柴苓汤对慢性环孢素A肾病大鼠肾脏细胞增殖的影响
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摘要
目的：探讨细胞增殖在环孢素A(CsA)慢性肾损伤中的作用及中药复方制剂柴苓汤对其的影响，为临床防治CsA肾毒性提供实验依据。
方法：
第一部分：柴苓汤对慢性环孢素A肾病大鼠肾功能及肾组织病理形态学的影响
1动物分组、模型复制及药物治疗
40只雌性SD大鼠随机分为四组，即对照组(Control)、模型组(CsA)、柴苓汤组(CsAC)及缬沙坦组(CsAV)，每组10只。采用CsA30mg/(kg·d)经口灌服28天的方法，建立慢性CsA肾病大鼠模型。对照组每日灌胃给与等容量橄榄油。造模的同时，两治疗组分别给予柴苓汤3g/(kg·d)和缬沙坦10mg/(kg·d)治疗，模型组和对照组则每日给予等容量生理盐水。
2指标检测
2.1体重及尿液检测每周末将所有大鼠移入代谢笼中，分别测量每只大鼠体重、24小时尿量及尿肌酐。
2.2肾脏病理形态学检测切取大鼠左侧肾脏组织，4%多聚甲醛固定，常规制作石蜡切片后分别行HE、Masson及PAS染色，观察肾组织病理形态改变，并参照Katafuchi评分标准从炎性细胞浸润、间质纤维化、肾小管萎缩三方面对肾小管间质损伤程度进行分析。
2.3肾功能检测实验第29天，所有大鼠被处死，取血以检测大鼠血尿素氮(BUN)及血清肌酐(SCr),并计算出肌酐清除率(CCr)。
3统计分析
数据资料以均数±标准差（±s）表示，使用SPSS16.0统计软件进行统计，组间比较采用单因素方差分析。显著性差异水平以0.05和0.01为标准。
第二部分：柴苓汤对慢性环孢素A肾病大鼠肾脏增殖细胞核抗原表达的影响
1动物分组、模型复制、药物治疗及统计分析方法同第一部分。
2指标检测实验第29天，取大鼠部分肾脏组织，分别采用免疫组化、Western-Blot、RT-PCR及流式细胞术四种方法检测大鼠肾脏PCNA表达。
2.1免疫组化：采用SABC法，以光镜下肾脏组织出现棕黄色颗粒为阳性表达。
2.2Western Blot:切取100mg-70℃冰箱中保存的肾组织，提取蛋白并定量，依次进行电泳，转膜，免疫反应及显色。最后用富士LAS-4000LIMINESCENT图像分析仪拍照。
2.3RT-PCR：根据Trizol RNA抽提试剂盒要求，切取100mg液氮保存的肾组织，提取肾皮质总RNA，并反转录合成cDNA进行PCR反应。取10L产物在1%琼脂糖凝胶中电泳，紫外灯下拍照，应用BIO-PROFIF凝胶图像分析系统进行分析。
2.4流式细胞术：取70%酒精固定的肾组织，制作肾间质、肾小管单细胞悬液后，离心沉淀2分钟。取0.1ml单细胞悬液(细胞密度为1×106个/mL)，加入碘化丙啶，4℃冰箱避光染色30min，以500目铜网过滤。采用美国Beckton Dickinson公司FACScan-420型流式细胞仪检测PCNA荧光指数(FI)、细胞凋亡率及细胞周期，并根据公式(PI)=(S+G2M)/(G0/G1+S+G2M)×100%计算出增殖指数(PI)。
第三部分：柴苓汤对慢性环孢素A肾病大鼠肾脏结缔组织生长因子表达的影响
1动物分组、模型复制、药物治疗及统计分析方法同第一部分。
2指标检测实验第29天，取大鼠部分肾脏组织，分别采用免疫组化、Western-Blot、RT-PCR检测大鼠肾脏CTGF的表达。操作步骤同第二部分。
第四部分：柴苓汤对慢性环孢素A肾病大鼠肾脏纤维连接蛋白表达的影响
1动物分组、模型复制、药物治疗及统计分析方法同第一部分。
2指标检测实验第29天，取大鼠部分肾脏组织，分别采用免疫组化、Western-Blot、RT-PCR检测大鼠肾脏FN的表达。操作步骤同第二部分。
结果：
第一部分：柴苓汤对慢性环孢素A肾病大鼠肾功能及肾组织病理形态学的影响
1体重及尿量检测结果
实验第一、二周，所有大鼠体重均有所增加，但同时段各组间比较无明显差异（P＞0.05）。随着实验时间的延长，模型组大鼠体重增长减缓，至第四周末，模型组体重较前一周反而下降，且与同时段两治疗组相比有显著差异（P＜0.01）。实验的前3周，四组大鼠24小时尿量比较无明显差异（P＞0.05）。然而至实验第4周末，模型组尿量较其余三组显著减少（P＜0.05）。
2病理形态学检测结果
HE染色结果：对照组未见明显异常；模型组肾间质大量炎性细胞浸润，同时可见肾小管上皮细胞坏死、脱落；柴苓汤组及缬沙坦组肾脏损伤程度明显轻于模型组。Masson染色结果：对照组肾间质可见少量胶原成分。模型组肾间质胶原成分大量沉积，并呈条带状分布；柴苓汤及缬沙坦组肾间质胶原成分较模型组明显减少。半定量分析结果表明，模型组肾小管间质损伤各项参数积分值均明显高于对照组（P＜0.01）。与模型组相比，两治疗组各项参数积分值则明显降低（P＜0.05，P＜0.01）。PAS染色结果：模型组肾小球基底膜增厚，细胞基质沉积增多，系膜细胞增生明显。柴苓汤组及缬沙坦组大鼠肾组织病理损伤较模型组显著减轻。
3肾功能检测结果
与对照组比较，模型组血肌酐、尿素氮水平明显升高（P＜0.01），但同时该组肌酐清除率显著降低（P＜0.01）。柴苓汤组及缬沙坦组血肌酐、尿素氮水平较模型组明显降低（P＜0.01），肌酐清除率则均较模型组明显升高（P＜0.05）。两治疗组间比较无明显统计学差异（P＞0.05）。
第二部分：柴苓汤对慢性环孢素A肾病大鼠肾脏增殖细胞核抗原表达的影响
1免疫组化结果
对照组仅见少量PCNA阳性细胞。模型组阳性细胞较对照组明显增多（P＜0.01）。柴苓汤及缬沙坦组PCNA阳性细胞较模型组显著减少（P＜0.01），但两治疗组相比无明显统计学差异（P＞0.05）。
2Western-Blot结果
对照组肾组织PCNA蛋白仅少量表达，与之相比，模型组表达增强（P＜0.01）。经柴苓汤及缬沙坦治疗，PCNA蛋白表达被下调（P＜0.05），两治疗组相比无明显差异（P＞0.05）。
3RT-PCR结果
与对照组相比，模型组PCNAmRNA表达明显增强（P＜0.01）。柴苓汤及缬沙坦均可下调PCNAmRNA高表达（P＜0.05），两治疗组相比无明显统计学差异（P＞0.05）。
4流式细胞术结果
4.1荧光指数模型组荧光指数明显高于对照组（P＜0.01）；与模型组比较，柴苓汤组及缬沙坦组荧光指数显著降低（P＜0.01）。
4.2细胞周期模型组处于S期的细胞比例较对照组增多（P＜0.01），而处于G0/G1期的细胞比例较对照组减少（P＜0.01）；柴苓汤及缬沙坦组处于S期的细胞比例较模型组明显降低（P＜0.01），处于G0/G1期的细胞比例较模型组增多（P＜0.01）。各组G2/M期细胞比例相对接近，组间比较无统计学差异（P＞0.05）。
4.3增殖指数、凋亡率与对照组比较，模型组肾脏细胞增殖指数明显升高（P＜0.01），凋亡率较对照组也有所增加，但两组凋亡率的差异没有统计学意义（P＞0.05）。模型组凋亡率/增殖指数的比值显著低于对照组（P＜0.01)；与模型组比较，柴苓汤组及缬沙坦组增殖指数均明显降低（P＜0.01），凋亡率则分别升高（P＜0.01，P＜0.05)，凋亡率/增殖指数的比值也提高（P＜0.01）。
第三部分：柴苓汤对慢性环孢素A肾病大鼠肾脏结缔组织生长因子表达的影响
1免疫组化结果
对照组大鼠肾脏组织CTGF蛋白表达为弱阳性。模型组CTGF蛋白表达明显增强，主要表达在肾小管上皮细胞及间质细胞。经柴苓汤及缬沙坦治疗后，CTGF蛋白表达显著减弱。
2Western-Blot结果
对照组大鼠肾组织CTGF蛋白表达量很少，与之相比，模型组则显著增强，柴苓汤组及缬沙坦组CTGF蛋白表达较模型组明显减少。
3RT-PCR结果
与对照组相比，模型组CTGFmRNA表达明显增强（P＜0.01），柴苓汤组及缬沙坦组CTGFmRNA表达较模型组明显降低（P＜0.05）。
第四部分：柴苓汤对慢性环孢素A肾病大鼠肾脏纤维连接蛋白表达的影响
1免疫组化结果
对照组FN蛋白少量表达，主要见于肾小管上皮细胞；模型组FN蛋白表达较对照组明显增强；经柴苓汤及缬沙坦治疗后，FN蛋白表达明显减少。
2Western-Blot结果
对照组肾组织FN蛋白仅少量表达，与之相比，模型组表达增强，柴苓汤及缬沙坦均可显著下调FN的高表达。
3RT-PCR结果
与对照组相比，模型组FNmRNA表达明显增强（P＜0.01）。柴苓汤组及缬沙坦组FNmRNA表达均显著减少（P＜0.01）。
结论：
1柴苓汤可减轻CsA诱导的肾脏病理损害，改善肾功能。
2柴苓汤可通过下调慢性CsA肾病大鼠肾组织PCNA的高表达及调节细胞周期，抑制细胞增殖活动，延缓肾间质纤维化的进程。
3柴苓汤可抑制慢性CsA肾病大鼠肾组织中CTGF蛋白及基因的表达，从而延缓肾间质纤维化进程。
4柴苓汤可抑制慢性CsA肾病大鼠肾组织中FN的合成及表达，减少细胞外基质沉积，减轻肾间质纤维化程度。
AIM: To explore the role of cell proliferation in chronic renal injuryinduced by cyclosporine A(CsA) and the effect of Chailing Decoction on cellproliferation, then to provide the experimental basis for prevention andtreatment of cyclosporine nephrotoxicity.
METHODS:
PartⅠ: Effects of Chailing Decoction on Renal Function and Pathomo-rphology in Rats with Chronic Cyclosporine A Nephropathy (CCN).
1. Animal Grouping, Model Establishment and Medication
Forty female rats were randomized equally into4groups: Control group(Control), model group(CsA),Chailing Decoction group(CsAC)and Valsartangroup(CsAV), and each group had10rats. The rat models with CCN wereinduced by gavage with cyclosporine A30mg/(kg·d) for28days. The rats incontrol group received orally the same dosage of olive oil. At the same time,the rats in two treatment groups were treated respectively with ChailingDecoction3g/(kg·d) and Valsartan10mg/(kg·d), while the rats in CsA groupand Control group received physical saline at equal quantity.
2. Index Detection
2.1The detections of weight and urine All rats were put intometabolism cages at the end of each week, then the weight，urine outputduring24hours and urine creatinine were measured respectively.
2.2The detection of kidney pathomorphology Left kidneys of ratswere fixed by4%paraformaldehyde, and then routinely made into paraffinsections respectively for HE, Masson and PAS staining, thus to observe thekidneys’ pathology changes. The damage degree of kidney tubule andinterstitium were analyzed from three aspects of inflammatory cells infiltration,interstitial fibrosis and tubular atrophy according to Katafuchi standard for evaluation.
2.3The detection of renal function On the twenty-ninth day ofexperiment, all animals were sacrificed and specimens of blood were got todetect the contents of Blood Urea Nitrogen(BUN)and Serum Creatinine(SCr),then the creatinine clearance rate(CCr)were calculated.
3. Statistical Analyses
The data were expressed as mean±standard deviation (SD). SPSS16.0software was adopted: One way ANOVA was apply to comparation amongmany groups. All results are considered significant at P＜0.05and P＜0.01.
PartⅡ: Effect of Chailing Decoction on the Expression of ProliferatingCell Nuclear Antigen (PCNA) in Kidney of Rats with CCN.
1. The methods of animal grouping，model establishment, medication andstatistics were same to PartⅠ.
2. Index Detection
On the twenty-ninth day of experiment, some of the kidney tissues of ratswere harvested to detect the expression of PCNA respectively byImmunohistochemical staining, Western Blot, RT-PCR and Flow cytometry.
2.1Immunohistochemical Staining: The method of SABC was adopted inour experiment. Appearance of tan particles under the light microscopy is thestandard of positive cells expression.
2.2Western Blot: The protein was extracted from100mg kidney tissuespreserved in refrigerator (-70℃) and quantitated, then electrophoresis, transfer,immune reaction and developing were proceed in turn. Finally, we tookpictures with Fuji LAS-4000LIMINESCENT image analyzer.
2.3RT-PCR: According to Trizol RNA extraction kit requirements,100mg specimen was taken from the kidney tissue preserved in liquid nitrogen.The total RNA of kidney cortex was extracted and then synthesized intocDNA by reverse transcriptase for PCR. The10L products wereelectrophoresed on1%agarose gel, and then analyzed with BIO-PROFIF Gelimage analysis system after being taken pictures under UV lamp.
2.4Flow Cytometry: Single-celled suspension of renal interstitium and tubule was made from kidney tissues fixed in70%alcohol and then wastreated for2minutes by centrifuge. After adding propidium iodide,0.1mlsuspension liquid (cell density:1x106/ml) mentioned above was dyed for30min in4℃refrigerator on the premise of preventing light, then was filteredwith500mesh copper nets. Fluorescence index (FI) of PCNA、cell apoptosisrate and cell cycle were detected by FACScan-420flow cytometry (BecktonDickinson Company, American). Then proliferation index (PI) was calculatedaccording to the following formula:
PI=(S+G2M)/(G0/G1+S+G2M)×100%
Part Ⅲ: Effect of Chailing Decoction on the Expression of ConnectiveTissue Growth Factor (CTGF) in Kidney of Rats with CCN.
1. The methods of animal grouping，model establishment, medicati-on and statistics were same to PartⅠ.
2. Index Detection
On the twenty-ninth day of experiment, some of the kidney tissues of ratswere harvested to detect the expression of CTGF respectively byimmunohistochemical staining, Western Blot and RT-PCR. The steps same topartⅡ.
Part Ⅳ: Effect of Chailing Decoction on the Expression of Fibronectin(FN) in Kidney of Rats with CCN.
1. The methods of animal grouping，model establishment, medication andstatistics were same to PartⅠ.
2. Index Detection
On the twenty-ninth day of experiment，some of the kidney tissues of ratswere harvested to detect the expression of FN respectively byimmunohistochemical staining, Western Blot and RT-PCR. The steps same topartⅡ.
RESULTS:
PartⅠ: Effects of Chailing Decoction on Renal Function and Pathomorp-hology in Rats with CCN.
1. The Result of Weight and Urine
In the first two weeks of the experiment, all rats' weights were increased,but there were no significant differences among groups at the same period(P＞0.05).With the extension of time，the growth of rats' weight in CsA group wasslowed down. On the4th weekend, the rats' weight in CsA group was lowerinstead than that on the previous week, and there was significant differencewhen comparing with the two treatment groups(P＜0.01).. In the three weeksof the experiment, there was no obvious difference in urine output during24hours among four groups(P＞0.05). However, the urine output of CsA groupdecreased evidently compared with that of the other three groups on the4thweekend(P＜0.05).
2. The Result of Pathomorphology
HE staining showed no abnormalities in control group. In CsA group, alarge of inflammatory cells infiltrated in renal interstitial space, at the sametime, the necrosis and detachment of the renal tubules epithelial cells wereobserved. The lesion of kidneys in CsAC and CsAV group was better than thatof CsA group. Masson staining showed that a little collagen deposited in renalinterstitium in control group, but was increased and distributed just likestrip-shaped in CsA group. The collagen compositions in CsAC and CsAVgroup were lower than that in CsA group. Semi-quantitative analysis showedthat the integral value of parameters in renal tubulointerstitial damage in CsAgroup was obviously higher than that in control group(P＜0.01). Comparedwith CsA group, the integral values in two treatment groups significantlydecreased(P＜0.05，P＜0.01). Results of PAS: In CsA group, the glomerularbasement membrane was thickened, the matrix was increased, and themesangial cell proliferated obviously. Compared with CsA group, the renalpathological damage of rats in CsAC and CsAV group was reducedsignificantly.
3. The Result of Renal Function
Compared with control group, Scr and BUN of CsA group weresignificantly increased(P＜0.01), while Ccr was reduced in the meanwhile(P＜0.01).The levels of Scr and BUN in CsAC and CsAV group were obviously lower(P＜0.01), while Ccr were higher than that of CsA group(P＜0.05).There was no significant difference between the two treatment groups(P＞0.05).
PartⅡ: Effect of Chailing Decoction on the Expression of PCNA inKidney of Rats with CCN.
1The Results of Immunohistochemistry
In control group, there were a few PCNA positive cells. The number ofpositive cells was increased in CsA group than that in control group(P＜0.01).Compared with CsA group, the positive cells in CsAC and CsAVgroupssignificantly decreased(P＜0.01), but there was no statistical differencebetween the two treatment groups(P＞0.05).
2The Results of Western Blot
In control group, a small amount of protein expression of PCNA inkidney tissue was observed，while the expression of PCNA in CsA group wasenhanced remarkably(P＜0.01). After treatment with Chailing Decoction andValsartan, the high expression of PCNA decreased(P＜0.05). There was noobvious difference between the two treatment groups(P＞0.05).
3The Results of RT-PCR
Compared with control group, the expression of PCNA mRNA wasenhanced obviously in CsA group(P＜0.01).The high expression of PCNAmRNA was down-regulated respectively by Chailing Decoction andValsartan(P＜0.05), but there was no significant difference between the twotreatment groups(P＞0.05).
4The Results of Flow Cytometry
4.1The Fluorescence Index (FI)
The FI in CsA group was much higher than that in control group(P＜0.01). Compared with CsA group, the FI in CsAC group and CsAV groupdecreased obviously (P＜0.01).
4.2Cell Cycle
Compared with control group, the cells in S-stage increased(P＜0.01),but the cells in G0/G1stage decreased in CsA group(P＜0.01). The S-stage percentage of CsAC and CsAV were lower(P＜0.01) and the G0/G1stagepercentage were higher than that in CsA group(P＜0.01). The G2/M stagepercentage was relatively close and there was no significant difference amongfour groups(P＞0.05).
4.3Proliferation Index (PI) and Rate of Apoptosis
The PI of renal cell in CsA group was much higher than that in controlgroup(P＜0.01) and the apoptosis rate was also increased in CsA group, butthe difference of apoptosis rate between the two groups mentioned above wasnot significant(P＞0.05). The ratio of apoptosis to proliferation was obviouslylower in CsA group than that in control group(P＜0.01). After ChailingDecoction and Valsartan treatment, the PI of renal cell significantlydecreased(P＜0.01), the apoptosis rate respectively raised (P＜0.01，P＜0.05),the ratio of apoptosis to proliferation was increased too (P＜0.01).PartⅢ: Effect of Chailing Decoction on the Expression of CTGF inKidney of Rats with CCN.
1The Results of Immunohistochemistry
The expression of CTGF protein was extremely weak in control group,but was significantly enhanced in CsA group，especially in renal tubularepithelial cells and interstitial cells. The high expression of CTGF protein wasrespectively down-regulated obviously by Chailing Decoction and Valsartan.
2The Results of Western Blot
There was weak expression of CTGF protein in control group but wasup-regulated remarkably in CsA group. The expressions of CTGF protein inCsAC and CsAV group were obviously lower than that in CsA group.
3The Results of RT-PCR
Compared with control group, the expression of CTGF mRNA in CsAgroup was significantly increased(P＜0.01).The expressions of CTGF mRNAin CsAC and CsAV group were obviously lower than that in CsA group (P＜0.05).
Part Ⅳ: Effect of Chailing Decoction on the Expression of FN inKidney of Rats with CCN
1The Results of Immunohistochemistry
The protein expression of FN in control group was weak，and onlyconcentrated in renal tubular epithelial cells. Compared with control group,the protein expression of FN obviously strengthened in CsA group. Aftertreatment of Chailing Decoction and Valsartan, the expression of FNdecreased.
2The Results of Western-Blot
There was a little of FN protein expression in control group. The proteinexpression of FN in CsA group was higher than that in control group, and thehigh expression of FN was notablely down-regulated by Chailing Decoctionand Valsartan.
3The Results of RT-PCR
Compared with control group, the expressions of FN mRNA in CsAgroup obviously strengthen(P＜0.01), the expression of FN mRNA wasremarkably reduced in CsAC and CsAV group (P＜0.05, P＜0.01).
Conclusions:
1Chailing Decoction has the effects of reducing the kidney pathologicaldamage induced by CsA and improving renal function.
2Chailing Decoction plays a role in inhibiting the cell proliferation bydown-regulating the over expression of PCNA induced by CsA and regulatingthe cell cycle, thus retarding the progress of renal interstitial fibrosis (RIF).
3Chailing Decoction has the effect of inhibiting the over expression ofCTGF induced by CsA in protein and gene level, hence delaying the progressof RIF.
4Chailing Decoction plays a role in reducing the deposition ofextracellular matrix (ECM) by inhibiting the synthesis and expression of FN,and then alleviating the degree of RIF.
方法：
第一部分：柴苓汤对慢性环孢素A肾病大鼠肾功能及肾组织病理形态学的影响
1动物分组、模型复制及药物治疗
40只雌性SD大鼠随机分为四组，即对照组(Control)、模型组(CsA)、柴苓汤组(CsAC)及缬沙坦组(CsAV)，每组10只。采用CsA30mg/(kg·d)经口灌服28天的方法，建立慢性CsA肾病大鼠模型。对照组每日灌胃给与等容量橄榄油。造模的同时，两治疗组分别给予柴苓汤3g/(kg·d)和缬沙坦10mg/(kg·d)治疗，模型组和对照组则每日给予等容量生理盐水。
2指标检测
2.1体重及尿液检测每周末将所有大鼠移入代谢笼中，分别测量每只大鼠体重、24小时尿量及尿肌酐。
2.2肾脏病理形态学检测切取大鼠左侧肾脏组织，4%多聚甲醛固定，常规制作石蜡切片后分别行HE、Masson及PAS染色，观察肾组织病理形态改变，并参照Katafuchi评分标准从炎性细胞浸润、间质纤维化、肾小管萎缩三方面对肾小管间质损伤程度进行分析。
2.3肾功能检测实验第29天，所有大鼠被处死，取血以检测大鼠血尿素氮(BUN)及血清肌酐(SCr),并计算出肌酐清除率(CCr)。
3统计分析
数据资料以均数±标准差（±s）表示，使用SPSS16.0统计软件进行统计，组间比较采用单因素方差分析。显著性差异水平以0.05和0.01为标准。
第二部分：柴苓汤对慢性环孢素A肾病大鼠肾脏增殖细胞核抗原表达的影响
1动物分组、模型复制、药物治疗及统计分析方法同第一部分。
2指标检测实验第29天，取大鼠部分肾脏组织，分别采用免疫组化、Western-Blot、RT-PCR及流式细胞术四种方法检测大鼠肾脏PCNA表达。
2.1免疫组化：采用SABC法，以光镜下肾脏组织出现棕黄色颗粒为阳性表达。
2.2Western Blot:切取100mg-70℃冰箱中保存的肾组织，提取蛋白并定量，依次进行电泳，转膜，免疫反应及显色。最后用富士LAS-4000LIMINESCENT图像分析仪拍照。
2.3RT-PCR：根据Trizol RNA抽提试剂盒要求，切取100mg液氮保存的肾组织，提取肾皮质总RNA，并反转录合成cDNA进行PCR反应。取10L产物在1%琼脂糖凝胶中电泳，紫外灯下拍照，应用BIO-PROFIF凝胶图像分析系统进行分析。
2.4流式细胞术：取70%酒精固定的肾组织，制作肾间质、肾小管单细胞悬液后，离心沉淀2分钟。取0.1ml单细胞悬液(细胞密度为1×106个/mL)，加入碘化丙啶，4℃冰箱避光染色30min，以500目铜网过滤。采用美国Beckton Dickinson公司FACScan-420型流式细胞仪检测PCNA荧光指数(FI)、细胞凋亡率及细胞周期，并根据公式(PI)=(S+G2M)/(G0/G1+S+G2M)×100%计算出增殖指数(PI)。
第三部分：柴苓汤对慢性环孢素A肾病大鼠肾脏结缔组织生长因子表达的影响
1动物分组、模型复制、药物治疗及统计分析方法同第一部分。
2指标检测实验第29天，取大鼠部分肾脏组织，分别采用免疫组化、Western-Blot、RT-PCR检测大鼠肾脏CTGF的表达。操作步骤同第二部分。
第四部分：柴苓汤对慢性环孢素A肾病大鼠肾脏纤维连接蛋白表达的影响
1动物分组、模型复制、药物治疗及统计分析方法同第一部分。
2指标检测实验第29天，取大鼠部分肾脏组织，分别采用免疫组化、Western-Blot、RT-PCR检测大鼠肾脏FN的表达。操作步骤同第二部分。
结果：
第一部分：柴苓汤对慢性环孢素A肾病大鼠肾功能及肾组织病理形态学的影响
1体重及尿量检测结果
实验第一、二周，所有大鼠体重均有所增加，但同时段各组间比较无明显差异（P＞0.05）。随着实验时间的延长，模型组大鼠体重增长减缓，至第四周末，模型组体重较前一周反而下降，且与同时段两治疗组相比有显著差异（P＜0.01）。实验的前3周，四组大鼠24小时尿量比较无明显差异（P＞0.05）。然而至实验第4周末，模型组尿量较其余三组显著减少（P＜0.05）。
2病理形态学检测结果
HE染色结果：对照组未见明显异常；模型组肾间质大量炎性细胞浸润，同时可见肾小管上皮细胞坏死、脱落；柴苓汤组及缬沙坦组肾脏损伤程度明显轻于模型组。Masson染色结果：对照组肾间质可见少量胶原成分。模型组肾间质胶原成分大量沉积，并呈条带状分布；柴苓汤及缬沙坦组肾间质胶原成分较模型组明显减少。半定量分析结果表明，模型组肾小管间质损伤各项参数积分值均明显高于对照组（P＜0.01）。与模型组相比，两治疗组各项参数积分值则明显降低（P＜0.05，P＜0.01）。PAS染色结果：模型组肾小球基底膜增厚，细胞基质沉积增多，系膜细胞增生明显。柴苓汤组及缬沙坦组大鼠肾组织病理损伤较模型组显著减轻。
3肾功能检测结果
与对照组比较，模型组血肌酐、尿素氮水平明显升高（P＜0.01），但同时该组肌酐清除率显著降低（P＜0.01）。柴苓汤组及缬沙坦组血肌酐、尿素氮水平较模型组明显降低（P＜0.01），肌酐清除率则均较模型组明显升高（P＜0.05）。两治疗组间比较无明显统计学差异（P＞0.05）。
第二部分：柴苓汤对慢性环孢素A肾病大鼠肾脏增殖细胞核抗原表达的影响
1免疫组化结果
对照组仅见少量PCNA阳性细胞。模型组阳性细胞较对照组明显增多（P＜0.01）。柴苓汤及缬沙坦组PCNA阳性细胞较模型组显著减少（P＜0.01），但两治疗组相比无明显统计学差异（P＞0.05）。
2Western-Blot结果
对照组肾组织PCNA蛋白仅少量表达，与之相比，模型组表达增强（P＜0.01）。经柴苓汤及缬沙坦治疗，PCNA蛋白表达被下调（P＜0.05），两治疗组相比无明显差异（P＞0.05）。
3RT-PCR结果
与对照组相比，模型组PCNAmRNA表达明显增强（P＜0.01）。柴苓汤及缬沙坦均可下调PCNAmRNA高表达（P＜0.05），两治疗组相比无明显统计学差异（P＞0.05）。
4流式细胞术结果
4.1荧光指数模型组荧光指数明显高于对照组（P＜0.01）；与模型组比较，柴苓汤组及缬沙坦组荧光指数显著降低（P＜0.01）。
4.2细胞周期模型组处于S期的细胞比例较对照组增多（P＜0.01），而处于G0/G1期的细胞比例较对照组减少（P＜0.01）；柴苓汤及缬沙坦组处于S期的细胞比例较模型组明显降低（P＜0.01），处于G0/G1期的细胞比例较模型组增多（P＜0.01）。各组G2/M期细胞比例相对接近，组间比较无统计学差异（P＞0.05）。
4.3增殖指数、凋亡率与对照组比较，模型组肾脏细胞增殖指数明显升高（P＜0.01），凋亡率较对照组也有所增加，但两组凋亡率的差异没有统计学意义（P＞0.05）。模型组凋亡率/增殖指数的比值显著低于对照组（P＜0.01)；与模型组比较，柴苓汤组及缬沙坦组增殖指数均明显降低（P＜0.01），凋亡率则分别升高（P＜0.01，P＜0.05)，凋亡率/增殖指数的比值也提高（P＜0.01）。
第三部分：柴苓汤对慢性环孢素A肾病大鼠肾脏结缔组织生长因子表达的影响
1免疫组化结果
对照组大鼠肾脏组织CTGF蛋白表达为弱阳性。模型组CTGF蛋白表达明显增强，主要表达在肾小管上皮细胞及间质细胞。经柴苓汤及缬沙坦治疗后，CTGF蛋白表达显著减弱。
2Western-Blot结果
对照组大鼠肾组织CTGF蛋白表达量很少，与之相比，模型组则显著增强，柴苓汤组及缬沙坦组CTGF蛋白表达较模型组明显减少。
3RT-PCR结果
与对照组相比，模型组CTGFmRNA表达明显增强（P＜0.01），柴苓汤组及缬沙坦组CTGFmRNA表达较模型组明显降低（P＜0.05）。
第四部分：柴苓汤对慢性环孢素A肾病大鼠肾脏纤维连接蛋白表达的影响
1免疫组化结果
对照组FN蛋白少量表达，主要见于肾小管上皮细胞；模型组FN蛋白表达较对照组明显增强；经柴苓汤及缬沙坦治疗后，FN蛋白表达明显减少。
2Western-Blot结果
对照组肾组织FN蛋白仅少量表达，与之相比，模型组表达增强，柴苓汤及缬沙坦均可显著下调FN的高表达。
3RT-PCR结果
与对照组相比，模型组FNmRNA表达明显增强（P＜0.01）。柴苓汤组及缬沙坦组FNmRNA表达均显著减少（P＜0.01）。
结论：
1柴苓汤可减轻CsA诱导的肾脏病理损害，改善肾功能。
2柴苓汤可通过下调慢性CsA肾病大鼠肾组织PCNA的高表达及调节细胞周期，抑制细胞增殖活动，延缓肾间质纤维化的进程。
3柴苓汤可抑制慢性CsA肾病大鼠肾组织中CTGF蛋白及基因的表达，从而延缓肾间质纤维化进程。
4柴苓汤可抑制慢性CsA肾病大鼠肾组织中FN的合成及表达，减少细胞外基质沉积，减轻肾间质纤维化程度。
AIM: To explore the role of cell proliferation in chronic renal injuryinduced by cyclosporine A(CsA) and the effect of Chailing Decoction on cellproliferation, then to provide the experimental basis for prevention andtreatment of cyclosporine nephrotoxicity.
METHODS:
PartⅠ: Effects of Chailing Decoction on Renal Function and Pathomo-rphology in Rats with Chronic Cyclosporine A Nephropathy (CCN).
1. Animal Grouping, Model Establishment and Medication
Forty female rats were randomized equally into4groups: Control group(Control), model group(CsA),Chailing Decoction group(CsAC)and Valsartangroup(CsAV), and each group had10rats. The rat models with CCN wereinduced by gavage with cyclosporine A30mg/(kg·d) for28days. The rats incontrol group received orally the same dosage of olive oil. At the same time,the rats in two treatment groups were treated respectively with ChailingDecoction3g/(kg·d) and Valsartan10mg/(kg·d), while the rats in CsA groupand Control group received physical saline at equal quantity.
2. Index Detection
2.1The detections of weight and urine All rats were put intometabolism cages at the end of each week, then the weight，urine outputduring24hours and urine creatinine were measured respectively.
2.2The detection of kidney pathomorphology Left kidneys of ratswere fixed by4%paraformaldehyde, and then routinely made into paraffinsections respectively for HE, Masson and PAS staining, thus to observe thekidneys’ pathology changes. The damage degree of kidney tubule andinterstitium were analyzed from three aspects of inflammatory cells infiltration,interstitial fibrosis and tubular atrophy according to Katafuchi standard for evaluation.
2.3The detection of renal function On the twenty-ninth day ofexperiment, all animals were sacrificed and specimens of blood were got todetect the contents of Blood Urea Nitrogen(BUN)and Serum Creatinine(SCr),then the creatinine clearance rate(CCr)were calculated.
3. Statistical Analyses
The data were expressed as mean±standard deviation (SD). SPSS16.0software was adopted: One way ANOVA was apply to comparation amongmany groups. All results are considered significant at P＜0.05and P＜0.01.
PartⅡ: Effect of Chailing Decoction on the Expression of ProliferatingCell Nuclear Antigen (PCNA) in Kidney of Rats with CCN.
1. The methods of animal grouping，model establishment, medication andstatistics were same to PartⅠ.
2. Index Detection
On the twenty-ninth day of experiment, some of the kidney tissues of ratswere harvested to detect the expression of PCNA respectively byImmunohistochemical staining, Western Blot, RT-PCR and Flow cytometry.
2.1Immunohistochemical Staining: The method of SABC was adopted inour experiment. Appearance of tan particles under the light microscopy is thestandard of positive cells expression.
2.2Western Blot: The protein was extracted from100mg kidney tissuespreserved in refrigerator (-70℃) and quantitated, then electrophoresis, transfer,immune reaction and developing were proceed in turn. Finally, we tookpictures with Fuji LAS-4000LIMINESCENT image analyzer.
2.3RT-PCR: According to Trizol RNA extraction kit requirements,100mg specimen was taken from the kidney tissue preserved in liquid nitrogen.The total RNA of kidney cortex was extracted and then synthesized intocDNA by reverse transcriptase for PCR. The10L products wereelectrophoresed on1%agarose gel, and then analyzed with BIO-PROFIF Gelimage analysis system after being taken pictures under UV lamp.
2.4Flow Cytometry: Single-celled suspension of renal interstitium and tubule was made from kidney tissues fixed in70%alcohol and then wastreated for2minutes by centrifuge. After adding propidium iodide,0.1mlsuspension liquid (cell density:1x106/ml) mentioned above was dyed for30min in4℃refrigerator on the premise of preventing light, then was filteredwith500mesh copper nets. Fluorescence index (FI) of PCNA、cell apoptosisrate and cell cycle were detected by FACScan-420flow cytometry (BecktonDickinson Company, American). Then proliferation index (PI) was calculatedaccording to the following formula:
PI=(S+G2M)/(G0/G1+S+G2M)×100%
Part Ⅲ: Effect of Chailing Decoction on the Expression of ConnectiveTissue Growth Factor (CTGF) in Kidney of Rats with CCN.
1. The methods of animal grouping，model establishment, medicati-on and statistics were same to PartⅠ.
2. Index Detection
On the twenty-ninth day of experiment, some of the kidney tissues of ratswere harvested to detect the expression of CTGF respectively byimmunohistochemical staining, Western Blot and RT-PCR. The steps same topartⅡ.
Part Ⅳ: Effect of Chailing Decoction on the Expression of Fibronectin(FN) in Kidney of Rats with CCN.
1. The methods of animal grouping，model establishment, medication andstatistics were same to PartⅠ.
2. Index Detection
On the twenty-ninth day of experiment，some of the kidney tissues of ratswere harvested to detect the expression of FN respectively byimmunohistochemical staining, Western Blot and RT-PCR. The steps same topartⅡ.
RESULTS:
PartⅠ: Effects of Chailing Decoction on Renal Function and Pathomorp-hology in Rats with CCN.
1. The Result of Weight and Urine
In the first two weeks of the experiment, all rats' weights were increased,but there were no significant differences among groups at the same period(P＞0.05).With the extension of time，the growth of rats' weight in CsA group wasslowed down. On the4th weekend, the rats' weight in CsA group was lowerinstead than that on the previous week, and there was significant differencewhen comparing with the two treatment groups(P＜0.01).. In the three weeksof the experiment, there was no obvious difference in urine output during24hours among four groups(P＞0.05). However, the urine output of CsA groupdecreased evidently compared with that of the other three groups on the4thweekend(P＜0.05).
2. The Result of Pathomorphology
HE staining showed no abnormalities in control group. In CsA group, alarge of inflammatory cells infiltrated in renal interstitial space, at the sametime, the necrosis and detachment of the renal tubules epithelial cells wereobserved. The lesion of kidneys in CsAC and CsAV group was better than thatof CsA group. Masson staining showed that a little collagen deposited in renalinterstitium in control group, but was increased and distributed just likestrip-shaped in CsA group. The collagen compositions in CsAC and CsAVgroup were lower than that in CsA group. Semi-quantitative analysis showedthat the integral value of parameters in renal tubulointerstitial damage in CsAgroup was obviously higher than that in control group(P＜0.01). Comparedwith CsA group, the integral values in two treatment groups significantlydecreased(P＜0.05，P＜0.01). Results of PAS: In CsA group, the glomerularbasement membrane was thickened, the matrix was increased, and themesangial cell proliferated obviously. Compared with CsA group, the renalpathological damage of rats in CsAC and CsAV group was reducedsignificantly.
3. The Result of Renal Function
Compared with control group, Scr and BUN of CsA group weresignificantly increased(P＜0.01), while Ccr was reduced in the meanwhile(P＜0.01).The levels of Scr and BUN in CsAC and CsAV group were obviously lower(P＜0.01), while Ccr were higher than that of CsA group(P＜0.05).There was no significant difference between the two treatment groups(P＞0.05).
PartⅡ: Effect of Chailing Decoction on the Expression of PCNA inKidney of Rats with CCN.
1The Results of Immunohistochemistry
In control group, there were a few PCNA positive cells. The number ofpositive cells was increased in CsA group than that in control group(P＜0.01).Compared with CsA group, the positive cells in CsAC and CsAVgroupssignificantly decreased(P＜0.01), but there was no statistical differencebetween the two treatment groups(P＞0.05).
2The Results of Western Blot
In control group, a small amount of protein expression of PCNA inkidney tissue was observed，while the expression of PCNA in CsA group wasenhanced remarkably(P＜0.01). After treatment with Chailing Decoction andValsartan, the high expression of PCNA decreased(P＜0.05). There was noobvious difference between the two treatment groups(P＞0.05).
3The Results of RT-PCR
Compared with control group, the expression of PCNA mRNA wasenhanced obviously in CsA group(P＜0.01).The high expression of PCNAmRNA was down-regulated respectively by Chailing Decoction andValsartan(P＜0.05), but there was no significant difference between the twotreatment groups(P＞0.05).
4The Results of Flow Cytometry
4.1The Fluorescence Index (FI)
The FI in CsA group was much higher than that in control group(P＜0.01). Compared with CsA group, the FI in CsAC group and CsAV groupdecreased obviously (P＜0.01).
4.2Cell Cycle
Compared with control group, the cells in S-stage increased(P＜0.01),but the cells in G0/G1stage decreased in CsA group(P＜0.01). The S-stage percentage of CsAC and CsAV were lower(P＜0.01) and the G0/G1stagepercentage were higher than that in CsA group(P＜0.01). The G2/M stagepercentage was relatively close and there was no significant difference amongfour groups(P＞0.05).
4.3Proliferation Index (PI) and Rate of Apoptosis
The PI of renal cell in CsA group was much higher than that in controlgroup(P＜0.01) and the apoptosis rate was also increased in CsA group, butthe difference of apoptosis rate between the two groups mentioned above wasnot significant(P＞0.05). The ratio of apoptosis to proliferation was obviouslylower in CsA group than that in control group(P＜0.01). After ChailingDecoction and Valsartan treatment, the PI of renal cell significantlydecreased(P＜0.01), the apoptosis rate respectively raised (P＜0.01，P＜0.05),the ratio of apoptosis to proliferation was increased too (P＜0.01).PartⅢ: Effect of Chailing Decoction on the Expression of CTGF inKidney of Rats with CCN.
1The Results of Immunohistochemistry
The expression of CTGF protein was extremely weak in control group,but was significantly enhanced in CsA group，especially in renal tubularepithelial cells and interstitial cells. The high expression of CTGF protein wasrespectively down-regulated obviously by Chailing Decoction and Valsartan.
2The Results of Western Blot
There was weak expression of CTGF protein in control group but wasup-regulated remarkably in CsA group. The expressions of CTGF protein inCsAC and CsAV group were obviously lower than that in CsA group.
3The Results of RT-PCR
Compared with control group, the expression of CTGF mRNA in CsAgroup was significantly increased(P＜0.01).The expressions of CTGF mRNAin CsAC and CsAV group were obviously lower than that in CsA group (P＜0.05).
Part Ⅳ: Effect of Chailing Decoction on the Expression of FN inKidney of Rats with CCN
1The Results of Immunohistochemistry
The protein expression of FN in control group was weak，and onlyconcentrated in renal tubular epithelial cells. Compared with control group,the protein expression of FN obviously strengthened in CsA group. Aftertreatment of Chailing Decoction and Valsartan, the expression of FNdecreased.
2The Results of Western-Blot
There was a little of FN protein expression in control group. The proteinexpression of FN in CsA group was higher than that in control group, and thehigh expression of FN was notablely down-regulated by Chailing Decoctionand Valsartan.
3The Results of RT-PCR
Compared with control group, the expressions of FN mRNA in CsAgroup obviously strengthen(P＜0.01), the expression of FN mRNA wasremarkably reduced in CsAC and CsAV group (P＜0.05, P＜0.01).
Conclusions:
1Chailing Decoction has the effects of reducing the kidney pathologicaldamage induced by CsA and improving renal function.
2Chailing Decoction plays a role in inhibiting the cell proliferation bydown-regulating the over expression of PCNA induced by CsA and regulatingthe cell cycle, thus retarding the progress of renal interstitial fibrosis (RIF).
3Chailing Decoction has the effect of inhibiting the over expression ofCTGF induced by CsA in protein and gene level, hence delaying the progressof RIF.
4Chailing Decoction plays a role in reducing the deposition ofextracellular matrix (ECM) by inhibiting the synthesis and expression of FN,and then alleviating the degree of RIF.
引文
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