Studing Of The Pathway Of Signal Conduction Of JAK/STAT And Effect Of Matrine On STAT3 Signaling Molecule In Adriamycin-induced Glomerular Sclerosis Rat

Primary nephrotic syndrome is one of usual diseases of childhood.The disease incidence of it holds secondary of pediatric urinary system diseases in hospital.Its pathologic type is mainly the minimal change,but the most of these cases are likely to recur or relapse,which can aggravate renal lesion and lead to glomerular sclerosis finally.Renal fibrosis is the common pathway and main pathologic foundation of the various kinds of kidney disease that develop into chronic renal failure at last,and also is the irreversible pathologic change of the various glomerulopathy terminal stage, including glomerular sclerosis,nephric tubule atrophia and lesion,abnor increase and excessive sedimentation of ECM and so on.Its pathogenesy is yet not clear completely.Because of difficult treatment and poor prognosis,it is more important for clinic to explore mechanism of glomerular sclerosis and measure of prevention and crue.In the initial stage,glomerular sclerosis shows focus and segment;and in later stage,it displays diffuse globular distribution.Its pathologic process divides into glomerulus hypertrophy stage,mesenterium sclerosis stage and blood vessel obliteration stage. This process is the common pathway of many kidney disease,which leads kidney to chronic renal failure finally.JAKs/STATs signal transduction pathway,which has become a foucs of studing cytokines,is the essential way that cytokines fulfill their biological function.As the target protein of JAKs,STATs belong to transcription factor family located in periplast.In this family,STAT3 is a important member.It is a transcription factor activated by cytokines and polypeptide ligand.Researchs indicate that STAT3 pathway participates not only renal physiological process but also many kinds of Renal glomerular disease process.Matrine,a alkaloid abstracted and extracted from dry root of Sophora flavescens Ait, has versatile pharmacologic action,such as antisepticising,antiinflammating,cruing rheumatism,anti- tumor,anti-anaphylaxis,anti-arhythmia,detumescence and diuresis, and adjustment of immune ang biology.At present,studis reveal that marine can foncation to inhibit synthesis of collagenoblast and collagen,and to prevent and crue hepatic fibrosis of experimental rat.But how to funcation is not clear yet. experimental model of rat glomerulosclerosis induced by adriamycin is more stable.It is charecaterized by plentiful albuminuria and chronical and progressive renal lesion, which is similar to humanbeing' s kidney disease.In this study,we try to observe the change of STAT1,STAT3,TGF-β1 and COL-Ⅳ, and the expression of JAK2,STAT1,STAT3mRNA in cource of glomerular sclerosis of rats kidney,and investigate the role of pathway of signal conduction of JAK/STAT.At the same time,we survey the change of STAT3,TGF-β1 STAT3 proteinum expression and STAT3,PIAS mRNA expression in the nephridial tissue of Adriamycin-induced glomerular sclerosis rat treated by matrine.Through this study, we aim at approach the action and mechanism of matrine in cruing glomerular sclerosis,and seek a new approach to prevent and crue it.PartⅠStudy of the pathway of signal conduction of JAK/STAT in Adriamycin-induced glomerular sclerosis rat AIM To dynamically observe the change of signaling molecule of pathway of signal conduction of JAK/STA in adriamycin-induced glomerulosclerosis in rats,and explore its action and mechanisms.METHODS Thirty male Wistar rats were randomly divided into tow groups:model group and control group.Rats adriamycin nephropathy was induced by complete excision of left kidney and a single tail intravenousinjection of adriamycin(5 mg/kg); control group are similarly operated but only intravenousinjected the same does distilled water.5 rats in each group were sacrificed every 2 weeks.Serum creatinine (Scr)and 24 hour urine protein excretion(TP/24h)were measured at 2,4 and 6 weeks. A semiquantitative score was used to evaluate the degree of glomerular lesions. Immunohistochemistry was to examine the expression of STAT1,STAT3, TGF-β1 and COL-Ⅳ.Finally,the expressions of JAK2,STAT1 and STAT3 mRNA were measured by real-time quantitative RT-PCR.RESULTS At 6 week,not only Scr(68.3±8.3μmol/L),BuN(25.8±3.8μmol/L) and TP/24h(58.9±9.7mg/d)but also GSI(glomerulosclerosis index)(71.7±11.2%) in model group increased progressively,and were significantly higher than those of control group(123.3±20.9μmol/L),(14.8±2.0μmol/L),(9.6±1.1mg/d),(17.6±2.5%),(p＜0.05);Immunohistochemistry staining indicated that COL-Ⅳand TGF-β1 expression gradually increased in model group(TGF-β1:2.196±0.394%;COL-Ⅳ:5.26±1.66) and higher than those of control group(TGF-β1:0.017±0.005%; STAT3:7.64±1.25%)(p＜0.05)at 6 week,which indicates the experimental model is successful.At same time the proteinum expression of STAT3(19.6±2.6%)also generally raises,but expression of STAT1(6.76±1.1%)is not evidently altered in model group compared with that of control group(7.64±1.2%)(p＜0.05),(5.73±1.0%).The gene expression of JAK2,STAT1 and STAT3 at mRNA level in model group(7.28±1.53,4.13±0.34,5.06±0.26 times to control group)was increased, compared with those of the control group and showed developing change(p＜0.05). CONCLUSION Pathway of signal conduction of JAK/STA participates the formative process of glomerular sclerosis.Accompanied with expression increase of JAK2,STAT1,STAT3,TGF-β1 and COL-Ⅳ,this pathway maybe one of very important way to mediate plentiful ECM sedimentation,and generation and development of renal fibrosis.As to STAT1 proteinum expression does not enhance obviously while its expression of mRNA accelerates,we think it is because body remains compensation ability,and the expression of protein and mRNA are not consistant fully.PartⅡEffect of matrine on STAT3 signaling molecule in Adriamycin-induced glomerular sclerosis ratAIM To dynamically observe the effect of matrine on STAT3 and PIAS3 in adriamycin-induced glomerulosclerosis in rats and explore its protective mechanisms.METHODS fourty-five male Wistar rats were randomly divided into three groups: model group,control group and experiment group.Adriamycin nephropathy was induced by complete excision of left kidney and a single tail intravenousinjection of adriamycin(5 mg/kg);control group only are operated but not intravenousinjected adriamycin;experiment group are treated by matrine 100 mg/kg·d after accomplishment of Adriamycin nephropathy.5 rats in each group were sacrificed every 2 weeks.A semiquantitative score was used to evaluate the degree of glomerular lesions.Immunohistochemistry was to examine the expression of STAT3 and TGF-β1.Finally,the expressions of STAT3 and PIAS3 mRNA were measured by real-time quantitative RT-PCR.RESULTS GSI(glomerulosclerosis index)in model group increased progressively(71.7±11.2%),and were higher than that of control group(17.6±2.5%) and experiment group(44.9±8.9%)(p＜0.01)at 6 week;Immunohistochemistry staining indicated that STAT3 and TGF-β1 expression gradually increased in model group(TGF-β1:2.196±0.394%;STAT3:19.58±2.66)(p＜0.01)and higher than control group(TGF-β1:0.017±0.005%;STAT3:7.64±1.25%)(p＜0.01)and experiment group(TGF-β1:10.750±0.089%;STAT3:14.90±1.66%)(p＜0.01)at 6 week.The gene expression of STAT3 at mRNA level in model group(5.06±0.26 times to control group)was increased compared with the control group and experiment group(3.49±0.39 times to control group)(p＜0.01)and showed developing change.On the contrary,PIAS3 mRNA expression in model was decreased.CONCLUSION Matrine has depressive role on the developing of glomerular sclerosis in Adriamycin-induced nephropathy rats.Its mechanism is considered to be relevant to intervention of the pathway of signal conduction of JAK/STAT: up-regulate PIAS3 and down-regulate STAT3.At the same time the expression of TGF-β1 was decreased concomitantly.Conculusions1.Pathway of signal conduction of JAK/STA participates formative process of glomerular sclerosis.Its signaling molecules,such as JAK2,STAT1,STAT3,are the more important links.2.TGF-β1,COL-Ⅳmaybe directly stimulate JAK2,STAT1 and STAT3 to activate, through which to mediate formation of glomerular sclerosis;or by Pathway of signal conduction of JAK/STA,TGF-β1 and COL-Ⅳ,which can cause fibrosis, are produced.3.Matrine can up-regulate expression of PIAS3 and down-regulate expression of STAT3 and has funcation of anti-fibrotic.Its mechanism maybe is played by Pathway of signal conduction of JAK/STA.