| ObjectiveIn hypertension-induced renal damage,oxidative stress affects podocyte apoptosis and segmental glomerulosclerosis,thus affecting glomerular permeability.Oxidative stress impairs the proximal and middle renal tubules.Oxidative stress impairs the epithelial mesenchymal transformation of glomerular mesangial cells,promoting the accumulation of myofibroblasts,leading to excessive secretion of fibrogenic factors and extracellular matrix proteins,and causing the remodeling of renal tubulointerstitial extracellular matrix.Sestrin2 is a class of evolutionarily highly conserved stress-inducing proteins.When cells are under stress,their expression is up-regulated,which protects against oxidative stress.In the first part of the experimental study,unilateral nephrectomy and microosmotic pump infusion of angiotensin Ⅱ were used to make hypertensive renal damage model.Qian Yang Yu Yin Granules(QYYYG)were used to intervene hypertension-induced renal damage.To observe the changes of blood pressure,heart rate,blood urea nitrogen,urine ACR and renal pathological changes after the intervention of QYYYG.The changes of QYYYG in interfering with Sestrin2 signaling pathway were observed.In the second part of the clinical study,the changes of serum Sestrin2 protein in patients with early hypertensive renal damage were observed,and the anti-oxidative effect of QYYYG in patients with early hypertensive renal damage was explored.Method1.Experimental study1)A model of renal damage in spontaneously hypertensive rats(SHR)was established by unilateral nephrectomy and angiotensin perfusion with osmotic pump.WKY rats were used as a control group.2)WKY was used as the normal control group,and the SHR after modeling was randomly divided into 4 groups according to systolic blood pressure,namely the model group(Model),valsartan group(Valsartan),QYYYG high-dose group(QYYYG(H)),QYYYG Low-dose Group(QYYYG(L)),intragastric intervention,once a day for 8 weeks,to observe the effects of QYYYG on weight,blood pressure,heart rate,blood urea nitrogen,microalbumin to creatinine ratio(ACR),kidney tissue morphology(HE staining)and degree of fibrosis(Masson staining)of SHR.3)After the intervention of QYYYG,WB,IHC and RT-PCR were used,the effects of Sestrin2-AMPK-mTOR-HIF-1α signal pathway,Sestrin2-Nrf2-HO-1 signal pathway,and Sestrin2-NOX4 signal pathway were observed in oxidative stress.2.Clinical studyTo explore the effect of QYYYG on the expression of serum Sestrin2 in patients with hypertension and early renal damage.1)Comparing the general demographic data of the two groups:gender,age,height,weight,waist circumference,BMI.2)Observing the effect of QYYYG on serum alanine aminotransferase.3)Observing the effect of QYYYG on urine microalbumin and creatinine ratio.4)Observing the effect of QYYYG on serum creatinine.5)Observing the effect of QYYYG on systolic blood pressure.6)Observing the effect of QYYYG on serum Sestrin 2.8)Observing the effect of QYYYG on serum ROS.Result1.Experimental study1)After unilateral nephrectomy and microosmotic pump perfusion with Angiotensin Ⅱ(Ang Ⅱ)for 2 weeks,the systolic blood pressure and heart rate of SHR increased significantly.The blood BUN and urine ACR of SHR increased.Through optical microscope,thickening of artery wall,infiltration of plasma,degeneration of hyaluronium,ischemic contraction and hardening of basement membrane of glomerular capillaries,atrophy of glomerulus,infiltration of multifocal inflammatory cells in renal interstitium and obvious fibrosis were observed.Unilateral nephrectomy and microosmotic pump perfusion with Ang Ⅱ can successfully establish hypertensive renal damage model in SHR.2)QYYYG can lower SBP and DBP of SHR after modeling,QYYYG can decrease BUN and ACR of SHR after modeling,QYYYG can improve the pathological damage of kidney tissue in SHR after modeling,reduce inflammation infiltration and vitreous degeneration,improve thickening of renal artery blood vessel wall and glomerular capillary basement membrane ischemia,and finally reduce kidney tissue fibrosis.3)In immunohistochemical experiment,QYYYG high-dose group can enhance the expression of SESN2 protein,activate the expression of AMPKa phosphorylation level,and then inhibit the mTOR phosphorylation level,further suppress the substream protein of HIF-la expression.4)In immunohistochemical experiment,QYYYG high-dose group can enhance the expression of SESN2 protein,activate the expression level of Nrf2 protein,and further enhance the expression of downstream protein HO-1.5)In immunohistochemical experiment,QYYYG high-dose group can enhance the expression of SESN2 protein and inhibit the expression level of NOX4 protein.6)In western blot experiment,QYYYG may have the ability to activate SESN2 protein,activate AMPK phosphorylation,and inhibit mTOR protein phosphorylation,and finally inhibit the downstream molecule HIF-la protein expression to achieve anti-oxidative stress.7)In western blot experiment,QYYYG high-dose group can enhance the expression of SESN2 protein,activate the expression of Nrf2 protein,and at the same time enhance the expression of downstream molecule HO-1 protein,thereby achieving anti-oxidative stress.8)In western blot experiment,QYYYG high-dose group can enhance the expression of SESN2 protein,inhibit the expression of NOX4 protein,and thus achieving anti-oxidative stress.9)QYYYG high-dose group can increase SENS2 gene expression after intervention,QYYYG high-dose and low-dose group can significantly inhibit HIF-la gene expression level after intervention,QYYYG low-dose group can significantly increase the HO-1 gene expression level after intervention,and QYYYG high-dose and low-dose group could significantly inhibit the expression level of NOX4 gene after intervention.2.Clinical study1)After 12 months of QYYYG intervention,compared with the control group,there were no significant differences in systolic blood pressure,diastolic blood pressure,heart rate,serum creatinine,eGFR,ACR.2)Before treatment,there was no significant difference in serum SESN2 concentration between the two groups(P>0.05).After treatment,there was no significant difference in serum SESN2 concentration between the two groups(P>0.05).Before treatment,there was no significant difference between the two groups in serum ROS concentration(P>O.05).After treatment,there was no significant difference between the two groups in serum ROS concentration(P>0.05).After 12 months of QYYYG intervention,the concentrations of SESN2 in treatment group increased,there was a significant difference before and after treatment(P<0.01),the concentrations of SESN2 in control group also increased,there was a significant difference before and after treatment(P<0.01).Conclusion1)After unilateral nephrectomy and osmotic pump perfusion of Angiotensin Ⅱ,renal damage of SHR can be established in a short time.2)After intervention of QYYYG,it can reduce systolic and diastolic blood pressure after SHR modeling,improve renal function,reduce ACR,and improve histological morphology fibrosis of rat kidneys,and the local oxidative stress response in rat kidneys is improved through the SESN2--AMPK-mTOR-HIF-1α protein expression,the SESN2-Nrf2-HO-1 protein expression,and the SESN2-NOX4 protein expression.This experiment provides scientific research ideas for oxidative stress in hypertensive renal injury,and provides reference recommendations for prevention and treatment in clinical practice.3)The serum levels of SESN2 and ROS in the two groups of patients with hypertension and early renal damage increased significantly after 12 months of treatment,and there was no obvious abnormality in renal function,however,the oxidative stress response caused by hypertension is still progressing.SESN2 and ROS can be used as indicators to reflect the oxidative stress of early hypertensive renal injury. |
