| 1.Objective:To observe the clinical efficacy of Shendigranules on Chronic glomerulonephritis(CGN)with spleen and kidney deficiency,and to further discuss the interventional effect of m Tor C1-S6 /4EBP1 signaling pathway in renal tissue of patients with Mesangial proliferative glomerulonephritis(MSPGN)as glomerulonephritis.2.Methods:choose 99 cases of spleen and kidney deficiency syndrome in patients with CGN(preliminary design of 90 cases with < 15% off and eliminate specimen quantity)as the research object,99 patients were divided into three sets: taking western medicine valsartan,valsartan set and particle set taking ginseng to particle,the joint set of two kinds of medicine with clothing,including valsartan once per day,once a grain,and particle three times a day,every time a pack,are oral.In this experiment,9 patients were excluded because they did not meet the conditions,and a total of 90 patients completed the experiment,including 30 patients in the valsartan set,28 patients in the Shendi granule set,and 32 patients in the combined set.The treatment course of the three sets was 12 weeks.In addition,the selection of 10 patients with mesangial proliferative glomerulonephritis kidney(renal pathological),1 cases of normal renal tissue(hamartoma resection specimens),to observe the renal tissue of m TORC1,S6,4 ebp1,P62,Beclin 1,LC3-Ⅱ expression.3.The result:3.1 Comparison of TCM syndrome scores: there was no significant difference between the three sets before the drug intervention(P>0.05),and the three sets all decreased to a certain extent after the drug intervention.The decrease rate of the combined set and Shendi granule set was much higher than that of the valsartan set(P<0.05),but there was no significant difference between the combined set and Shendi granule set(P>0.05).3.2 Comparison of the total effective rate of TCM syndromes: 90.7% in the combined set,85.7% in the Shendi granule set and 50% in the valsartan set.The efficacy of valsartan set was significantly worse than that of Shendi granule set and combined set(P<0.05),and there was no significant difference between the combined set and Shendi granule set(P>0.05).3.3 Comparison of the total effective rate of clinical disease efficacy: the combined set was 90.6%,the Shendi granule set was 89,3%,and the valsartan set was 60%.The efficacy of valsartan set was significantly worse than that of Shendi granule set and combined set(P<0.05),and there was no significant difference between the combined set and Shendi granule set(P>0.05).3.4 Quantitative comparison of 24 h urine protein before and after drug intervention:make a comparison with before drug intervention: the quantitative level of 24 h urine protein in the 4 weeks,8 weeks and 12 weeks in each set was significantly reduced,with significant differences(P<0.05),and the quantitative level of 24 h urine protein was significantly affected by the time of drug intervention,with a negative correlation between the two.Before and 4 weeks of drug intervention,there was no significant difference between the three intervention sets(P>0.05).At week 8,there was a significant difference among the three sets,and the combination set had the largest decrease(P<0.05).At the 12 th week,the decrease rate of the combined set and Shendi granule set was much higher than that of the valsartan set(P<0.05),but there was no significant difference between the Shendi granule set and the combined set(P>0.05).3.5 Renal function(SCR,EGFR): Combined with the renal function of patients before drug intervention,there was no significant difference between sets and within sets after 12 weeks of drug intervention(P>0.05),and renal function fluctuated within44~133umol/L.3.6 Expression of m TORC1,S6,4EBP1,P62,Beclin 1,LC3-Ⅱ in renal tissues3.6.1 Expression of m TORC1,S6 and 4EBP1 in renal tissues: make a comparison with normal renal tissues,the expression levels of m TORC1,S6 and 4EBP1 in renal tissues of patients with mesangial proliferative glomerulonephritis were significantly increased(P<0.05).3.6.2 P62 in the kidney tissues,Beclin 1,the expression of LC3-Ⅱ:make a comparison with normal kidney tissues,mesangial proliferative glomerulonephritis patients significantly increased the amount of P62 expression in kidney tissues(P <0.05),while Beclin 1,significantly reduce the amount of LC3-Ⅱ expression(P <0.05).3.7 Safety indicators: After 12 weeks of drug intervention,the safety indicators of all patients in each set were within the normal range.4.Conclusion4.1 Shendi granules can significantly reduce the quantitative level of 24 h urinary protein in patients with CGN and alleviate the clinical symptoms of patients.Combined use with valsartan can enhance the efficacy.4.2 Activated m TORC1-S6/4EBP1 signaling pathway in renal tissues of mesangial proliferative glomerulonephritis patients decreased autophagy activity.4.3 No adverse reactions occurred in CGN patients after taking Shendi granules,which proved that Shendi granules had good safety.Purpose 1.The intervention effect of Shendi granules on autophagy in mesangial hyperplastic rats was studied based on m TORC1-S6/4EBP1 signaling pathway.Method 2.Sixty 2-month-old male SPF SD rats with body weight of 200±50ug were selected without special intervention.After feeding for 7 days,they were randomly divided into 5 sets according to sham operation set,model set,valsartan set,Shendi granule set and combined set.A total of 48 rats in the model set,valsartan set,Shendi granule set and combined set were anesthetized and their left kidney was removed.A single injection of Thy-1 antibody(333ug/ rat)was performed 14 days after surgery for model replication.7 days after antibody injection,the model was determined by the urine protein detection of rats.The rats with successful model replication(positive urinary protein)were immediately given drug intervention 1 week later,in which the Shendi granule set was given the corresponding dose of Shendi granule water by gavage;The valsartan set was gavaged with valsartan capsule solution,while the combined set was gavaged with two solutions simultaneously.The sham operation set and the model set were given normal saline intragastric administration.Each time4 ml qd was used for 3 months.Serum Creatinine(SCR)and 24-hoururine protein(24h Upro)were measured according to 24 h urine,abdominal aorta blood and right kidney tissue of rats.Protein immunoblot(Western Blot,WB),Reverse transcription-polymerase Chain Reaction(Reverse Transcription-Polymerase Chain Reaction,rt-pcr)method to detect renal tissues m TORC1,S6,4 ebp1,P62,Beclin 1,LC3-Ⅱ expression level;Immunofluorescence three dye detection LC3-Ⅱ+m TORC1 + S6,LC3-Ⅱ+ m TORC1 + 4 ebp1 expression in the kidney tissues(confocal laser scanning microscopy imaging);Hematoxylin eosin(HE),Masson and Periodicacid-Schiff(PAS)staining were performed to observe the pathological changes of renal tissue in rats of each set.Results 3.3.1 make a comparison with the model set and each with drug interpose sets,the rats in the sham operation set were in good overall condition,with high motor sensitivity,white hair color,large amount,and no abnormal diet.After successful model replication,other sets of rats suffered from depression,poor diet,dark and dull hair color,and generally poor condition.After 12 weeks of intervention,the general condition of each with drug interpose sets improved to a certain extent.make a comparison with the Shendi granule set,the general condition of rats in the valsartan set was significantly worse,while there was no significant difference in the general condition between the combined set and the Shendi granule set.3.2 Urinary protein changes: make a comparison with the sham operation set,the urinary protein indexes in the other sets increased significantly(P<0.05).After three months of drug intervention,make a comparison with the model set,the urinary protein indexes in each intervention set decreased significantly(P<0.05).make a comparison with the valsartan set,the urinary protein indexes in the Shendi granule set decreased significantly(P<0.05),and the decrease in the combination set was the most obvious(P<0.05).3.3 The expression of p-m TORC1/m TORC1,p-S6/S6 and p-4EBP1/4EBP1 was detected by Western blot:3.3.1 P-m TORC1 /m TORC1: make a comparison with the sham operation set,the values of this index in other sets were significantly increased(P<0.05);make a comparison with the model set,the values of p-m TORC1/m TORC1 in each with drug interpose sets decreased to different degrees(P<0.05),but there was no significant difference in the degree of decrease between each with drug interpose sets(P>0.05).3.3.2 P-S6 /S6: make a comparison with the sham operation set,this index was significantly increased in the other sets(P<0.05);make a comparison with model set,P-S6 /S6 in with drug interpose sets decreased in different degrees(P<0.05).There were significant differences in p-m TORC1/m TORC1 among all with drug interpose sets(P<0.05),and the combination set showed the greatest decrease(P<0.05).3.3.3 P-4EBP1/4EBP1: make a comparison with the sham operation set,this index was significantly increased in the other sets(P<0.05);make a comparison with model set,P-4EBP1/4EBP1 values of with drug interpose sets were decreased in different degrees(P<0.05).P-4EBP1/4EBP1 was significantly different among all with drug interpose sets(P<0.05).Among them,the combination set had the largest decrease degree(P<0.05).3.4 using Western blot method to detect P62,Beclin 1,LC3-Ⅱ expression in:3.4.1 P62: make a comparison with the sham operation set,this index in the other sets was significantly increased(P<0.05);make a comparison with the model set,the values of P62 in each with drug interpose sets were decreased in different degrees(P<0.05).P62 values were significantly different among with drug interpose sets(P<0.05).There was no significant difference between Shendi granules set and Valsartan set in the degree of decrease of this index(P>0.05).Among the three sets,the degree of decrease was the largest in the combined set(P<0.05).3.4.2 Beclin 1: make a comparison with the sham operation set,Beclin 1 in the model set showed a significantly decreased trend(P<0.05);make a comparison with Beclin 1 model set,the values of with drug interpose sets were increased to different degrees(P<0.05).Beclin 1 expression level increased significantly among with drug interpose sets(P<0.05).The combination set had the largest increase(P<0.05).Rule 3.4.3 LC3-Ⅱ: LC3-Ⅱ in model set make a comparison with control set,the trend of numerical value decreased significantly(P<0.05);The with drug interpose sets LC3-Ⅱ expression quantity and control,numerical different levels increase(P<0.05);The with drug interpose sets LC3-Ⅱ expression quantity make a comparison with model set,have different degree of numerical increase(P<0.05);LC3-Ⅱ expression quantity contrast among different drug intervention way,growth degree has obvious difference(P<0.05);The combination set had the largest increase(P<0.05).3.5 in the detection of renal tissue by rt-pcr method m TORC1,S6,4ebp1,P62,Beclin1,LC3-Ⅱ expression in:3.5,1 m TORC1 m RNA: make a comparison with sham operation set,this index in other sets was significantly increased(P<0.05);make a comparison with model set,m TORC1 m RNA values in with drug interpose sets were decreased in different degrees(P<0.05).There was no significant difference in the degree of decrease between each with drug interpose sets(P<0.05).3.5.2 S6 m RNA: make a comparison with sham operation set,this index was significantly increased in other sets(P<0.05);make a comparison with the model set,the S6 m RNA value of each with drug interpose sets decreased in different degrees(P<0.05);S6m RNA was significantly different among all with drug interpose sets(P<0.05),and the combination set had the greatest decrease degree(P<0.05).3.5.3 4EBP1 m RNA: make a comparison with the sham operation set,this index was significantly increased in the other sets(P<0.05);make a comparison with the model set,the 4EBP1 m RNA of each intervention set decreased in different degrees(P<0.05).There were significant differences in 4EBP1 m RNA among all with drug interpose sets(P<0.05),and the combination set showed the greatest decreasing degree(P<0.05).3.5.4 P62 m RNA: make a comparison with sham operation set,this index was significantly increased in other sets(P<0.05);make a comparison with model set,P62 m RNA in with drug interpose sets decreased in different degrees(P<0.05).make a comparison with each with drug interpose sets,there was no significant difference in the decrease degree of this index between the Shendi granule set and the valsartan set(P>0.05),and the combination set had the greatest decrease degree among the three sets(P<0.05).3.5.5 Beclin 1m RNA: Beclin 1m RNA in the model set was significantly decreased make a comparison with the sham set(P<0.05);Beclin 1m RNA of each with drug interpose sets was increased in different degrees make a comparison with that of the model set(P<0.05).There was no significant difference in the increase degree of this index between Shendi granule set and Valsartan set(P>0.05),and the combination set had the largest increase degree among the three sets(P<0.05).3.6 renal tissue LC3-Ⅱ+ m TORC1+ S6,LC3-Ⅱ+ m TORC1+ 4ebp1 immune fluorescent dye expression in three3.6.1 LC3-Ⅱ + m TORC1+ S6: LC3-Ⅱ,m TORC1,S6 were in control set were reflected,LC3-Ⅱ appears as green fluorescent,S6 appears as red fluorescence,m TORC1 appears as an orange fluorescence;The colors of m TORC1 and S6 in the model set were brighter than those in the other sets.model set LC3-Ⅱ color than the rest of the several sets of gloom.The expression of m TORC1 and S6 in each with drug interpose sets decreased to different degrees make a comparison with the model set,and the expression of m TORC1 and S6 in the combination set was the weakest.The with drug interpose sets LC3-Ⅱ expression showed different degree enhancement make a comparison with model set,of which express the strongest joint set.3.6.2 LC3-Ⅱ + m TORC1 + 4 ebp1: LC3-Ⅱ,m TORC1,4ebp1 were in control set were reflected,LC3-Ⅱ appears as green fluorescent,4ebp1 appears as red fluorescence,m TORC1 appears as an orange fluorescence;make a comparison with sham operation set and each with drug interpose sets,m TORC1 and 4EBP1 in model set were brighter.make a comparison with the control set,the with drug interpose sets,model set LC3-Ⅱ color is bleak.make a comparison with the model set,the color of m TORC1 and 4EBP1 in each intervention set decreased to different degrees,and the expression of m TORC1 and 4EBP1 in the combination set was the weakest.make a comparison with model set,the intervention set showed different degree of LC3-Ⅱ color enhancement,of which express the strongest joint set.3.7 Kidney pathological results of experimental ratsIn the sham operation set,relatively complete glomerular structure was observed,and no obvious mesangial cell proliferation and mesangial matrix deposition were observed in the mesangial area.The capillary lumen of the glomerulus was round and regular,with no obvious stenosis and no adhesion in the balloon.make a comparison with the sham operation set,a large number of mesangial cells were proliferated in the model set,and the mesangial matrix was also deposited in the mesangial area.The glomerular capillary lumen was malformed and narrow,and the proliferated mesangial cells even exceeded half of the lumen diameter of the capillaries.The proliferation of glomerular mesangial cells and the deposition of mesangial matrix were decreased to different degrees in each intervention set,and the combination set was the weakest among the three sets.4.Conclusion4.1 Shendi granules can reduce the level of urinary protein in rats with mesangial proliferative glomerulonephritis,and combined use with valsartan can increase the effect.4.2 The autophagy activity of renal tissue in Thy-1 MSPGN rat model was significantly decreased,which was associated with the activation of m TORC1-S6/4EBP1 Signaling pathways.4.3 Shendi granules can reduce the pathological damage of renal tissue in rats with mesangial proliferative glomerulonephritis,which may be related to the inhibition of m TORC1-S6/4EBP1 signaling pathway activation in vivo,and the enhancement of autophagy response. |
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