| 1.Objective To bear out the clinical efficacy of Shendi Granule on chronic glomerulonephritis?CGN?patients with deficiency of spleen and kidney,and to observe the effect of PI3K-AKT-mTORC1 signal on autophagy in mesangial proliferative glomerulonephritis?Ms PGN?patients.2.Methods 66 cases of CGN deficiency of spleen and kidney?60 cases are expected,the number of cases of rejection and shedding is increased by less than 15%?were selected as the study object,and randomly divided into valsartan group,Shendi granule group and combined drug group,22 cases each.In the course of the experiment,6 cases were removed,60 cases were completed,20 cases in each group.Among them,valsartan group: take valsartan 80mg/day?in the morning?;Shendi granule group: take Shendi granule three times a day,one bag a time,with warm water;joint drug group: take Shendi granule three times a day,one bag a time,with warm water,and at the same time,take valsartan 80mg/day?in the morning?;all three groups are treated for 12 weeks.Before and after treatment,the integral value of TCM syndrome,24-hour urine protein quantitative?24-hour Upro?,renal function?SCR,bun,EGFR?were observed.Meanwhile,the clinical disease effect and TCM syndrome effect of the three groups were compared.In addition,10 cases of mesangial proliferative glomerulonephritis and one case of normal renal tissue?most of the patients with left renal hamartoma?were collected to detect the expression of PI3 K,AKT,mTORC1,VDAC1,Beclin 1,LC3-and p62 proteins in renal tissue,and the ?number of autophagy bodies of mesangial cells and the morphological changes of mitochondria in renal tissue were observed by transmission electron microscope.3.Results 3.1 clinical disease efficacy: the total effective rate of valsartan group was 60%,that of Shendi granule group was 85%,and that of combined drug group was 95%.There was statistically significant difference between Shendi granule group and valsartan group?P<0.05?;there was statistically significant difference between valsartan group and combined drug group?P<0.05?;there was no significant difference between Shendi granule group and combined drug group?P >0.05?.3.2 curative effect of TCM syndrome: the total effective rate of valsartan is 50%,that of Shendi granule group is 90%,and that of combined drug group is 90%.There is statistical significance between Shendi granule group and valsartan group?P<0.05?;there is statistical significance between valsartan group and combined drug group?P< 0.05?;there is no significant difference between Shendi granule group and combined drug group?P>0.05?.3.3 TCM syndrome score: before treatment,there was no notable distinction among the three groups?P>0.05?;after 12 weeks,the syndrome score of each group was decreased in varying degrees?P<0.05?,among which,the score of Shendi granule group was significantly lower than that of valsartan group?P < 0.05?,and there was no significant difference between Shendi granule group and combined drug group?P>0.05?.3.4 24-hour Upro comparison: compared with the pre-treatment,24-hour Upro comparison of patients in the fourth week,the eighth week and the twelfth week showed significant difference?P < 0.05?,and the curative effect was time-dependent.Before treatment and at the same time point in the fourth week,there was no significant difference between the groups?P > 0.05?;at the 8th and 12 th week,the Shendi granule group showed more effective result in comparison with the valsartan group?P < 0.05?,and the effect of combination group was the best?P < 0.05?.3.5 renal function: before and after therapy,there was no large dissimilarity between the groups and within the group?P < 0.05?,and the renal function indexes?SCR,EGFR,BUN?were within the normal range.3.6 expression of PI3 K,AKT and m T?1?ORC1 in renal tissue: compared with normal renal tissue,the expression of PI3 K,AKT and mTORC1 in Ms PGN was significantly higher?P<0.05?;?2?Expression of VDAC,Beclin 1,LC3-and p62 protein in renal tissue: compared ?with normal renal tissue,the expression of VDAC1,Beclin 1 and LC3-in Ms P?GN dropped off prominently?P<0.05?.Besides,the expression of p62 protein increased significantly?P<0.05?.3.7 observation on the number of autophagy of mesangial cells and the morphology of mitochondria in renal tissue: the number of autophagy of mesangial cells in patients with mesangial proliferative glomerulonephritis was significantly lower than that in normal renal tissue.In the patients with mesangial proliferative glomerulonephritis,the mitochondria of mesangial cells were swollen,widened,partially fused and disordered.3.8 safety indicators: after 12 weeks of drug intervention,the safety indicators of patients in each group did not change significantly.4.Results 4.1 Shendi granule can effectively alleviate the clinical manifestations of CGN patients with spleen and kidney deficiency syndrome,reduce urinary protein,and have synergistic effect with valsartan.4.2 the activation of pi3k-akt-mtorc1 pathway inhibited the autophagy activity.4.3 in patients with mesangial proliferative glomerulonephritis,there is mitochondrial dysfunction of mesangial cells,in which autophagy activity decreases.4.4 there was no adverse drug reaction in the process of taking Shendi granules,indicating that the drug was safe.1.Objective Based on the PI3K-AKT-mTORC1 signal pathway,the regulatory effect of Shendi Granule on mitochondrial autophagy in mesangial proliferative glomerulonephritis?Ms PGN?rats was studied,and the morphological changes of mitochondria in mesangial cells were observed by transmission electron microscope.2.Methods Sixty 2-month-old male SPF grade SD rats with a weight of 200±50g were classified into five groups at random: sham operation group,model group,valsartan group,Shendi granule group and combined drug group,each group of which included 12 rats.Apart from the sham operation group,the left kidney of the rats in the other groups was excised after anesthesia.Two weeks after operation,a single tail vein injection of anti-Thy-1 antibody?333ug/rat?was used to replicate the Ms PGN rat model.One week after the injection of the antibody,the urine protein of the rats was detected,and the urine protein of the rats was detected as?+?++by the urine protein test strip,then the anti Thy-1 nephritis model was successful.After 2 weeks of antibody injection;Shendi granule group was given 4.0g/kg·D?4ml?of Shendi granule water solution;Rats were fed with 20mg/kg·D?4ml?of valsartan capsule suspension in the valsartan group;The combined group was given 4.0g/kg·D + valsartan capsule suspension 20mg/kg·D?4 ml?of Shendi granule water solution,;The other groups were given 4 ml of normal saline for 12 weeks,the urine of rats during 24 hours was gotten in the metabolic cage,and the blood of abdominal aorta and right kidney were taken aseptically.The serum creatinine?SCR?and 24-hour urine protein?24-hour Upro?were measured.Western blot?WB?and reverse transcription polymerase chain reaction?RT-PCR?were used,The expression levels of PI3 K,AKT,mTORC1,p62,VDAC,Beclin 1 and LC3-?were detected by RT-PCR,and PI3K+AKT+mTORC1,VDAC+p62+Beclin were detected by immunofluorescence 1.The expression in renal tissue?laser scanning confocal microscopy?;the number of autophagy and the morphological changes of mitochondria of mesangial cells in renal tissue were watched by adopting the method of transmission electron microscopy;In addition,the pathological changes of renal tissue were observed by hematoxylin eosin?he?,Masson and periodic acid Schiff?PAS?staining.3.Results 3.1 the rats in the sham operation group had good activity and mental state,glossy hair,and normal diet;the rats in the other groups had poor mental state,gray hair,irritable,reduced diet,and lighter weight than those in the normal group.After continuous gavage treatment in Shendi granule group,valsartan group and combination group,the mental state and diet of rats gradually improved.Among them,the hair luster and diet of Shendi granule group were better than that of valsartan group,and there was no obvious distinction between the combined group and Shendi granule group.3.2 changes of urinary protein: compared with sham operated rats,the 24-hour Upro level of rats in each group raised obviously?P<0.05?.In comparison with the model group,the 24-hour Upro level in the Shendi granule group,the Valsartan group,and the combined drug group dropped off?P<0.05?,and the Shendi granule group dropped off obviously?P<0.05?,especially in the combined drug group?P<0.05?.3.3 expression of p-PI3K/PI3 K,p-AKT/AKT,p-mTORC1/mTORC1?Western blot method?:?1?p-PI3K/ PI3K: In comparison with the sham operation group,the model group and each drug treatment group had obvious higher expression of p-PI3K/PI3K?P < 0.05?;In comparison with the model group,the expression of p-PI3K/PI3 K in each treatment group was significantly lower?P<0.05?;there was no significant difference between each treatment group?P>0.05?.?2?P-AKT/AKT: In comparison with the sham operation group,the expression of p-AKT/ AKT in the model group was obviously higher than that in the drug treatment group?P<0.05?;In comparison with the model group,the expression of p-AKT/AKT in the treatment group was obviously lower?P<0.05?;there was a difference in the expression of p-AKT/AKT between the treatment groups?P<0.05?,especially in the combination group?P<0.05?.?3?p-mTORC1/mTORC1: In comparison with the sham operation group,the expression of p-mTORC1/mTORC1 in the model group and each drug treatment group was obviously higher?P<0.05?;In comparison with the model group,the expression of p-mTORC1/mTORC1 in each treatment group was obviously lower?P<0.05?;there was no significant difference between each treatment group?P>0.05?.3.4 expression of p62,Beclin 1,LC3-and VDAC1?Western blot method?:??1?P62: In comparison with the sham operation group,the expression of p62 protein in the model group was obviously higher than that in the drug treatment group?P<0.05?;In comparison with the model group,the expression of p62 protein in the treatment group was obviously lower?P<0.05?;the combination group was the lowest?P<0.05?;there was no obvious distinction between the Valsartan group and the Shendi granule group?P>0.05?.?2?Beclin 1: In comparison with the sham operation group,the expression of Beclin 1 in the model group obviously dropped off?P<0.05?,and the expression of Beclin 1 in each drug treatment group raised significantly?P<0.05?;In comparison with the model group,the expression of Beclin 1 in each treatment group raised significantly?P<0.05?;among them,the combination drug group was the most significant?P<0.05?.?3?LC3-?: In comparison with the sham operation group,the expression of LC3-?in the model group dropped off obviously?P<0.05?,and the expression of LC3-?protein in each drug treatment group raised obviously?P<0.05?;In comparison with the model group,Beclin in each treatment group raised obviously?P< 0.05?1 protein expression was obviously up-regulated?P<0.05?,especially in the combination group?P<0.05?;In comparison with valsartan group,Shendi granule group was obviously up-regulated?P<0.05?.?4?VDAC1: In comparison with the sham operation group,the expression of VDAC1 in the model group dropped off?P<0.05?,and the expression of VDAC1 in the drug treatment groups raised obviously?P<0.05?;In comparison with the model group,the expression of VDAC1 in the treatment groups raised significantly?P<0.05?;in the combined group,the expression was most obviously increased?P<0.05?;In comparison with the Valsartan group,the expression of VDAC1 in the Shendi granule group raised significantly?P<0.05?.3.5 expression of PI3 K,AKT,mTORC1,VDAC1,Beclin 1 and p62 m RNA in renal tissue?RT-PCR?:?1?PI3K m RNA: : In comparison with the sham operation group,the expression of PI3 K m RNA in the model group raised obviously?P<0.05?,and there was no obvious distinction between the drug treatment groups?P>0.05?;In comparison with the model group,the expression of PI3 K m RNA in the treatment groups dropped off obviously?P<0.05?;there was no obvious difference between the treatment groups?P>0.05?.?2?AKT m RNA: In comparison with sham operation group,AKT m RNA expression in model group and drug treatment group raised significantly?P<0.05?;In comparison with model group,AKT m RNA expression in treatment group dropped off obviously?P<0.05?;there was no obvious difference between treatment groups?P>0.05?.?3?mTORC1 m RNA: In comparison with the sham operation group,the expression of mTORC1 m RNA in the model group raised obviously?P<0.05?,and there was no difference between the drug treatment groups?P>0.05?;In comparison with the model group,the expression of mTORC1 m RNA in the treatment groups dropped off obviously?P<0.05?;there was no obvious distinction between the treatment groups?P>0.05?.?4?VDAC m RNA: In comparison with the sham operation group,the expression of VDAC m RNA in the model group dropped off obviously?P<0.05?,and the expression of VDAC m RNA in each drug treatment group raise significantly?P<0.05?;In comparison with the model group,the expression of vdac1 mrna in each treatment group raised obviously?P<0.05?;the combination drug group was the most obvious?P<0.05?;there was no significant difference between valsartan group and Shendi granule group?P<0.05?.?5?P62m RNA: In comparison with the sham operation group,the expression of P62 m RNA in the model group and each drug treatment group raised significantly?P<0.05?;In comparison with the model group,the expression of P62 m RNA in each treatment group dropped off obviously?P<0.05?;among them,the combination group dropped off most significantly?P<0.05?;there was no obvious distinction between valsartan group and Shendi granule group?P<0.05?.?6?Beclin 1m RNA: In comparison with the sham operation group,the expression of Beclin 1m RNA in the model group dropped off obviously?P<0.05?,and the expression of Beclin 1m RNA in each drug treatment group raised significantly?P<0.05?;In comparison with the model group,the expression of Beclin 1m RNA in each treatment group raised obviously?P<0.05?There was no obvious difference between valsartan group and Shendi granule group?P<0.05?.3.6 expression of PI3 K + AKT + mTORC1,VDAC1 + p62 + Beclin 1 in renal tissue?1?PI3K+AKT+mTORC1: PI3 K,AKT and mTORC1 were all expressed in the sham operation group,showing green,red and orange fluorescence respectively;the expression of the three was significantly enhanced in the model group,showing bright green,red and orange fluorescence;the expression of PI3 K,AKT and mTORC1 in each drug intervention group was weaker than that in the model group in different degrees,among which the expression of the combined drug group was the weakest.?2?VDAC1+p62+Beclin 1: p62 showed red fluorescence in the sham operation group,which was enhanced in the model group and the drug intervention groups;VDAC1 and Beclin 1 were expressed in the sham operation group,which were bright green and orange fluorescence,respectively;the expression of VDAC1 and Beclin 1 in the model group was significantly weakened,which was orange and red fluorescence,VDAC1 and Beclin in the drug intervention groups in comparison with the model group,the expression of 1 was enhanced in different degrees,especially in the combination group,with bright green and orange fluorescence.3.7 electron microscopic examination of mitochondria of mesangial cells in renal tissue In the sham operation group,the mitochondria were normal in shape and arranged in order;in the model group,the mitochondria were swollen,broad and deformed,and the arrangement of mitochondria cristae was disordered.The arrangement of mitochondria in valsartan group was more orderly than that in model group,and the vacuolation of mitochondria was observed;the morphology and arrangement of mitochondria in Shendi granule group was obviously improved than that in model group;only a few mitochondria in combination group were swollen,and the cristae of mitochondria were observed,and the arrangement of mitochondria was the most orderly in treatment group.3.8 renal pathological observation of experimental rats In the sham operation group,the glomerular structure was normal,mesangial cells did not proliferate,extracellular matrix did not increase significantly,the morphology of glomerular capillary cavity was normal,no stenosis or adhesion was found in the balloon,and no thickening of basement membrane was found.In the model group,the mesangial cells and mesangial matrix proliferated diffusely,and the capillaries were narrow.Compared with the model group,the pathological injury degree of Shendi granule group,valsartan group and combined drug group was improved;mesangial cell proliferation and inflammatory cell infiltration were seen in Shendi granule group and losartan group,but mesangial matrix proliferation was not obvious,and basement membrane thickness was lighter than the model group.Only mild inflammatory cell infiltration and mesangial cell proliferation were observed,4.Conclusion 4.1 Shendi granule can not only obviously diminish the urinary protein level of Ms PGN rats,but has synergistic effect with valsartan as well.4.2 Shendi granule can inhibit the activity of pi3k-akt-mtorc1 in renal tissue of Ms PGN rats,thus up regulating the level of mitochondrial autophagy.4.3 Shendi granule can improve the mitochondrial dysfunction of mesangial cells in Ms PGN rats by up regulating mitochondrial autophagy activity.4.4 Shendi granule can reduce the pathological damage of renal tissue in Ms PGN rats,but one of its mechanisms is to inhibit the activity of pi3k-akt-mtorc1,up regulate the level of mitochondrial autophagy and improve the dysfunction of mitochondria in mesangial cells. |
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