肾络通对梗阻性肾病大鼠氧化应激和细胞凋亡的影响
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摘要
梗阻性肾病是因尿液排出障碍引发的肾脏结构和功能损伤的泌尿外科常见疾病，其典型病理改变为炎性细胞浸润、细胞增殖与凋亡、胶原成分异常沉积及肾间质纤维化。目前临床上对梗阻性肾病的治疗主要为解除梗阻，恢复尿路通畅，但是很多患者在梗阻因素解除后肾功能仍不能恢复，甚至继续恶化，因此，研究梗阻性肾病的发病机制可为临床治疗提供理论依据。最近的研究表明，梗阻性肾病与氧自由基引起的氧化应激和肾小管上皮细胞凋亡有密切联系。梗阻造成肾积水继而肾盂内压力升高，引发肾脏血流动力学改变。肾小管血供减少诱发肾小管上皮细胞缺血缺氧性坏死和细胞凋亡，同时缺氧导致大量氧自由基释放，进一步加重肾小管上皮细胞的氧化损伤；肾血流量的减少还可导致肾小球滤过率下降和肾素-血管紧张素-醛固酮系统(Renin-angiotensin-aldosterone, RAAS)激活，该系统激活后产生大量氧自由基和核因子-κB(Nuclear factor-κＢ, NF-κＢ)，诱导促凋亡信号释放，导致肾小管上皮细胞凋亡。实验研究证实应用抗氧化药物可减轻肾脏氧化损伤、抑制肾小管上皮细胞凋亡，改善肾脏功能，说明氧化应激可能参与了细胞凋亡进程。此外，梗阻介导的缺血性氧化损伤诱发肾小管上皮细胞凋亡可能与多种促凋亡通路密切相关。本次实验针对UUO(Unilateral ureteral obstruction单侧输尿管结扎)模型中氧化应激与细胞凋亡可能相关机制，给予特异性抗氧化剂Tempol治疗，同时给予化瘀通络方肾络通，观察MDA、8-OhdG、8-iso-PGF2α等氧化损伤指标及Caspase-12、caspase-9、Bcl-2、Bax等凋亡相关因子的表达，初步探讨中药对梗阻性肾病大鼠氧化应激和细胞凋亡的保护作用及作用机制。第一部分肾络通对梗阻性肾病大鼠氧化损伤和肾脏组织病理形态学的
影响
方法：实验动物自由进食和饮水，温度条件控制在(22±2)℃。适应性饲养1WK后，48只Wistar大鼠随机分为假手术组（Sham group）、UUO组(UUO group)、Tempol组(Tempol group)、肾络通组(SLT group)，每组12只。各组大鼠10%水合氯醛注射后，于左侧中腹部切开皮肤，游离左侧输尿管，分别在输尿管上1/3处和中1/3处用丝线结扎，切断输尿管，逐层缝合皮肤。假手术组仅将输尿管游离但不结扎离断。术后第10天肾脏明显肿大，输尿管扩张，肾盂明显积水，模型复制成功。术后开始给药，Tempol组以Tempol30mg/(kg·d)入水瓶(避光)自由饮用；肾络通组给予肾络通煎剂26g/(kg·d)入水瓶饮用，假手术组及UUO组给予等容量生理盐水饮用，均每天1次。实验动物入代谢笼收集尿液，备检。连续干预10天。10天后，各组大鼠称重后断头处死，留取血液标本，备检；切取梗阻侧肾脏，除去被膜后称重，部分肾组织置于4％多聚甲醛固定，行HE、Masson染色。
采用SPSS13.0for windows统计软件进行统计分析，计量资料以均数±标准差(x±s)表示。多样本均数间比较采用One-way ANOVA检验，组间比较采用LSD-t检验。所有假设检验方法均以0.05和0.01为显著性水平标准。
结果：
1各组大鼠肾组织病理形态学改变
HE染色显示，假手术组有少量炎性细胞浸润，肾小管上皮细胞排列整齐；UUO组肾间质大量炎性细胞浸润，肾小管明显扩张，上皮细胞变性、浊肿、坏死、脱落；Tempol组及肾络通组亦可见肾小管扩张及上皮细胞变性坏死等，但炎性细胞浸润明显减少。Masson染色显示，假手术组可见少量胶原成分表达，主要位于肾小球/肾小管基底膜及肾间质；UUO组肾脏结构紊乱，胶原成分显著增多，以肾间质为主；Tempol组及肾络通组胶原纤维表达较假手术组增多，但与UUO组相比则明显减少。PAS染色显示，UUO组肾小球基底膜明显增厚，肾小球内糖原沉积增多，肾间质大量炎性细胞浸润，肾小管部分萎缩，管腔闭塞或扩张，肾间质变宽。假手术组无上述改变，Tempol组及肾络通组肾小球糖原沉积减少，可见或多或少炎性细胞浸润，肾小球基底膜轻度增厚，肾小管扩张，但较UUO组减轻。
2各组大鼠血清MDA和血清、尿液8-OhdG及8-iso-PGF2α水平比较
与假手术组比较，UUO组血清MDA和血清、尿液8-OHdG含量均明显升高，差异有统计学意义(P<0.01, P<0.05)；与UUO组比较，Tempol组及肾络通组血清MDA和血清、尿液8-OhdG含量下降，差异有统计学意义(P<0.01, P<0.05)。
与假手术组比较，UUO组尿液8-iso-PGF2α含量明显升高，差异有统计学意义(P<0.01)；与UUO组比较，Tempol组及肾络通组尿液8-iso-PGF2α含量下降，差异有统计学意义(P<0.01)。与假手术组比较，UUO组血清8-iso-PGF2α含量有升高趋势，但差异无统计学意义(P>0.05)；与UUO组比较，Tempol组及肾络通组尿液8-iso-PGF2α含量有下降趋势，但差异无统计学意义(P>0.05)。
第二部分肾络通对梗阻性肾病大鼠细胞凋亡的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时留取肾脏标本，部分肾组织置于4％多聚甲醛固定，用于光镜和TUNEL检测细胞凋亡。统计学方法同第一部分。
结果：
1光镜观察肾脏细胞凋亡
1000倍油镜下可观察到UUO组大鼠肾脏细胞凋亡，细胞核染成紫蓝色，细胞核形态发生改变，表现为核浓缩、核裂解。
2TUNEL法检测肾脏细胞凋亡
假手术组可见少量阳性细胞，UUO组阳性凋亡细胞显著增多(P<0.01)；与UUO组比较，Tempol组及肾络通组阳性细胞数明显减少(P<0.05)，其中Tempol组下降稍明显，但与肾络通组比较无统计学意义。第三部分肾络通对梗阻性肾病大鼠细胞凋亡Caspase通路的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时切取梗阻侧肾脏，部分肾组织4％多聚甲醛固定用于免疫组化，部分肾组织-70℃保存用于提取蛋白。统计学方法同第一部分。
结果：
1免疫组化检测Caspase12、Caspase9的表达
假手术组caspase12呈弱表达；UUO组表达明显增强，其中以髓质扩张的肾小管上皮细胞胞浆表达最为显著；Tempol组及肾络通组亦可见到阳性表达，呈局部中度表达，但较UUO组明显减弱。
假手术组caspase9呈弱表达，主要表达于肾小管上皮细胞胞浆；UUO组表达明显增强，主要表达于肾小管上皮细胞胞浆，部分附着于细胞膜，呈棕黄色或棕褐色颗粒；Tempol组及肾络通组亦可见Caspase9阳性表达，较假手术组为甚，但与UUO组比较则明显减轻。
2Western Blot检测Caspase12、Caspase9蛋白表达
与假手术组比较，UUO组caspase12, caspase9蛋白表达明显上调；与UUO组比较，Tempol组及肾络通组其表达均被下调；与Tempol组相比，肾络通组caspase9未见明显差异，但caspase12有显著性差异。
第四部分肾络通对梗阻性肾病大鼠细胞凋亡因子Bcl-2、BaX表达的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时切取梗阻侧肾脏，部分肾组织4％多聚甲醛固定用于免疫组化，部分肾组织-70℃保存用于提取mRNA及蛋白。统计学方法同第一部分。
结果：
1Real-time PCR检测Bcl-2、Bax及Bax/Bcl-2mRNA表达
与假手术组比较，UUO组中Bax及Bax/Bcl-2mRNA表达上调，Bcl-2mRNA表达下调，差异有统计学意义(P<0.01)。与UUO组比较，Tempol组及肾络通组Bax及Bax/Bcl-2mRNA表达显著下调，而Bcl-2mRNA表达上调，其中以Tempol组作用更为明显，差异有统计学意义(P<0.01)。
2免疫组化检测肾组织Bcl-2、Bax表达
假手术组可见Bcl-2阳性表达，主要表达于肾小管和皮髓交界处，肾间质也可见少量表达，呈棕黄色或棕褐色颗粒；UUO组其表达明显下降，主要表达于近曲小管；与UUO组比较，Tempol组及肾络通组Bcl-2表达明显增强。
假手术组可见Bax阳性表达，主要表达于肾小管上皮细胞胞浆，呈棕黄色或棕褐色颗粒；与假手术组比较，UUO组其表达明显增强，主要表达于肾小管上皮细胞胞浆；与UUO组比较，Tempol组及肾络通组Bax表达明显下降。
3Western Blot检测肾组织Bcl-2、Bax蛋白表达
与假手术组比较，UUO组大鼠Bax表达上调，Bcl-2表达下调，差异有统计学意义(P<0.01)。与UUO组比较，Tempol组及肾络通组大鼠Bax表达显著下调，而Bcl-2表达上调，其中以Tempol组作用更为明显，差异有统计学意义(P<0.01, P<0.05)。
结论：
1采用单侧输尿管结扎复制梗阻性肾病肾间质纤维化动物模型，肾脏病理显示大量炎性细胞浸润、输尿管扩张明显及胶原成分大量沉积。肾络通可改善UUO大鼠肾组织间质纤维化病理损伤程度。
2肾络通可下调MDA、8-OhdG、8-iso-PGF2α等氧化损伤指标的表达，改善UUO大鼠氧化应激状态，起到一定的抗氧化损伤的作用。
3肾络通可以通过抑制肾脏固有细胞过度凋亡，维持细胞凋亡和增殖之间的动态平衡，从而减轻UUO诱导的肾脏损伤，起到肾脏保护作用。
4氧化应激通过激活Caspase家族上游效应介质Caspase-12及Caspase-9实现凋亡信号的启动和传导。肾络通可以通过下调Caspase-12、Caspase-9蛋白表达来抑制肾小管上皮细胞凋亡，从而发挥肾脏保护作用。
5Bcl-2家族在细胞凋亡信号传导过程中发挥重要作用，其中Bcl-2为抑制凋亡因子，Bax为促进凋亡因子，两者共同调控细胞存亡。肾络通可以通过上调Bcl-2mRNA及蛋白表达，下调Bax mRNA蛋白表达，且下调Bax/Bcl-2mRNA表达，减少输尿管梗阻后肾小管上皮细胞凋亡，起到肾脏保护作用。
Obstructive nephropathy which is caused by urination disorder is acommon urology surgical disease and leads to kidney structure and functioninjury.Its typical pathological changes are inflammatory cells infiltration, cellproliferation, apoptosis, collagen deposition abnormally and renal interstitialfibrosis. Recently, mainly clinical treatment for obstructive nephropathy is torelieve obstruction and let urinary tract unobstructed. But many patientscannot restore renal function or even worse when the obstruction factors lifted.Therefore, we can provide theoretical basis for clinic-al treatment if we studythe pathogenesis of obstructive nephropathy. Recent studies have shown thatrenal tubularepithelial cell apoptosis correlates with obstructive nephropathyand oxidative stress caused by oxygen radicals. The renal pelvis pressureincreased by uronephrosis which is caused by obstruction, and then the renalhemodynamic changed. Decreased kidney tubules blood supply lead tohypoxic-ischemic necrosis and apoptosis of renal tubular epithelialcells. At thesame time a large number of oxygen radicals releas when lack of oxygen, andfurther oxidative damage of renal tubular epithelial cells aggravated;decreased kidney blood supply can also lead to decreased glomerulus filtrationrate and activated RAAS. Once the system activated, it produced a largenumber of oxygen radicals and NF-κＢ, released promoting apoptosis signaland induced renal tubular epithelial cell apoptosis. Experimental studyconfirmed that the application of antioxidant drugs can reduce oxidativedamage of kidney, inhibited of renal tubular epithelial cells apoptosis,improved renal function. All of them show that oxidative stress may beinvolved in the process of apoptosis. In addition, the ischemic oxidativedamage mediated by obstruction induced renal tubular epithelial cellapoptosis.It may be closely related to a variety of promoting apoptosis pathway. This research aimed at the mechanism between oxidative stress andapoptosis in UUO model. The treated groups were respectively given specificantioxidant Tempol and disperse blood stasis,ditan and dredgecollateral ricipe shenluotong. Observe oxidative damage index like MDA,8-OhdG,8-iso-PGF2α and the apoptosis related factors like Caspase-12,Caspase-9, Bcl-2, Bax. Discusse the mechanism and protection oftraditional Chinese medicine on obstructive nephropathy rats of oxidatie stressand apoptosis.
PartⅠ E ffect ofShenluotong on oxidative stress and kidney tissuepathological morphology in obstructive nephropathy rats
Methods: the experimental animals eat and drink freely, temperaturecontroled in (22±2)℃. After one week to adapt,48Wistar rats randomlydivided into Sham group, UUO group, Tempol group, SLT group, and eachgroup wad12rats. After10%chloral hydrate injected with each group, makea incision in the left side of the abdominal skin, free the left ureter, ligatedwith a silk thread respectively on ureteral1/3and middle1/3, cut off the ureter,r suture the skin layer by laye. The sham group take the same operationwithout being ligated.. After10days the kidney enlarged significantly, ureterexpansed, hydronephrosis obvious, model copyed successfully. Medicine wasgiven after operation, Tempol group and SLT group were respectively givenTempol30mg/(kg·d) and SLT decoction26g/(kg·d) into the water to drink,control and UUO groups were given the same dosage of saline to drink1timea day. The urine was collected in metabolic cages for text. Continuousintervention for10days. Beheaded after weigh the rats10days later, retainblood specimens for text. Cut down the obstruction kidney, weigh the tissueafter remove integument, part of the kidneys were fixed in4%paraformaldehyde for HE and Masson staining.
The data were expressed as mean±standard deviation SPSS13.0softwarewas adopted: One way ANOVA was applyed to comparation among manygroups. All results are considered significant at P＜0.05and P＜0.01. LSD-ttest was applied to comparison awong groups. All hypothesis test method for 0.05and0.01significant level.
Results:
1The changes of kidney Pathomorphology
HE staining measured, it was a little inflammative cells infiltration inSham group, regularly arranged tubular epitheliar cells in tubule; in UUOgroup, a large number of inflammative cells infiltration, obvious expand intubule, renal tubular epitheliar cells were denatured, edema, necrosis andecclasis; there were also expand and denatured in Tempol and SLT group,but the inflammative cells infiltration decreased obviously. Masson stainingshowed that there was a little collagen deposit of renal interstitium in Shamgroup; in Tempol and SLT groups were significantly increased, but it wasobviously reduced compared with the UUO group. PAS staining measured,there was a significant glycogen deposit of glomerular and renal interstitiumin UUO group; in Tempol and SLT groups were significantly increased.
2The comparison of MDA, serum,8-OhdG in urine and8-iso-PGF2α levelin each Groups of rats
Compared with sham group, the level of serum MDA, serum and urine8-OhdG was significantly higher in UUO group(P<0.01, P<0.05); Comparedwith UUO group, the level of serum MDA, serum and urine8-OhdG wassignificantly lower in Tempol and SLT groups(P<0.01, P<0.05).
Compared with sham group, the content of urine8-iso-PGF2α wassignificantly higher in UUO group(P<0.01)；compared with UUO group, thecontent of urine8-iso-PGF2α was significantly lower in Tempol and SLTgroups (P<0.01). Compared with sham group, the trend of urine8-iso-PGF2αcontent was rising in UUO group, but there was no statistically significantdifference between the two groups(P>0.05); compared with UUO group, thetrend of urine8-iso-PGF2α content was descending in Tempol and SLT groups,but there was no statistically significant difference(P>0.05).
PartⅡ Effect of Shenluotong on apoptpsis in obstructive nephropathyrats
Methods: The methods of animal grouping，model establishment, and medication were same to Part Ⅰ,10days totally.At the end of the experiment,some of the kidney tissues of rats were fixed in4%paraformaldehyde for lightmicroscope and detecting apoptosis by TUNEL. The statistics was same toPart Ⅰ.
Results:
1Observed renal apoptosis by light microscope
Apoptosis can be observed in UUO group by1000times theoilmicroscopically cell nucleus dyed purple blue, the nuclear shape haschanged, its shape was nuclear enrichment and cracking.2Observed renal apoptosis by TUNEL
There were a small amount of positive cells in Sham group; in UUOgroup, positive apoptosis cells increased significantly (P <0.01); comparedwith UUO group, positive cells was significantly reduced in Tempol and SLTgroups (P <0.05), and Tempol group declined obviously, but there was nostatistical significance compared with SLT group.
Part Ⅲ Effect of Shenluotong on apoptpsis of Caspase pathway inobstructive nephropathy rats
Methods: The methods of animal grouping， model establishment,medication and were same to Part Ⅰ,10days totally. At the end of theexperiment, some of the kidney tissues of rats were fixed in4%paraformaldehyde for immunohistochemistry, part of the kidney kept at-70℃for extracting protein. The statistics was same to Part Ⅰ.
Result:
1The expression of Caspase12、Caspase9by immunohistochemistry
The expression of caspase12was weak in Sham group; the expression inUUO group was obviously enhanced, expansion in medulla of renal tubularepithelial cells cytoplasm was the most significant; Tempol and SLT groupsalso could see a local moderate positive expression, but compared with theUUO group has decreased significantly.
The result showed that caspase9was weak in Sham group and mainlyexpressed in renal tubular epithelial cell cytoplasm; in UUO group was obviously enhanced, mainly expressed in renal tubular epithelial cellcytoplasm and part attached to the cell membrane, present a tan or browngranules; Tempol and SLT groups also could see positive expression,compared with the Sham group was more serious, but compared with UUOgroup was significantly relieved.
2The expression of Caspase12、Caspase9by Western blot
Compared with Sham group, the expression of caspase12and caspase9were up-regulate obviously in UUO group; Compared with UUO group,theexpression in Tempol and SLT groups were down-regulate; Compared withTempol group, the expression of caspase9has no difference in SLT group, butthe expression of caspase12has significant difference (P＞0.05).
Part Ⅳ Effect of Shenluotong on apoptpsis of Bcl-2、BaX in obstructivenephropathy rats
Methods: The methods of animal grouping， model establishment,medication and were same to Part Ⅰ,10days totally.At the end of theexperiment, some of the kidney tissues of rats were fixed in4%paraformaldehyde for immunohistochemistry, part of the kidney kept at-70℃for extracting mRNA and protein. The statistics was same to Part Ⅰ.
Result:
1The expression of Bcl-2、Bax and Bax/Bcl-2mRNA by Real-time PCR
Compared with Sham group, the expression of Bax and Bax/Bcl-2mRNA were up-regulate in UUO group, Bcl-2mRNA was down-regulate,there was statistical significance(P<0.01); Compared with UUO group, theexpression in Tempol and SLT groups were down-regulate, but Bcl-2mRNAwas up-regulate, the expression obvious in Tempol group especially,andthere was statistical significance(P<0.01).
2The expression of Bcl-2、Bax by immunohistochemistry in kidney tissue
The expression of Bcl-2was positive in Sham group，mainly in renaltubular and corticomedullary border, a small amount in renal interstitium andit presented a tan or brown granules; the expression in UUO group wasobviously decreased and the mainly area was proximal convoluted tubule; compared with UUO group, the expression of Tempol and SLT groups weredecreased significantly.
The expression of Bax was positive in Sham group，mainly in renaltubular epithelial cells cytoplasm and it presented a tan or brown granules;compared with Sham group, the expression in UUO group was obviouslydecreased and mainly in renal tubular epithelial cells cytoplasm; comparedwith UUO group, the expression of Tempol and SLT groups were decreasedsignificantly.
3The expression of Caspase12、Caspase9by Western blot
Compared with Sham group, the expression of Bax was up-regulate andthe Bcl-2was down-regulate, there was statistical significance(P<0.01);Compared with UUO group,the Bax expression in Tempol and SLT groupswere down-regulate obviously, while the Bcl-2expression was up-regulate,especially in Tempol group,and there was statistical significance(P<0.01,P<0.05).
Conclusions:
1Using UUO copy obstructive nephropathy renal interstitial fibrosisanimal model, renal pathology showed a large number of inflammatory cellsinfiltration, ureter expansed obviously and a large number of collagendeposited. The degree of renal tissue fibrosis pathological damage in UUO ratcan be improved by Shenluotong.
2The expression of Oxidative damage index such as MDA、8-OhdG、8-iso-PGF2α could be down-regulate by Shenluotong, which can also impoveoxidative stress status in UUO rats and effect on oxidative damage.
3Shenluotong can maintain the dynamic balance between cell apoptosisand value-added by inhibit the over expression of apoptosis in kidney inherentcells, so as to relieve the kidney damage which is induced by UUO, and thencan play a role in kidney protection.
4oxidative stress realized apoptosis signal started and conducted byactivated Caspase family upstream medium Caspase-12and Caspase–9.Shenluotong plays a role in kidney protection by down-regulate the expression of Caspase-9and Caspase-12to inhibit the renal tubular epithelial cellapoptosis.
5The Bcl-2family play an important role in the process of apoptosissignal transduction, in which the Bcl-2is inhibit apoptosis factor and Bax ispromote apoptosis factor, both two regulat cell’s survive and dye.Shenluotong can reduce renal tubular epithelial cell apoptosis after ureteralobstructed by up-regulate the expression of Bcl-2mRNA and protein,down-regulate the expression of Bax mRNA and protein, and down-regulatethe expression of Bax/Bcl-2mRNA, so as to have effect on kidneyprotection.
影响
方法：实验动物自由进食和饮水，温度条件控制在(22±2)℃。适应性饲养1WK后，48只Wistar大鼠随机分为假手术组（Sham group）、UUO组(UUO group)、Tempol组(Tempol group)、肾络通组(SLT group)，每组12只。各组大鼠10%水合氯醛注射后，于左侧中腹部切开皮肤，游离左侧输尿管，分别在输尿管上1/3处和中1/3处用丝线结扎，切断输尿管，逐层缝合皮肤。假手术组仅将输尿管游离但不结扎离断。术后第10天肾脏明显肿大，输尿管扩张，肾盂明显积水，模型复制成功。术后开始给药，Tempol组以Tempol30mg/(kg·d)入水瓶(避光)自由饮用；肾络通组给予肾络通煎剂26g/(kg·d)入水瓶饮用，假手术组及UUO组给予等容量生理盐水饮用，均每天1次。实验动物入代谢笼收集尿液，备检。连续干预10天。10天后，各组大鼠称重后断头处死，留取血液标本，备检；切取梗阻侧肾脏，除去被膜后称重，部分肾组织置于4％多聚甲醛固定，行HE、Masson染色。
采用SPSS13.0for windows统计软件进行统计分析，计量资料以均数±标准差(x±s)表示。多样本均数间比较采用One-way ANOVA检验，组间比较采用LSD-t检验。所有假设检验方法均以0.05和0.01为显著性水平标准。
结果：
1各组大鼠肾组织病理形态学改变
HE染色显示，假手术组有少量炎性细胞浸润，肾小管上皮细胞排列整齐；UUO组肾间质大量炎性细胞浸润，肾小管明显扩张，上皮细胞变性、浊肿、坏死、脱落；Tempol组及肾络通组亦可见肾小管扩张及上皮细胞变性坏死等，但炎性细胞浸润明显减少。Masson染色显示，假手术组可见少量胶原成分表达，主要位于肾小球/肾小管基底膜及肾间质；UUO组肾脏结构紊乱，胶原成分显著增多，以肾间质为主；Tempol组及肾络通组胶原纤维表达较假手术组增多，但与UUO组相比则明显减少。PAS染色显示，UUO组肾小球基底膜明显增厚，肾小球内糖原沉积增多，肾间质大量炎性细胞浸润，肾小管部分萎缩，管腔闭塞或扩张，肾间质变宽。假手术组无上述改变，Tempol组及肾络通组肾小球糖原沉积减少，可见或多或少炎性细胞浸润，肾小球基底膜轻度增厚，肾小管扩张，但较UUO组减轻。
2各组大鼠血清MDA和血清、尿液8-OhdG及8-iso-PGF2α水平比较
与假手术组比较，UUO组血清MDA和血清、尿液8-OHdG含量均明显升高，差异有统计学意义(P<0.01, P<0.05)；与UUO组比较，Tempol组及肾络通组血清MDA和血清、尿液8-OhdG含量下降，差异有统计学意义(P<0.01, P<0.05)。
与假手术组比较，UUO组尿液8-iso-PGF2α含量明显升高，差异有统计学意义(P<0.01)；与UUO组比较，Tempol组及肾络通组尿液8-iso-PGF2α含量下降，差异有统计学意义(P<0.01)。与假手术组比较，UUO组血清8-iso-PGF2α含量有升高趋势，但差异无统计学意义(P>0.05)；与UUO组比较，Tempol组及肾络通组尿液8-iso-PGF2α含量有下降趋势，但差异无统计学意义(P>0.05)。
第二部分肾络通对梗阻性肾病大鼠细胞凋亡的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时留取肾脏标本，部分肾组织置于4％多聚甲醛固定，用于光镜和TUNEL检测细胞凋亡。统计学方法同第一部分。
结果：
1光镜观察肾脏细胞凋亡
1000倍油镜下可观察到UUO组大鼠肾脏细胞凋亡，细胞核染成紫蓝色，细胞核形态发生改变，表现为核浓缩、核裂解。
2TUNEL法检测肾脏细胞凋亡
假手术组可见少量阳性细胞，UUO组阳性凋亡细胞显著增多(P<0.01)；与UUO组比较，Tempol组及肾络通组阳性细胞数明显减少(P<0.05)，其中Tempol组下降稍明显，但与肾络通组比较无统计学意义。第三部分肾络通对梗阻性肾病大鼠细胞凋亡Caspase通路的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时切取梗阻侧肾脏，部分肾组织4％多聚甲醛固定用于免疫组化，部分肾组织-70℃保存用于提取蛋白。统计学方法同第一部分。
结果：
1免疫组化检测Caspase12、Caspase9的表达
假手术组caspase12呈弱表达；UUO组表达明显增强，其中以髓质扩张的肾小管上皮细胞胞浆表达最为显著；Tempol组及肾络通组亦可见到阳性表达，呈局部中度表达，但较UUO组明显减弱。
假手术组caspase9呈弱表达，主要表达于肾小管上皮细胞胞浆；UUO组表达明显增强，主要表达于肾小管上皮细胞胞浆，部分附着于细胞膜，呈棕黄色或棕褐色颗粒；Tempol组及肾络通组亦可见Caspase9阳性表达，较假手术组为甚，但与UUO组比较则明显减轻。
2Western Blot检测Caspase12、Caspase9蛋白表达
与假手术组比较，UUO组caspase12, caspase9蛋白表达明显上调；与UUO组比较，Tempol组及肾络通组其表达均被下调；与Tempol组相比，肾络通组caspase9未见明显差异，但caspase12有显著性差异。
第四部分肾络通对梗阻性肾病大鼠细胞凋亡因子Bcl-2、BaX表达的影响
方法：动物分组、模型复制、给药方法同第一部分，共10天。实验结束时切取梗阻侧肾脏，部分肾组织4％多聚甲醛固定用于免疫组化，部分肾组织-70℃保存用于提取mRNA及蛋白。统计学方法同第一部分。
结果：
1Real-time PCR检测Bcl-2、Bax及Bax/Bcl-2mRNA表达
与假手术组比较，UUO组中Bax及Bax/Bcl-2mRNA表达上调，Bcl-2mRNA表达下调，差异有统计学意义(P<0.01)。与UUO组比较，Tempol组及肾络通组Bax及Bax/Bcl-2mRNA表达显著下调，而Bcl-2mRNA表达上调，其中以Tempol组作用更为明显，差异有统计学意义(P<0.01)。
2免疫组化检测肾组织Bcl-2、Bax表达
假手术组可见Bcl-2阳性表达，主要表达于肾小管和皮髓交界处，肾间质也可见少量表达，呈棕黄色或棕褐色颗粒；UUO组其表达明显下降，主要表达于近曲小管；与UUO组比较，Tempol组及肾络通组Bcl-2表达明显增强。
假手术组可见Bax阳性表达，主要表达于肾小管上皮细胞胞浆，呈棕黄色或棕褐色颗粒；与假手术组比较，UUO组其表达明显增强，主要表达于肾小管上皮细胞胞浆；与UUO组比较，Tempol组及肾络通组Bax表达明显下降。
3Western Blot检测肾组织Bcl-2、Bax蛋白表达
与假手术组比较，UUO组大鼠Bax表达上调，Bcl-2表达下调，差异有统计学意义(P<0.01)。与UUO组比较，Tempol组及肾络通组大鼠Bax表达显著下调，而Bcl-2表达上调，其中以Tempol组作用更为明显，差异有统计学意义(P<0.01, P<0.05)。
结论：
1采用单侧输尿管结扎复制梗阻性肾病肾间质纤维化动物模型，肾脏病理显示大量炎性细胞浸润、输尿管扩张明显及胶原成分大量沉积。肾络通可改善UUO大鼠肾组织间质纤维化病理损伤程度。
2肾络通可下调MDA、8-OhdG、8-iso-PGF2α等氧化损伤指标的表达，改善UUO大鼠氧化应激状态，起到一定的抗氧化损伤的作用。
3肾络通可以通过抑制肾脏固有细胞过度凋亡，维持细胞凋亡和增殖之间的动态平衡，从而减轻UUO诱导的肾脏损伤，起到肾脏保护作用。
4氧化应激通过激活Caspase家族上游效应介质Caspase-12及Caspase-9实现凋亡信号的启动和传导。肾络通可以通过下调Caspase-12、Caspase-9蛋白表达来抑制肾小管上皮细胞凋亡，从而发挥肾脏保护作用。
5Bcl-2家族在细胞凋亡信号传导过程中发挥重要作用，其中Bcl-2为抑制凋亡因子，Bax为促进凋亡因子，两者共同调控细胞存亡。肾络通可以通过上调Bcl-2mRNA及蛋白表达，下调Bax mRNA蛋白表达，且下调Bax/Bcl-2mRNA表达，减少输尿管梗阻后肾小管上皮细胞凋亡，起到肾脏保护作用。
Obstructive nephropathy which is caused by urination disorder is acommon urology surgical disease and leads to kidney structure and functioninjury.Its typical pathological changes are inflammatory cells infiltration, cellproliferation, apoptosis, collagen deposition abnormally and renal interstitialfibrosis. Recently, mainly clinical treatment for obstructive nephropathy is torelieve obstruction and let urinary tract unobstructed. But many patientscannot restore renal function or even worse when the obstruction factors lifted.Therefore, we can provide theoretical basis for clinic-al treatment if we studythe pathogenesis of obstructive nephropathy. Recent studies have shown thatrenal tubularepithelial cell apoptosis correlates with obstructive nephropathyand oxidative stress caused by oxygen radicals. The renal pelvis pressureincreased by uronephrosis which is caused by obstruction, and then the renalhemodynamic changed. Decreased kidney tubules blood supply lead tohypoxic-ischemic necrosis and apoptosis of renal tubular epithelialcells. At thesame time a large number of oxygen radicals releas when lack of oxygen, andfurther oxidative damage of renal tubular epithelial cells aggravated;decreased kidney blood supply can also lead to decreased glomerulus filtrationrate and activated RAAS. Once the system activated, it produced a largenumber of oxygen radicals and NF-κＢ, released promoting apoptosis signaland induced renal tubular epithelial cell apoptosis. Experimental studyconfirmed that the application of antioxidant drugs can reduce oxidativedamage of kidney, inhibited of renal tubular epithelial cells apoptosis,improved renal function. All of them show that oxidative stress may beinvolved in the process of apoptosis. In addition, the ischemic oxidativedamage mediated by obstruction induced renal tubular epithelial cellapoptosis.It may be closely related to a variety of promoting apoptosis pathway. This research aimed at the mechanism between oxidative stress andapoptosis in UUO model. The treated groups were respectively given specificantioxidant Tempol and disperse blood stasis,ditan and dredgecollateral ricipe shenluotong. Observe oxidative damage index like MDA,8-OhdG,8-iso-PGF2α and the apoptosis related factors like Caspase-12,Caspase-9, Bcl-2, Bax. Discusse the mechanism and protection oftraditional Chinese medicine on obstructive nephropathy rats of oxidatie stressand apoptosis.
PartⅠ E ffect ofShenluotong on oxidative stress and kidney tissuepathological morphology in obstructive nephropathy rats
Methods: the experimental animals eat and drink freely, temperaturecontroled in (22±2)℃. After one week to adapt,48Wistar rats randomlydivided into Sham group, UUO group, Tempol group, SLT group, and eachgroup wad12rats. After10%chloral hydrate injected with each group, makea incision in the left side of the abdominal skin, free the left ureter, ligatedwith a silk thread respectively on ureteral1/3and middle1/3, cut off the ureter,r suture the skin layer by laye. The sham group take the same operationwithout being ligated.. After10days the kidney enlarged significantly, ureterexpansed, hydronephrosis obvious, model copyed successfully. Medicine wasgiven after operation, Tempol group and SLT group were respectively givenTempol30mg/(kg·d) and SLT decoction26g/(kg·d) into the water to drink,control and UUO groups were given the same dosage of saline to drink1timea day. The urine was collected in metabolic cages for text. Continuousintervention for10days. Beheaded after weigh the rats10days later, retainblood specimens for text. Cut down the obstruction kidney, weigh the tissueafter remove integument, part of the kidneys were fixed in4%paraformaldehyde for HE and Masson staining.
The data were expressed as mean±standard deviation SPSS13.0softwarewas adopted: One way ANOVA was applyed to comparation among manygroups. All results are considered significant at P＜0.05and P＜0.01. LSD-ttest was applied to comparison awong groups. All hypothesis test method for 0.05and0.01significant level.
Results:
1The changes of kidney Pathomorphology
HE staining measured, it was a little inflammative cells infiltration inSham group, regularly arranged tubular epitheliar cells in tubule; in UUOgroup, a large number of inflammative cells infiltration, obvious expand intubule, renal tubular epitheliar cells were denatured, edema, necrosis andecclasis; there were also expand and denatured in Tempol and SLT group,but the inflammative cells infiltration decreased obviously. Masson stainingshowed that there was a little collagen deposit of renal interstitium in Shamgroup; in Tempol and SLT groups were significantly increased, but it wasobviously reduced compared with the UUO group. PAS staining measured,there was a significant glycogen deposit of glomerular and renal interstitiumin UUO group; in Tempol and SLT groups were significantly increased.
2The comparison of MDA, serum,8-OhdG in urine and8-iso-PGF2α levelin each Groups of rats
Compared with sham group, the level of serum MDA, serum and urine8-OhdG was significantly higher in UUO group(P<0.01, P<0.05); Comparedwith UUO group, the level of serum MDA, serum and urine8-OhdG wassignificantly lower in Tempol and SLT groups(P<0.01, P<0.05).
Compared with sham group, the content of urine8-iso-PGF2α wassignificantly higher in UUO group(P<0.01)；compared with UUO group, thecontent of urine8-iso-PGF2α was significantly lower in Tempol and SLTgroups (P<0.01). Compared with sham group, the trend of urine8-iso-PGF2αcontent was rising in UUO group, but there was no statistically significantdifference between the two groups(P>0.05); compared with UUO group, thetrend of urine8-iso-PGF2α content was descending in Tempol and SLT groups,but there was no statistically significant difference(P>0.05).
PartⅡ Effect of Shenluotong on apoptpsis in obstructive nephropathyrats
Methods: The methods of animal grouping，model establishment, and medication were same to Part Ⅰ,10days totally.At the end of the experiment,some of the kidney tissues of rats were fixed in4%paraformaldehyde for lightmicroscope and detecting apoptosis by TUNEL. The statistics was same toPart Ⅰ.
Results:
1Observed renal apoptosis by light microscope
Apoptosis can be observed in UUO group by1000times theoilmicroscopically cell nucleus dyed purple blue, the nuclear shape haschanged, its shape was nuclear enrichment and cracking.2Observed renal apoptosis by TUNEL
There were a small amount of positive cells in Sham group; in UUOgroup, positive apoptosis cells increased significantly (P <0.01); comparedwith UUO group, positive cells was significantly reduced in Tempol and SLTgroups (P <0.05), and Tempol group declined obviously, but there was nostatistical significance compared with SLT group.
Part Ⅲ Effect of Shenluotong on apoptpsis of Caspase pathway inobstructive nephropathy rats
Methods: The methods of animal grouping， model establishment,medication and were same to Part Ⅰ,10days totally. At the end of theexperiment, some of the kidney tissues of rats were fixed in4%paraformaldehyde for immunohistochemistry, part of the kidney kept at-70℃for extracting protein. The statistics was same to Part Ⅰ.
Result:
1The expression of Caspase12、Caspase9by immunohistochemistry
The expression of caspase12was weak in Sham group; the expression inUUO group was obviously enhanced, expansion in medulla of renal tubularepithelial cells cytoplasm was the most significant; Tempol and SLT groupsalso could see a local moderate positive expression, but compared with theUUO group has decreased significantly.
The result showed that caspase9was weak in Sham group and mainlyexpressed in renal tubular epithelial cell cytoplasm; in UUO group was obviously enhanced, mainly expressed in renal tubular epithelial cellcytoplasm and part attached to the cell membrane, present a tan or browngranules; Tempol and SLT groups also could see positive expression,compared with the Sham group was more serious, but compared with UUOgroup was significantly relieved.
2The expression of Caspase12、Caspase9by Western blot
Compared with Sham group, the expression of caspase12and caspase9were up-regulate obviously in UUO group; Compared with UUO group,theexpression in Tempol and SLT groups were down-regulate; Compared withTempol group, the expression of caspase9has no difference in SLT group, butthe expression of caspase12has significant difference (P＞0.05).
Part Ⅳ Effect of Shenluotong on apoptpsis of Bcl-2、BaX in obstructivenephropathy rats
Methods: The methods of animal grouping， model establishment,medication and were same to Part Ⅰ,10days totally.At the end of theexperiment, some of the kidney tissues of rats were fixed in4%paraformaldehyde for immunohistochemistry, part of the kidney kept at-70℃for extracting mRNA and protein. The statistics was same to Part Ⅰ.
Result:
1The expression of Bcl-2、Bax and Bax/Bcl-2mRNA by Real-time PCR
Compared with Sham group, the expression of Bax and Bax/Bcl-2mRNA were up-regulate in UUO group, Bcl-2mRNA was down-regulate,there was statistical significance(P<0.01); Compared with UUO group, theexpression in Tempol and SLT groups were down-regulate, but Bcl-2mRNAwas up-regulate, the expression obvious in Tempol group especially,andthere was statistical significance(P<0.01).
2The expression of Bcl-2、Bax by immunohistochemistry in kidney tissue
The expression of Bcl-2was positive in Sham group，mainly in renaltubular and corticomedullary border, a small amount in renal interstitium andit presented a tan or brown granules; the expression in UUO group wasobviously decreased and the mainly area was proximal convoluted tubule; compared with UUO group, the expression of Tempol and SLT groups weredecreased significantly.
The expression of Bax was positive in Sham group，mainly in renaltubular epithelial cells cytoplasm and it presented a tan or brown granules;compared with Sham group, the expression in UUO group was obviouslydecreased and mainly in renal tubular epithelial cells cytoplasm; comparedwith UUO group, the expression of Tempol and SLT groups were decreasedsignificantly.
3The expression of Caspase12、Caspase9by Western blot
Compared with Sham group, the expression of Bax was up-regulate andthe Bcl-2was down-regulate, there was statistical significance(P<0.01);Compared with UUO group,the Bax expression in Tempol and SLT groupswere down-regulate obviously, while the Bcl-2expression was up-regulate,especially in Tempol group,and there was statistical significance(P<0.01,P<0.05).
Conclusions:
1Using UUO copy obstructive nephropathy renal interstitial fibrosisanimal model, renal pathology showed a large number of inflammatory cellsinfiltration, ureter expansed obviously and a large number of collagendeposited. The degree of renal tissue fibrosis pathological damage in UUO ratcan be improved by Shenluotong.
2The expression of Oxidative damage index such as MDA、8-OhdG、8-iso-PGF2α could be down-regulate by Shenluotong, which can also impoveoxidative stress status in UUO rats and effect on oxidative damage.
3Shenluotong can maintain the dynamic balance between cell apoptosisand value-added by inhibit the over expression of apoptosis in kidney inherentcells, so as to relieve the kidney damage which is induced by UUO, and thencan play a role in kidney protection.
4oxidative stress realized apoptosis signal started and conducted byactivated Caspase family upstream medium Caspase-12and Caspase–9.Shenluotong plays a role in kidney protection by down-regulate the expression of Caspase-9and Caspase-12to inhibit the renal tubular epithelial cellapoptosis.
5The Bcl-2family play an important role in the process of apoptosissignal transduction, in which the Bcl-2is inhibit apoptosis factor and Bax ispromote apoptosis factor, both two regulat cell’s survive and dye.Shenluotong can reduce renal tubular epithelial cell apoptosis after ureteralobstructed by up-regulate the expression of Bcl-2mRNA and protein,down-regulate the expression of Bax mRNA and protein, and down-regulatethe expression of Bax/Bcl-2mRNA, so as to have effect on kidneyprotection.
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