Effect Of Shenluotong On Oxidative Stress And Apoptosis In Obstructive Nephropathy Rats

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 ratsMethods: 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 PathomorphologyHE 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 ratsCompared 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 nephropathyratsMethods: 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 microscopeApoptosis 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 TUNELThere 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 ratsMethods: 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 immunohistochemistryThe 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 blotCompared 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 ratsMethods: 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 PCRCompared 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 tissueThe 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 blotCompared 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.