| Part 1:Literature researchAccording to the statistics,the number of people diagnosed with diabetes has reached 425 million by 2017 and is expected to rapidly increase to 629 million in 2045.Among them,patients diagnosed with type 2 diabetesaccount for a very large proportion.With the high morbidity of diabetes,the number of the infected with diabetic nephropathy grows increasingly.With rapid progress and high mortality rate,diabetic nephropathy brings serious burden to individuals,families and society.Therefore,it is of great social significance and economic value to find an effective way to prevent and treatment diabetic nephropathy.Comprehensive effects of multiple factors many cause the complex pathogenesis of diabetic nephropathy(DN).From the research on inflammatory response and the morbidity and progression of diabetic nephropathy,it was found that the up-regulated expressions of various inflammatory factors,such as NF-?B,TGF-?1,MCP-1 and TNF-a,can not only directly lead to the injury of kidney tissues,especially podocytes,but also damage the pancreatic tissues and induce insulin resistance in renal tissues,which promotes the occurrence and progression of diabetic nephropathy.Persistent existence of insulin resistance is the basic lesion of diabetic nephropathy.According to literature study,IRS1/PI3K/Akt signaling pathway mediates insulin signal transduction disorder of kidney tissues,which leads to insulin resistance,and affects renal perfusion,glomerular filtration and tissue inflammation fibrosis through adjusting the metabolism,growth pathways and renal microcirculation.According to the research progress about autophagy,autophagy response can participate in the regulation of the expression of inflammatory factors and affect the development of inflammatory cells.With the changes in the expression levels of autophagy proteins LC3II and P62,the autophagosomes form.Moreover,recent studies have shown that autophagy participates in the regulation of the sensitivity and response of renal tissues to insulin,which affects the transmission of insulin signals in renal tissues,and then regulates the degree of renal insulin resistance.By affecting the expression of IRS1/PI3K/Akt pathway,the occurrence of renal insulin resistance impedes the migration of GLUT4 to the cytomembrane,reduces the transport of serum glucose into the cell,destroys the glycometabolism in the body,and leads to the increase of urinary protein content,and further promotes the progression of diabetic nephropathy.At the same time,the autophagy of podocytes leads to the decrease of podocytes and the destruction of the cytoskeleton of podocytes.As the signaling protein downstream of PI3K/Akt,mTOR plays different biological effects through its mTORC1 and mTORC2 complexes and regulates autophagy by participating in IRS1/PI3K/Akt and PI3K/Akt/mTOR pathways.These two pathways that mTOR participates in affect the secretion function of islet cells in the body,transmission pathway of insulin signal,content of urinary protein,and changes in the structures and functions of renal podocytes,glomeruli and tubules.Therefore,being regulated by IRS1/PI3K/Akt pathway,the autophagy reaction of body participates in the regulation of the pathogenesis of diabetic nephropathy,including inflammation,insulin resistance and podocyte injury,plays a novel guiding role in basic research and clinical application of diabetic nephropathy,and puts forward new research goal for the action mechanism of the prevention and treatment of diabetic nephropathy by traditional Chinese medicine.Nowadays,modern medicine lacks precise and targeted treatment for diabetic nephropathy.Under the guidance of the holism concept and treatment based on syndrome differentiation,traditional Chinese medicine has a unique curative effect in the prevention and treatment of diabetic nephropathy.Jiangtang Sanhuang Tablet developed based on the pathogenesis of“deficiency of qi and yin and stagnated heat obstruction”is effective in the treatment of diabetes and the prevention and treatment of its complications.According to the pharmacological study on the Internet,itdefinitely proved that Jiangtang Sanhuang Tablet can slow the progression of diabetic nephropathy mainly by regulating the inflammatory reaction,glucose and lipid metabolism,insulin effect,oxidative phosphorylation,purine metabolism and other biological functions.Therefore,it is necessary to further study and clarify the action mechanism.Part 2:Experimental researchObjectiveThrough the step-by-step speculation and verification on critical links of DN pathological process,this research observes that Jiangtang Sanhuang Tablets affect the inflammatory damage in pancreatic tissue and kidney tissue to resist insulin,regulate IRS1/PI3K/Akt pathway expression through playing the role of autophagy regulation in the "tissue inflammation","insulin resistance" and "podocyte changes",and discusses the improvement mechanism of Jiangtang Sanhuang Tablet on DN.Methods65 SPF-grade male db/db mice aged 8 weeks and 15 db/m mice aged 8 weeks were fed in the IVC cages.After feeding them for 1 week to adapt to the environment,separate them according to 5 mice/IVC cage,and feed them freely.Test the blood glucose of the tail tip once a week,and then transfer them into the metabolic cage to fast without water,keep their urine for 24 hours and record the urine volume.Put the collected urine into 15 ml centrifuge tube,centrifuge it at 2000 r for 10 min,take clear liquid into 0.5 ml EP tube and store it in the refrigerator at-80?,detect the urinary protein with spectrophotometer in reserve.Random blood glucose of db/db mice aged 10 weeks was>16.7mmol/L,and their urine proteins were higher than that of the normal group.With the increase of water and food intake,it is judged that the DN model has been established,that is,the experimental intervention started.According to the random sampling method,with 13 mice for each group,65 db/db mice were divided into model group(M group),high doses of Jiangtang Sanhuang Tablet group(JH group),low doses of Jiangtang Sanhuang Tablet group(JL group),high doses of Rapamycin group(RH group),low doses of Rapamycin group(RL group).15 male db/m mice aged 8 weeks in the same cage were the normal group.Mice in each group were fed with SPF-grade fodder(cobalt-60 sterilization).Gastric perfusion was conducted on the mice in the normal group and M group with 1ml distilled water once a day.JH group:according to the conversion of drug doses of human and animal and the conversion of drug doses of animals with standard weights and animals with non-standard weights,gastric perfusion was conducted on them with physiological saline solution of Jiangtang Sanhuang Tablet(2.45g/kg)once a day.Low doses of Jiangtang Sanhuang Tablet group:according to the conversion method of drug doses(the method is the same as that used inthe JH group),gastric perfusion was conducted on them with physiological saline solution of Jiangtang Sanhuang Tablet(1.225g/kg)once a day.RH group:according to the conversion method of drug doses(the method is the same as that used inthe JH group),gastric perfusion was conducted on them with physiological saline solution of Rapamune(lmg/kg)once a day.RL group:according to the conversion method of drug doses(the method is the same as that used inthe JH group),gastric perfusion was conducted on them with physiological saline solution of Rapamune(0.3mg/kg)once a day for 8 weeks.Before the experiment,the general conditions of each group were recorded.The blood glucose of the tail tip was measured after fasting without water for 12h,and the urine was collected from the metabolic cage after 3 days,and urine protein for 24 hours was detected.During the intervention period,the mice were weighed every 2 weeks,and their water intake and food intake were recorded;their blood glucose of the tail tip was measured,and urine protein was detected.At the end of the experiment,fast mice without water for12 hour,then,pick their eyeballs and collect blood,collect serum by centrifugation,reserve it in the refrigerator at-80? for inspection.HE staining and immunohistochemistry were used to detect some pancreatic and renal tissues,and the pathomorphology and autophagosome distribution of renal tissues and podocytes were detected by transmission electron microscopy.The rest pancreatic and renal tissues were saved for Real-time PCR and Western blotting.ResultsGlucose and lipid metabolism:Fasting blood-glucose of db/m mice in the normal group showed no obvious change.Fasting blood-glucose of db/db mice in each group was gradually increased as the experiment went on.Fasting blood-glucose of mice in the model group significantly increased at the zeroth,second,fourth,sixth and eighth week,and the difference between them was of statistical significance(P<0.05).Compared with the model group,JH group showed a statistical difference from the fourth week(P<0.05)until the end of the experiment.Compared with the model group,JL group showed a statistical difference from the sixth week(P<0.05)until the end of the experiment,and the difference between the JL group and the JH group was of statistical significance(P<0.05).The blood glucose of mice in the JH group decreased more than that in the JL group at the sixth and eighth week(P<0.05).TG,TCHO and FFA of the mice in the model group were significantly increased,and the difference between the two groups in each indicator were of statistical significance(P<0.05).Compared with the model group,TG,TCHO and FFA levels of the JH group decreased(P<0.05)through the intervention of Jiangtang Sanhuang Tablet and rapamycin.The difference in the decline degree of FFA between the JL group and the JH group was of statistical significance,and the FFA level of JH group decreased significantly(P<0.05).The FFA level of RH group declined compared with that of the model group(P<0.05).The difference between the JH group and the RH group was not of statistical difference.Insulin resistance:the serum FINS level of db/db mice significantly increased compared with the normal group(P<0.05),and HOMA-IR enhanced(P<0.05).After the drug intervention,the FINS level of mice in the JH group(P<0.05)and JL group(P<0.05)decreased compared with that in the model group,and the FINS level of mice in the JH group decreased more(P<0.05).HOMA-IR level of mice in the JH(P<0.05)and JL groups(P<0.05)decreased compared with that in the model group(P<0.05),and insulin resistance level of mice in the JH group(P<0.05)decreased more.The FINS level of mice in the RH(P<0.05)and RL groups(P<0.05)significantly decreased compared with that in the model group,and the FINS level of mice in the RH group decreased more than that in the RL group(P<0.05).The HOMA-IR level of mice in the RH(P<0.05)decreased compared with that in the model group.The FINS level and insulin resistance level of mice in the RH group(P<0.05)decreased more than that in the JH group(P<0.05).HE staining of pancreas and kidney:compared with the normal group,the pancreatic lobule structure of mice in the model group was clear,and the acinar and ductal structures were normally distributed in the lobules.With the decreased number,irregular edge,unclear boundary,fibrous hyperblastosis,obvious islet fibrosis,incomplete structure,pancreas islet started atrophying,and ? cells of pancreas islet retrogressed;the distribution of a and ? cells was staggered and scattered;with thickened basilar membrane,partial glomerulus atrophy,decreased podocyte,the renal tissue of db/db mice in the model group showed mesangial hyperplasia;the pathological changes of Jiangtang Sanhuang Tablet group and rapamycin group were alleviated.Immunohistochemistry of pancreatic inflammatory factors:TGF-?1 in the pancreatic tissue of mice in the model group increased significantly compared with that in the normal group,and TGF-?1's expression intensity decreased after the intervention of Jiangtang Sanhuang Tablet and rapamycin.Among them,TGF-?1's expression intensity of mice in the Jiangtang Sanhuang Tablet group was weaker than that in the model group,and TGF-?1' s weakening extent of mice in the JH group was greater than that in the JL group.TGF-?1' s expression intensity of mice in the rapamycin group was weaker than that in the model group,and TGF-?1' s weakening extent of the mice in the RH group was greater than that in the JL group.NF-?B P65' s expression in pancreatic tissue of mice in the model group increased significantly compared with that in the normal group.Compared with the model group,NF-?B P65' s expression intensity of mice in the Jiangtang Sanhuang Tablet group decreased,and the decrease degree of mice in the JH group was greater than that in the GL group.NF-?B P65's expression of mice in the Rapamycin group decreased compared with that in the model group,and the expression intensity of mice in the RH group was greater than that in the RL group.WB detection of inflammatory factors in pancreas:the expression quantity of inflammatory factors TGF-?1(P<0.05)and NF-K B P65(P<0.05)in pancreas of db/db mice in the model group increased significantly compared with the normal group.After the intervention of Jiangtang Sanhuang Tablet and rapamycin,TGF-?1' s expression quantity in pancreatic tissue of mice in the JH(P<0.05)and JL groups(P<0.05)decreased compared with the model group,and the difference between JH group and JL group was not of statistical significance.After intervention,TGF-?1' s expression quantity of mice in the RH(P<0.05)and RL groups(P<0.05)decreased.Compared with the JH group,TGF-?1's expression quantity of the RH group decreased,and the decline degree of the RH group was greater than that in the JH group(P<0.05).After the intervention,NF-?B P65' s expression quantity in pancreatic tissue of mice in the JH and RH group decreased compared with the model group,and the difference between the two groups was not of statistical significance.Renal function:serum creatinine and urea nitrogen levels of db/db mice in the model group increased compared with that in the normal group(P<0.05).Serum creatinine(P<0.05)and urea nitrogen levels of mice in the JH group showed a downtrend compared with that in the model group(P<0.05).The serum creatinine level in the RH group decreased significantly(P<0.05).Urine protein of mice in the model group increased significantly compared with the normal group.From the second week of the experiment,urine protein of mice in the JH,RH and RL groups have been decreasing with a statistical significance(P<0.05)compared with that in the model group,and the decline degree of mice in the RH group was greater than that in the JH group with a statistical significance after intervention(P<0.05).The decline degree of urine protein of mice in the RH group was greater than that in the RL group(P<0.05).From the fourth week to the end of the experiment,the difference in decline degree of urine protein between the mice in the RH and JH group was of statistical significance(P<0.05),and the decline degree of mice in the RH group was greater than that the JH group(P<0.05).The decrease degree of urinary protein in the RH group was greater than that in the RL group(P<0.05).From the sixth week to the end of the experiment,the difference in the decrease degree of urine protein between the JL group and the JH group(P<0.05)was of statistical significance.Immunohistochemical staining of renal inflammatory factors:the expression of renal inflammatory factors of db/db mice enhanced compared with the normal group.Compared with the model group,TGF-?1' s expression intensity in the Jiangtang Sanhuang Tablet group and rapamycin group decreased,and the expression intensity of mice in the JH group was weaker than that in the JL group;the expression intensity of mice in the RH group was weaker than that in the RL group.NF-?B P65' s expression intensity in the kidney of db/db mice in the model group was significantly higher than that in the normal group,and NF-?B P65' s expression intensity in the kidney of db/db mice in the JH and JL group decreased compared with the model group;NF-?B P65' s expression intensity in the kidney of db/db mice in the RH and RL groups decreased compared with the model group.Immunohistochemical staining of renal IRS1:IRS1' s expression intensity of mice in the normal group was increased significantly compared with that in the model group,and IRS1' s expression intensity of mice in the JH and JL groups significantly increased compared with the model group.IRS1' s increase degree of mice in the JH was greater than that in the JL group.IRS1's expression intensity of mice in the RH group increased significantly,and the expression intensity of mice in the RL group was weaker than that in the RH group.Real-time PCR of renal inflammatory factor:TGF-?1 mRNA' s expression of mice in the model group significantly increased compared with the normal group(P<0.05).Compared with the model group,TGF-?1 mRNA's expression quantity of mice in the JH(P<0.05),JL(P<0.05),RH(P<0.05)and RL(P<0.05)groups decreased.Among them,the decrease degree of JH group was greater than that in the JL group.NF-?B P65 mRNA's expression of db/db mice in the model group increased significantly than that in the normal group(P<0.05).NF-?B P65 mRNA' s expression quantity of mice in the JH(P<0.05),JL(P<0.05),RH(P<0.05)and RL(P<0.05)groups decreased compared with the model group,the among them,the decrease degree of mice in the JH group was greater than that in the JL group.Real-time PCR detection of renal IRS1/PI3K/Akt pathway:the renal IRS1 mRNA expression of db/db mice in the model group decreased significantly compared with the normal group(P<0.05).IRS1 mRNA' s expression quantity of mice in the JH(P<0.05),JL(P<0.05)and RH(P<0.05)groups decreased compared with the model group,and the difference among them was not of statistical significance.PI3K mRNA' s expression of mice in the model group decreased compared with the normal group(P<0.05).PI3K mRNA' s expression level of mice in the JH(P<0.05)and RH(P<0.05)groups increased compared with the model group,but the difference between them was not of statistical difference.PI3K mRNA' s expression of mice in the RL(P<0.05)group increased compared with the model group.Akt mRNA' s expression quantity of mice in the model group decreased compared with the normal group(P<0.05).Akt mRNA' s expression quantity of mice in the JH(P<0.05),RH(P<0.05)and RL groups(P<0.05)increased compared with the model group and the increase degree in the RH group was greater than that in the JH group(P<0.05).TEM detection of renal podocyte:compared with the normal group,the mice in the model group mice had thickened basilar membrane of glomerulus,mesangial hyperplasia,decreased podocytes,widened foot process,visible foot process fusion,and decreased autophagosome.The mice in the RH group had increased podocytes,decreased width of podocytes,decreased foot process fusion,and increased autophagosomes.The mice in the JH group had increased podocytes and autophagosomes.Immunohistochemical detection of autophagy protein in pancreatic tissue:LC3II's expression intensity of mice in the model group decreased compared with the normal group,and LC3II protein's expression intensity of mice increased compared with the RH model after the intervention of Jiangtang Sanhuang Tablet and rapamycin.Among them,LC3II protein' s expression intensity of mice in the JH group was greater than that in the JL group,and LC3II protein expression of mice in the RH group was higher than that in the RL group.P62 protein' s expression intensity in pancreatic tissue of mice in the model group increased compared with the normal group but P62 protein' s expression intensity in pancreatic tissue of mice in the model group increased compared with the normal group,and P62 protein' s expression intensity of mice in the JH group was weaker than that in the JL group;P62 protein' s expression intensity of mice in the RH group was weaker than that in the RL group.WB detection of autophagy protein in pancreatic tissue:the pancreatic autophagy protein LC3II of mice in the model group decreased significantly(P<0.05)compared with that in the normal group(P<0.05),at the same time,the deposition of P62 protein increased in the two groups(P<0.05).LC3II expression of mice in the RH and RL group increased compared with the model group,at the same time,P62 protein expression of mice in both groups decreased(P<0.05).Among them,LC3II expression of mice in the RH group was higher(P<0.05),and the deposition of P62 protein of mice in both groups was less(P<0.05).LC3II expression of mice in the JH(P<0.05)and JL groups(P<0.05)increased compared with the model group,and the difference between the two groups was not of statistical significance.The deposition of P62 protein of mice in the JH group decreased(P<0.05).Immunohistochemical detection of autophagy protein in renal tissue:LC3II protein' s expression intensity in renal tissue of mice in the normal group was the highest,but that in the model group was the lowest.LC3II protein' s expression intensity of mice in the JH and JL groups increased compared with the model group,and LC3II protein's expression of mice in the JH group was higher than that in the JL group.LC3II protein' s expression intensity of mice in the RH group was greater than that in the RL group.P62 protein' s expression intensity in renal tissue of mice in the normal group was the weakest,and that in the model group enhanced compared with that in the normal group.P62 protein' s expression intensity of mice decreased compared with the model group after the intervention of Jiangtang Sanhuang Tablet and rapamycin.Among them,P62 protein' s expression intensity of mice in the JH group was weaker than that in the JL group,and P62 protein' s expression intensity of mice in the RH group was weaker than that in the RL group.Renal autophagy protein expression of Real-time PCR detection:renal LC3II of mice in the model group decreased significantly(P<0.05),and deposition of P62 protein increased(P<0.05)compared with the normal group(P<0.05).LC3II expression of mice in the JH group increased(P<0.05)and deposition of P62 protein decreased(P<0.05)compared with the model group.LC3II expression of mice in both groups increased(P<0.05)and P62 deposition decreased in both groups(P<0.05),among them,LC3II expression of mice in the RH group increased more than the JH group(P<0.05)and the RL group(P<0.05).Conclusion1.Jiangtang Sanhuang Tablets play a role of autophagy enhancement in the key pathogenesis of DN including "tissue inflammation","insulin resistance”and“podocyte changes".2.Jiangtang Sanhuang Tablets reduce inflammation and tissue structure damage,promote the normal play of insulin secretion function,protect the level before insulin receptor that is the upstream pathway of insulin signal transduction,lower the serum FINS level,improve HOMA-IR value and systemic insulin resistance from the source of insulin through enhancing the pancreas tissue autophagy of db/db mice.3.Jiangtang Sanhuang Tablets reduce inflammatory reaction,increases IRS1 expression which is the substrate of insulin receptor,protect the level of insulin receptor that is the insulin signal combined with the target,improve the integrity of renal insulin signaling conductive pathway and alleviate renal insulin resistance and renal pathological changes and improve renal function through enhancing the renal tissue autophagy.4.Jiangtang Sanhuang Tablets induce IRS1 to play its activation role in the upstream pathway,promote IRS1/PI3K/Akt pathway to improve the damage of renal podocyte structure,reduce the pathological progression of diabetic nephropathy,and exert the specific mechanism of Jiangtang Sanhuang Tablets to protect renal function by improving the impaired expression of IRS1/PI3K/Akt pathway mediated by insulin resistance in renal tissue. |
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