another 10 Wistar male rats were regard as normal control group. Apoptosis of renal cells， expression levels of TNF-α and NF-κB and levels of blood lipids， blood glucose were measured in all groups after four weeks and eight weeks.Results：Four and eight weeks after model was established， compared with normal control group and DMR group， there were significant increase in renal cells apoptosis [RCA， four weeks： (0.217±0.031)， (0.272±0.031) vs. (0.545±0.031)， eight weeks： (0.358±0.031)， (0.350±0.031) vs. (0.811±0.031)] and expressions of NF-κB p65 [OD： four weeks： (0.160±0.027)， (0.131±0.027) vs. (0.411±0.027)， eight weeks： (0.232±0.027)， (0.275±0.027) vs. (0.634±0.027)] and TNF-α [OD： four weeks： (0.242±0.027)， (0.275±0.027) vs. (0. 617±0.027)， eight weeks： (0.385±0.027)， (0.342±0.027) vs. (0.912±0.027)] in DM group (P<0.01 all). Correlation analysis indicated that there were positive correlations between renal NF-κB p65 and TNF-α， among RCA and TNF-α， NF-κB p65 (r=0.956， 0.953， 0.886，P<0.01 all). Conclusion：Renal cells apoptosis significant increase in rats with T2DM， but renal cells apoptosis significant decrease in DMR group， protection mechanism of rapamycin may be NF-κB、TNF-α inhibiting renal cells apoptosis.
Key words：Diabetes mellitus， type 2; Apoptosis; Sirolimus; Rats
Chronic complications of diabetic mellitus (DM) are main cause of disability and death in DM patients， in which diabetic nephropathy (DN) is one of most severe and common microvascular complications of DM. Recent studies found that apoptosis in the pathogenesis of diabetic nephropathy also plays an important role， with the increasing of number of apoptotic cells， the degree of proteinuria and renal function deterioration， but the mechanism is unclear. Nuclear factor (NF)-κB is an important factor regulating inflammatory reactions. Study indicates that rapamycin can inhibit the activation of NF-κB， so inhibits the expressions of some cytokines [such as tumor necrosis factor (TNF)-α] and chemotactic factor， plays anti-inflammatory effects . However， the role that rapamycin inhibited the activation of NF-κB and interfered cell apoptosis in diabetic kidney has not been reported in the literature. Therefore， this study applies generally accepted model of type 2 diabetes to observe expression of TNF-α and NF-κB in diabetic kidney tissue and its relationship with renal cell apoptosis and the intervention effect of rapamycin in order，and investigates possible mechanism of protection effect of rapamycin in order， to provide base for DN treatment.
1 Subjects and methods
1.1 Experimental materials
1.1.1 Experimental animals：A total of 10 Wistar rats with 200～400g of specific- pathogen free (SPF) grade (provided by experimental center of China Medical University) and 20 T2DM rats (Goto-Kakizaki， GK rats) with 300～400g (provided by Shanghai Slyke company) were selected.
1.1.2 Main reagents： Albumin kit and urine β2-microglubolin (β2-MG) kit were produced by American Biosource Company; NF-κB (P65) primary antibody was provided by Beijing Zhongshan Biology Company; following reagents were all provided by Wuhan Boshide Bioengineering Company： pathological and immunohistochemical reagents， TNF-α polyclonal antibody， TUNEL kit， brown DAB coloring kit， horseradish peroxidase labeled streptavidin working solution， immunohistochemistry kit， and fetal bovine serum.
1.1.3 Rapamycin was provided by Nanjing Debao Biochemical Equipment Co.ltd.
1.1.4 Main apparatus and equipments：Blood sugar analyzer was provided by USA Johnson & Johnson. Systematic microscope was type LEICA ICCA， and TD2000 image analysis system.
1.2 Experimental methods
1.2.1 Establishment of model and grouping：Fasting plasma glucose (FPG， fasting for 8h)≥7.8 mmol/L or 2h postprandial blood glucose (2hPG)≥11.1 mmol/L were regard as diagnostic standards for T2DM. Rats were divided into three groups： (1) DM group; including 10 GK rats; (2) DMR group (rapamycin treatment group， received rapamycin 6mg/kg， gavage， three days/week， after establishing DM model)， including 10 GK rats; (3) Normal control group; including 10 Wistar male rats， do not receive insulin hypoglycemic therapy throughout the experiment.
1.2.2 Detection of renal cell apoptosis by TUNEL method：Positive cells were defined as： nucleus stained to brown yellow， pyknosis or nuclear fragmentation and form was irregular. Image analysis system was used to measure mean absorbance of apoptotic cell. A total of 10 specimens from each group were analyzed under light microscope， and five random high power fields (×400) were selected from each slice for analysis. OTPIMAS 6.5 image analysis software was used to measure area of glomerulus and integrated optical density of glomerulus and renal interstitial.
1.2.3 Immunohistochemical detection of renal NF-κB， TNF-α expression：Positive signals were observed using SP method under light microscope. Image analysis system was used to measure mean absorbance of renal expressions of NF-κB and TNF-α.
1.3 Statistical analysis
SPSS 13.0 statistical software was used to perform statistical analysis. Measurement data were presented as mean ± standard deviation(x-±s). Single factor analysis of variance (ANOVA) was used to analyze difference among groups. For those with significant difference， Newman-Kuels q test was used to perform pairwise comparison. Pearson method was used to analyze relationship between NF-κB and TNF-α. Non-parametric Spearman method was used to analyze relationship among TNF-α， NF-κB and apoptosis. P<0.05 was regard as possessing significant difference.
2.1 Changes of blood glucose， blood triglyceride (TG) and blood total cholesterol (TC) in three groups
Level of blood glucose in DM group was significantly higher than that of normal control group (P<0.05)， and that of DMR group was significantly lower than that of DM group (P<0.05); TG and TC levels of DM group and DMR group were significantly higher than those of normal control group (P<0.05 all). There were no significant difference in TG and TC levels between DM group and DMR group， were shown in table 1.Table 1 Comparisons of blood glucose， TG and TC among three groups
2.2 Comparison of 24h urine albumin and β2-MG among three groups
Compared with NC group， there were significant increases in levels of urine albumin and β2-MG in DM group and DMR group (P<0.05 all). Levels of urine albumin and β2-MG in DMR group were significantly lower than those of DM group (P<0.05 all)， were shown in table 2. Table 2 Comparison of 24h excretion rate of urine albumin and urine β2-MG among three groups
2.3 Renal apoptosis in DM group and NC group by TUNEL method
There were no obvious apoptosis in renal tissues of rats in NC group; number of renal apoptotic cells gradually increased in DM group on four weeks and eight weeks (P<0.01). These were shown in figure 1(in inside front cover) and table 3.
2.4 Immunohistochemical expression of NF-κBp65 in NC group and DM group
Positive expression feature of NF-κBp65 is brown grains in nucleus or cytoplasm of renal cells. In NC group， there was a bit positive expression of NF-κBp65 in glomerulus and tubule， mainly in the latter;there was increased positive expression of NF-κBp65 on 4th week，and its expression significantly increased on 8th week in DM group(P<0.05 both)，were shown in figure 2(in inside front cover).
Semiquantitative analysis indicated that renal NF-κBp65 level significantly increased in DM group (P<0.01)， on 4 th week the NF-κBp65 level of renal tissue in DM group was significantly higher than that of NC group and DMR group (P<0.01). It further increased on 8 th week and it was significantly higher than that of normal control group and DMR group (P<0.01 both)， were shown in table 3.
2.5 Immunohistochemical expression of TNF-α in NC group and DM group
There was slight positive expression of TNF-α in renal tissue of rats in NC group; positive expression of TNF-α increased on 4th week and its expression significantly increased on 8th week in DM group (P<0.05 both)，were shown in figure 3(in inside front cover).
Semiquantitative analysis indicated that TNF-α expression gradually increased on 4th～ 8th weeks in rats’ renal tissues of DM group， and was all significantly higher than that of NC group and DMR group (P<0.01 all)， were shown in table 3.
2.6 Correlation among expressions of NF-κBp65， TNF-α and renal cell apoptosis in NC group and DM group
Spearman analysis indicated that expression of NF-κBp65 was positively correlated with TNF-α (r=0.956， P<0.01)， and positively correlated with renal cell apoptosis(r= 0.886， P<0.01)in renal tissue; and TNF-α was positively correlated with renal cell apoptosis (r=0.953， P<0.01); expression of NF-κBp65 and TNF-α were positively correlated with course of disease.
2.7 Influence of rapamycin on renal cell apoptosis and expressions of NF-κBp65 and TNF-α in DM rats
Compared with DM group， renal overexpressions of NF-κBp65 and TNF-α were significantly inhibited (P<0.01 both)， and number of renal apoptotic cells significantly decreased in DMR group on 4th and 8th weeks， were shown in table 3.Table 3 Renal cell apoptosis and expressions of NF-κBp65 and TNF-α in three groups
DN is a common chronic complication，one of general microvascular complications，and is second to large vascular complications for DM only. Its mechanism is still not clear and there is no effective early intervention method[5-9]. Study found that during development of DN， glomerular intrinsic cells and renal tubular epithelial cells gradually decreased. There′s evidence proving that the process of cell decrease was related with apoptosis. The present study indicated that there were few apoptotic cells in normal control group; compared with normal control group， renal apoptotic cells in DM group significantly increased on 4th and 8th weeks. It suggests that renal cell decrease is caused from renal cells apoptosis at least in part， and is one of causes of renal dysfunction. Enhanced expression of TNF-α can promote synthesis and release of inflammatory factors， make inflammatory cells aggregated and adhesive and lead to inflammatory injury of renal cells， and renal cell apoptosis.
When DN occurs， renal tissues can produce large amount of TNF-α entering into blood under oxidative stress and hyperglycemia etc.， lead up to a series of pathophysiological process， activating NF-κB via ribosome-inactivating protein(RIP)， and inducing renal cell apoptosis. NF-κB can efficiently induces expressions of many kinds of cytokines (such as TNF-α)， adhesion molecules， chemotactic factors and acute phase reactive proteins， also regulates expression of multiple genes as an important transcription factor mediating signal transduction in the cell， aggravating renal cell apoptosis.
The present study indicated that compared with normal control group，NF-κBp65 expression gradually increased in renal tissue of rats in DM group on 4th week and 8th weeks， was consistent and positively correlated with expression of TNF-α and changes of renal cell apoptosis， suggesting that TNF-α and NF-κBp65 in common mediate inflammatory reactions in renal tissue of DM， inducing renal cell apoptosis， and is one of important mechanisms of renal injure of DM.
Rapamycin is a new immunosuppressive agent with good effect， low toxicity and no renal toxicity， it's mainly applied in patients undergoing renal transplantation as immunosuppressive agent. In recent years， study found that it also possessed protection on kidney in DM patients， but there existed controversy on its effect on renal cell apoptosis in DM patients. Leiberthal et al found that rapamycin can inhibit proliferation and promote apoptosis in renal tubule of rats， but there was also study proving that rapamycin can reduce apoptosis of renal tubular epithelial cells. Now many clinical tests found that no obvious renal toxicity was found in therapeutic dose of rapamycin (6mg/kg). The present study indicated that overexpressions of TNF-α and NF-κB in renal tissue of DM rats were significantly inhibited after intervention with therapeutic dose of rapamycin. And rapamycin can lower blood glucose level， decrease loss of urinary albumin and urine β2-MG， decrease renal cell apoptosis， suggesting that rapamycin possesses protection on kidney in DM patients. Its effect on decreasing renal cell apoptosis may be correlated with overexpressions of TNF-α and NF-κB， therefore provided theory basis for treatment of DN with rapamycin.
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