Tangshen Recipe To Improve The Mechanism Of Diabetic Kidney Disease And Podocyte Damage

BackgroundDiabetic kidney disease(DKD)is one of the major microvascular complications of diabetic mellitus(DM).It is the leading cause of death and disability in DM patients in the world.The prevalence of DKD in the patients with type 2 DM is 10 to 40%in China.For a long time,strict control of blood pressure and blood sugar,especially the use of renin-angiotensin-aldosterone system inhibitors is the main treatment of DKD,but they have not effectively stopped the progress of DKD.Chinese medicine has played an important role in the treatment of DKD due to its multi-target and multi-system effect mechanism.With the support of the National 973 Project,on the basis of inheriting the experience of predecessors,mentor professor Li Ping combined with modern research results to develop a compound Chinese medicine preparation-Tangshen Formula(TSF)with Yiqi Rougan and Huoxue Tongluo as the main treatment rules.The research team conducted two multi-center,randomized,double-blind,placebo-controlled clinical trials of TSF in the treatment of DKD,evaluated its effectiveness and safety in the treatment of DKD,and proved that TSF can reduce the excretion of urinary protein and improve renal function.Several basic studies have found that the drug has anti-inflammatory and anti-fibrotic effects.However,little is known about the potential mechanism of TSF in reducing podocyte injury and improving proteinuria.Podocyte injury is the main mechanism of proteinuria and is considered to be an early feature and therapeutic target for predicting the development of DKD.Its unique actin cytoskeleton system is the key to maintaining the complex morphology and function of podocytes.Recent studies have found that the damage of the podocyte actin cytoskeleton systm can induce proteinuria.Transient Receptor Potential Canonical Channel 6(TRPC6)and cell-matrix adhesion protein talinl are important components of podocyte actin cytoskeleton system.The abnormal changes of TRPC6 are closely related to the occurrence of proteinuria and the progression of DKD.TRPC6-mediated calcium influx can cause abnormal shearing of talin1,which is of great significance in podocyte damage and proteinuria formation.Therefore,talinl plays a key role in the mechanism of podocyte injury.Based on the above research background,our research will use the db/db mice(spontaneous type 2 DM model and a classic animal model to study early DKD)to observe the effect and efficacy of different doses of TSF on DKD through in vivo pharmacodynamic evaluation optimal dosage;evaluate the effects of TSF on the related indexes of podocyte injury in db/db mice,and clarify the role and mechanism of TSF in protecting DKD podocytes.Combined with primary mice podocytes stimulated by advanced glycation end products(AGEs)in vitro experiments,the podocyte actin cytoskeleton system was used as the research target to further explore the mechanism of TSF in improving podocyte injury by regulating TRPC6/Talin1 pathway.In addition,db/db mice were used to observe the effect of TSF's blood monomer compound Neohesperidin on DKD.Objective(1)To screen the optimal therapeutic dose of TSF on improving DKD in db/db mice;(2)To explore the effects of TSF on DKD podocyte injury,podocyte actin cytoskeleton and TRPC6/Talin1 signaling pathway;(3)To observe the effect of Neohesperidin on DKD in db/db miceMethods(1)Experiment part I:To evaluate the therapeutic effect of TSF on DKD through db/db mice,and screen the optimal therapeutic dose.8-week-old male db/db mice and homogenic background db/m mice were fed with a normal diet.After 2 weeks adaptation,all mice were observed and measured basic weight and blood glucose,collected urine in metabolic cages,randomly divided into 6 groups and started drug intervention:db/m group,db/db group,db/db+TSF low-dose group(1.8 g/kg/day),db/db+TSF medium-dose group(3.6 g/kg/day)and db/db+TSF high-dose group(7.2 g/kg/day),and db/db+irbesartan group(22.5 mg/kg/day).Body weight was recorded weekly.In the 12th week before dissection,urine was collected with a metabolic cage.After 12 weeks of continuous gastric administration,blood was collected by cardiac puncture and detected by a fully automatic biochemical analyzer.Serum biochemical testing items included renal function,liver function and blood lipid-related indicators.The pathological damage of kidney and liver tissues in each group was evaluated.(2)Experiment part II:The role and mechanism of TSF in alleviating DKD podocyte injury were clarified by db/db mice.According to the results of Experiment part I,the best curative dose group of TSF was selected for the following experiment.Periodic acid-silver methenamine stain(PASM)staining and transmission electron microscope techniques were used to further observe and score damage to the glomerular,basement membrane and podocytes of mice in experiment part I.Immunohistochemistry,immunofluorescence co-staining,immunofluorescence,and western blot were used to evaluate the level and localization of podocyte cell-matrix adhesion protein talinl,TRPC6,podocyte marker proteins WT1 and nephrin in db/db mice.(3)Experiment part ?:To prove that TSF protects podocytes by regulating TRPC6/Talinl signaling pathway.In vitro,a model of primary mice podocyte injury induced by AGEs was establish,and the effects of AGEs and TSF on the ability of migration and the formation of lamellipodia and filopodia were observed through the wound healing assay.The expression of talin1,TRPC6,integrin ?1 and paxillin protein was detected by western blot.Observing the changes of the actin cytoskeleton patterns of primary mice podocytes in each group by the method of phalloidin staining.FACScan flow cytometry was used to observe the effect of AGEs,TSF and/or TRPC6 inhibitor SAR7334 on intracellular calcium ion levels in primary mice podocytes.The expression of TRPC6 in primary mice podocytes stimulated by AGEs was observed using fluorescent staining,as well as the nuclear positioning of nuclear factor of activated T cell(NFATC)2 and 3.The protective effect of TSF was evaluated in the primary mice podocyte induced by AGEs,and the blocking effect of TRPC6 agonist OAG on TSF was observed.(4)Experiment part ?:Using db/db mice to observe the effect of TSF's blood-infused monomer compound Neohesperidin on DKD.The second batch of 8-week-old male db/db mice and non-pathogenic db/m mice were purchased.The feeding and model confirmation methods are the same as in Experiment I.Randomly divided into 3 groups and started intragastric administration at the age of 10 weeks:db/m normal control group,db/db model control group,db/db+ Neohesperidin Group(100 mg/kg/day).During the experiment,weight was measured once a week,and fasting blood glucose was monitored every 4 weeks.The metabolic cage collects urine and measures proteinuria levels.After 12 weeks of continuous administration,the mice were fasted and collected,the blood was collected by cardiac puncture;the pathological damage of kidney and liver tissues in each group was evaluated.Results(1)Experiment part ?:It was proved that TSF could effectively improve the kidney damage of db/db mice,and the middle dose(3.6 g/kg/day)had the best curative effect.After 12 weeks of treatment with all doses of TSF,db/db mice could reduce their elevated serum creatinine and proteinuria to varying degrees,especially at medium and high doses;the effect was significant;kidney HE staining,PAS staining and MASSON Staining showed that the medium and high doses of TSF could significantly improve the glomerular hypertrophy,dilation of mesangial area,and tubulointerstitial injury manifested in the pathology of db/db mice.In addition,the medium-dose and high-dose of TSF could significantly reduce the body weight of db/db mice;effectively improve the elevated serum lipid levels of db/db mice;significantly reduce the elevated serum alanine aminotransferase and aspartate aminotransferase;liver HE staining and oil red O staining showed that TSF could significantly improve steatosis of liver tissue.(2)Experiment part ?:It was found that TSF could improve podocyte injury in db/db mice.TSF effectively improved the glomerular basement membrane thickening,foot process effacement and the reduction of podocyte number in db/db mice.db/db mice had significant WT1 reduction and talinl loss;immunofluorescence co-localization found that the loss of talinl in db/db mice was concentrated in podocytes,and there was significant podocyte TRPC6 overexpression;TSF could significantly inhibit the decrease of WT1,the loss of talin1 and the overexpression of TRPC6 in podocytes.(3)Experiment part ?:TSF could protect podocytes by regulating TRPC6/Talinl signaling pathway.The results of the wound healing experiment showed that the primary podocytes stimulated by AGEs showed increased cell migration and the formation of lamellipodia and filopodia;the phalloidin staining method found that AGEs could induce a significant reduction in the staining pattern of A-type and B-type actin cytoskeletons,while the C-type and D-type significantly increase;there was a significant rearrangement of the actin cytoskeleton;The results of FACScan flow cytometry found that AGEs could induced an increase of TRPC6-mediated calcium deposition;western blot and immunofluorescence results showed that TRPC6 was overexpressed,talin1,integrin ?1 and paxillin was reduced in primary podocytes stimulated by AGEs,and there was significant NFATC2 nuclear metastasis.TSF could alleviate the damage of primary mice podocytes stimulated by AGEs.(4)Experiment part IV:It was found that Neohesperidin could significantly reduce proteinuria in db/db mice.Neohesperidin could effectively reduce the increased body weight,reduce blood lipid levels of db/db mice,and alleviate liver tissue fatty degeneration;there was no effect of Neohesperidin on blood glucose levels in db/db mice.ConclusionOur research found that TSF could improve the kidney damage of db/db mice,and the middle dose has the best effect;this study innovatively found that TSF could maintain the stability of actin cytoskeleton system by regulating the podocyte TRPC6/Talin1 pathway,reduce podocyte injury and proteinuria.In addition,TSF's blood monomer compound Neohesperidin could reduce the proteinuria of DKD in db/db mice.The above results provided an experimental basis for the better application of TSF in the treatment of DKD.