| Objective:To observe the protective effect of sinomenine on IgA n ephropathy rats and its effects on podocyte injury, and pro vide experimental evidence of molecular biology for the cli nical treatment of IgA nephropathy.Method:1Male SD SPF rats60,1weeks after adaptive feeding,10were randomly selected as the normal control group, the other50rats were randomly divided into model group10,10in the control group, sinomenine, low, high dose group wit h10rats in each group, each rat to cattle serum albumin (BSA)(400mg/kg) every other day with40mg/ml by gavage for8weeks, subcutaneous injection of carbon tetrachloride0. lml+castor oil0.5ml for8weeks, sixth weeks to0.025%0.05mg lipopolysaccharide (0.2ml) via tail vein injection,3weeks in a row. In sixth,8weeks and24hours urine measur ement of urinary protein, urine red blood cell; interventio n began with ninth weeks, treatment for5weeks.2Sinomenine low dose group (30mg/kg), dose group of si nomenine in (60mg/kg), high dosage group (120mg/kg) rats we re orally given3mg/ml,6mg/ml,12mg/ml of sinomenine suspe nsion10ml/kg; positive control group (20mg/kg) rats were g iven valsartan10ml/kg; model group and the normal group by gavage given the same volume physiological saline. Interve ntion began in ninth weeks,1times a day, for5weeks.3Recorded the general condition of rats. Sixth,8,13weeks of24hours urine test24h urinary protein (24hupq) a nd urinary red blood cell count; Eighth,13over the weeken d,24hours after the last administration of blood creatini ne, urea nitrogen; the13weekend, renal tissue were observ ed pathological changes under light microscope; electron mi croscope detection of glomerular ultrastructural changes; d etection of Nephrin and α-actinin-4protein immunohistoch emistry; Determination of glomerular IgA deposition strengt h direct immunofluorescence; indirect immunofluorescence de tection of Nephrin and α-actinin-4protein; RT-PCR detect ion of Nephrin and α-actinin-4mRNA.4All data were analyzed by SPSS16.0statistics softwar e, the experimental data with the mean±standard deviatio n, single factor variance analysis with the comparison of m ultiple samples; comparison between groups were analyzed by LSD method.Result:1General situation:the rats in each treatment group hair color, diet intake and activity improved compared with model group.2Urine detection:in the model group, the Valsartan g roup, sinomenine group at sixth,8weeks24hupq, urine red blood cells were significantly higher than those in the nor mal group, the difference was statistically significant (P< 0.01). The thirteenth weekend, compared with the normal gro up, model group, urine red blood cell24hupq was significan tly increased (P<0.01); compared with the model group, the treatment group24hupq, urine red blood cells decreased sig nificantly (P<0.01); compared with valsartan group, no sign ificant difference of sinomenine in each dose group of24hu pq (P>0.05), has the obvious difference of sinomenine each dose group of urinary red blood cell count (P<0.01), and hi gh dose group was better than that of sinomenine in low dos e group, sinomenine (P<0.01).3Detection of blood:treatment of rats in each group before and after treatment, blood creatinine and urea nitro gen had no significant difference (P>0.05).4Nephrin and α-actinin-4detection:Nephrin and a-actinin-4immunohistochemistry and immunofluorescence aver age optical density (IOD) showed that, compared with the no rmal group, model group, Nephrin and a average optical den sity of-actinin-4expression decreased obviously (P<0.01); compared with the model group, the treatment group Nephrin and the average optical density of-actinin-4expression s ignificantly elevated (P<0.01); compared with valsartan gro up, sinomenine high dose group average optical density of N ephrin expression were significantly different (P<0.01), hi gh dose group and sinomenine average optical density of Nep hrin was significantly higher than that of sinomenine in lo w dose group,(P<0.01), Aoto dose group alpha average optic al density of-actinin-4showed no significant difference (P>0.05).5RT-PCR detection:compared with the normal group, mo del group, Nephrin significantly reduced the expression of mRNA and α-actinin-4mRNA (P<0.01); compared with the mod el group, the treatment group Nephrin mRNA and-actinin-4m RNA expression was significantly increased (P<0.01); compar ed with valsartan group, sinomenine each dose group of Neph rin mRNA and α-actinin-4mRNA expression had no significa nt differences (P>0.05).6Renal pathology:(1)PAS staining light microscope, compared with the normal group, the model group rats mesangial matrix increased, mesangial matrix; in the treatment group than in the model group improved in different degrees.(2)Immunofluorescence:rats in the model group IgA showed a mass deposition in the mesangial area, all treatment groups compared with model group, the fluorescence intensity of IgA are different degrees of weakening.(3)Electron microscope:normal rats basement membrane evenly, foot process clear, uniform matrix; model group showed obvious hyperplasia of mesangial matrix, electron-dense film deposition, visible apoptosis, fusion of foot processes; the treatment group partial foot process fusion and a small amount of electron dense deposits.Conclusion:The results of this study show:sinomenine could reduce24hour urinary protein content, reduce red blood cells, en hance the expression of Nephrin and mRNA protein, inhibit t he expression of α-actinin-4mRNA and protein; prompt sin omenine may through the change regulation of Nephrin and a-actinin-4mRNA, increase Nephrin protein expression, inhi biting the expression of α-actinin-4protein in many ways, reduce rat IgA nephropathy urine protein and urine red blo od cell, improve IgA nephropathy renal pathology in order t o play the role of prevention and control. |
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