Gastrin Is Involved In Salt-sensitive Hypertension Regulation By Inhibiting NHE3 In Intestine To Reduce Sodium Absorption&Cardiac-specific Dopamine D₅ Receptor Deficiency Cause Dilated Cardiomyopathy Through ROS Generation And Activation Of ERK

Objectives:Hypertension is a chronic disease that seriously endangers human health.With the increase of age,the incidence of hypertension is gradually increasing.Salt-sensitive hypertension accounts for up to 50%of hypertension and are likely to cause severe consequences such as cardiac hypertrophy,heart failure and renal insufficiency.The pathogenesis of salt-sensitive hypertension is complicated,in which sodium water metabolism disorder is one of the important mechanisms.The latest study found that gastrin not only regulates gastric acid secretion,but also plays an important role in the regulation of NHE3 and other sodium transporter in kidney.Intestinal NHE3 also plays an important role in sodium absorption,therefore limiting the sodium intake in intestinal tract may be a new target for the treatment of hypertension.In this study,we chemically linked gastrin to the surface of SiO2 microparticles,by entering the intestinal tract,the gastrin acts only on intestinal epithelial cells and is not absorbed into the blood.Therefore,we can evaluate the effect of gastrin on NHE3 in intestine and explore the role of gastrin on sodium water metabolism from a new perspective and also provides sufficient evidence for its role as a new target for the treatment of hypertension through animal experiments and cell experiments.Methods:In vivo study one:salt-sensitive Dahl rats and salt-resistant ss-13BN rats were divided into 3 groups respectively:control group(0.4%NaCl),high salt group(8%NaCl)and high salt+gastrin treatment group(8%NaCl+gastrin(0.2mg/kg/day));blood pressure was measured using tail cuff non-invasive method at the same time every day;urine of Dahl rats and ss-13BN rats was collected using metabolic cages and measure the levels of urinary sodium and urinary creatinine;serum synthetic gastrin presence was detected by mass spectrometry method;confocal was used to detect the direct or indirect interaction between gastrin receptor and NHE3 in the intestine brush border of Dahl rats;immunofluorescence was used to detect the change of NHE3 localization on brush border membrane of small intestine in Dahl salt-sensitive rats fed with high salt diet and the effect of gastrin on the change of NHE3 localization;Brush border membrane protein expression of NHE3,NHERF1,NHERF2,NHERF3,ezrin and IRBIT were detected by western blot.In vivo study two:CCKBR knock-out mice(CCKBR-/-)and wild-type mice(CCKBR+/+)were divided into two groups respectively:control group(0.4%NaCl)and high salt group(6%NaCl),blood pressure was detected by left carotid artery cannulation;urine of CCKBR-/-mice and CCKBR+/+ mice were collected using metabolic cages and measure the levels of urinary sodium and urinary creatinine;NHE3,NHERF1,NHERF2,NHERF3,ezrin and IRBIT expression on the brush border membrane were detected by western blot.In vitro study:we divided Caco-2 cells into five groups:control group,high salt group(high salt medium(increase NaCl concentration by 50mM)),high salt+gastrin group(high salt medium+gastrin(1000nM)),PLC inhibition group(high salt medium+PLC inhibitor(10?M)+gastrin(1000nM))and PKC inhibition group(high salt medium+PKC inhibitor(10?M)+gastrin(1000nM));Caco-2 cells of high salt+gastrin group were pretreated by gastrin for 1 hour and then stimulated by high salt,Caco-2 cells of PLC inhibition group were pretreated by PLC inhibitor(U73122)for 1 hour and then pretreated by gastrin for 1 hour and then stimulated by high salt,Caco-2 cells of PKC inhibition group were pretreated by PKC inhibitor(Go6983)for 1 hour and then pretreated by gastrin for 1 hour and then stimulated by high salt;NHE3,NHERF 1,NHERF2,NHERF3,ezrin and IRBIT expression on the cell membrane of five group cells were detected by western blot.Results:In vivo study one:Compared to control group of Dahl rats,blood pressure of high salt diet group was significantly higher(P<0.05),additional gastrin treatment significant block the high salt induced high blood pressure(P<0.05).However,there was no significant difference in blood pressure between control group,high salt diet group and gastrin treatment group of SS-13BN rats;urinary sodium excretion was increased in the high salt diet group of Dahl rats and SS-13BN rats(P<0.05),however the urinary sodium excretion of high salt diet group of SS-13BN rats was higher than Dahl rats with high salt diet(P<0.05);urinary sodium level of gastrin treatment group of Dahl rats and SS-13BN rats were decreased compared to high salt diet group of these two rats;confocal image shows that NHE3 and CCKBR co-localized on the brush border membrane;immunofluorescence result showes that NHE3 translocation to brush border membrane increased in high salt diet group of Dahl salt-sensitive rats when compared to control group;western blot result shows that NHE3,NHERF1,NHERF2,NHERF3,ezrin and IRBIT expression in the intestinal brush border membrane of high salt diet group of Dahl salt-sensitive rats were significant increase when compared to control group,gastrin treatment effectively decrease high salt induced proteins elevation(P<0.05).In vivo study two:systolic blood pressure of CCKBR-/-mice(0.4%NaCl)was significantly higher compared to CCKBR+/+ mice(0.4%NaCl)(P<0.05),the blood pressure of CCKBR-/-mice and CCKBR+/+ mice with high salt diet(8%NaCl)further increased separately,in which CCKBR-/-mice(8%NaCl)also have higher systolic blood pressure than CCKBR+/+ mice(8%NaCl)(P<0.05);urinary sodium of CCKBR-/-mice(0.4%NaCl)was decreased compared to CCKBR+/+ mice(0.4%NaCl)(P<0.05),high salt fed CCKBR-/-mice(8%NaCl)also have decreased urinary sodium than CCKBR+/+ mice(8%NaCl)(P<0.05);NHE3,NHERF1,NHERF2,NHERF3,ezrin and IRBIT expression in the intestinal brush border membrane of CCKBR-/-mice(0.4%NaCl)were increased compared to CCKBR+/+mice(0.4%NaCl),which proteins expression increased after fed with high salt diet.In vitro study shows that,NHE3,NHERF1,NHERF2,NHERF3,ezrin and IRBIT expression in the Caco-2 cells membrane increased in high salt group compared to control group,which were decreased in gastrin treatment group compared to high salt group,PLC and PKC inhibitor can significantly prevent gastrin induced down-reguation of these proteins expresison.Conclusion:Gastrin can inhibit the formation of macromolecular complexes(containing NHE3,NHERF 1,NHERF2,NHERF3,ezrin and IRBIT)and promotes NHE3 transfer from brush border membrane of intestinal villi to cytoplasm and reduce the expression of NHE3 on the intestinal brush border membrane,via PLC/PKC pathway,which maybe the noval target of salt sensitive hypertension treatment.Objective:Dilated cardiomyopathy(DCM)is a severe cardiomyopathy characterized by heart chamber expansion and impaired cardiac function with or without congestive heart failure.There are many factors that cause dilated cardiomyopathy,but the specific mechanism of its pathogenesis is still unclear.Our previous study found that dopamine D5 receptor knock out mice exhibit increased cardiac weight and cardiac hypertrophy.This shows that dopamine D5 receptor has a close relationship with the pathogenesis and development of cardiomyopathy.Therefore,our study is designed to investigate the specific mechanism of the dopamine D5 receptor effect in the development of dilated cardiomyopathy through animal experiments and cell experiments.Methods:Generate mice that express cardiac-specific hD5F173L(hD5F173L-TG)and hD5WT(hD5WT-TG)gene;construct cells that transfect with hD5F173L(H9c2-hD5F173L)and hD5WT CH9c2-hD5WT)gene.Cardiac function of hD5WT-TG mice and hD5F173L-TG mice was detected by echocardiography and evaluated the effect of apocynin on cardiac function of 3-month-old hD5F173L-TG mice.NADPH oxidase activity of hD5WT-TG,hD5F173L-TG mice,H9c2-hD5WT and H9c2-hD5F173L cells was detected by lucigenin method.ROS production of hD5WT-TG,hD5F173L-TG mice,H9c2-hD5WT and H9c2-hD5F173L cells was detected by ELISA method.The expression of P40phox,P47phox,Nrf2,Nrf2 downstream signaling molecules including NQO1 and HO1 cardiac injury markers including PLN,SerCa2,BCL-2 and BAX,signaling pathway molecules including p-ERK1/2,ERK1/2,p-JNK,JNK,p-P38 and P38 in the heart of hD5WT-TG and hD5F173L-TG mice were detected by Western blot and evaluated the effect of apocynin on these proteins.The expression of P40phox,P47phox,Nrf2,and Nrf2 downstream signaling molecules including NQO1 and HO1 in H9c2-hD5WT and H9c2-hD5F173L cells were detected by Western blot.The expression of Nrf2,BAX,BCL-2 and JNK in H9c2-hD5WT and H9c2-hD5F173L cells were detected by immunofluorescence and detect the effect of Nrf2 inhibitor(ML385)on BAX,BCL-2 and JNK expression.The interaction between Nrf2 and ubiquitinated proteins in 3 and 4-month-old hD5F173L-TG mice was detected by co-immunoprecipitation.The expression of P40phox,Nrf2,p-ERK1/2 and p-JNK in H9c2-hD5WT and H9c2-hD5F173L cells were detected by Western blot and detect the effect of PKA inhibitor(H89)and PKG inhibitor(KT5823)on these proteins.Results:In the animal experiments we found that compared to hD5WT-TG mice,LVESV,LVEDV,LVID;s and LVID;d increased in hDsF173L-TG mice,EF and FS decreased,the heart weight significantly increased,obvious fibrosis in the heart of hD5F173L-TG mice,but there is no significant difference between the blood pressure of hD5WT-TG mice and hD5F173-TG mice.After treatment of apocynin,the cardiac function of hD5F173L-TG mice was significantly improved compared to untreated group.Compared with hD5WT-TG mice,the cardiac NADPH oxidase activity,ROS production and the membrane expression of NADPH oxidase subunits P40phox and P47phox were significantly increased in hD5F173L-TG mice.After treatment of apocynin with hD5F173L-TG mice,the NADPH oxidase activity and the expression of P40phox and P47phox on the cell membrane was decreased.However,the decrease extent of ROS production is lower than the decrease extent of NADPH oxidase activity.In the cell experiments,NADPH oxidase activity,ROS production and basal protein expression of P40phox and P47phox were higher in H9c2-hD5F173L cells than H9c2-hD5WT cells.Nrf2,NQO1 and HOI expression increased in the 3-month-old hD5F173L-TG mice,but these proteins expression decreased in the 4-month-old hD5F173L-TG mice and apocynin treatment have no effect on these proteins expression.The ubiquitinated Nrf2 was slight decrease in the 3-month-old hD5F173L-TG mice,and significantly increased in the 4-month-old hD5F173L-TG mice.Nrf2 mainly expressed in plasma of H9c2-hD5WT cells,but its expression increased in the nucleus of H9c2-hD5F173L cells.PLN,SerCa2,BAX,BCL-2,p-JNK and p-ERK1/2 expression are significantly increased in hD5F173L-TG mice and apocynin significantly blocked the increase of the proteins expressionl PKG inhibitor significantly decreased the expression of p40phox,p-ERK1/2,and p-JNK in H9C2-hD5F173L cells.Conclusion:The results show that D5 receptor can inhibit the activity of NADPH oxidase,increase antioxidant protein expression via promoting Nrf2 translocate to nucleus.And then inhibit the effect of oxidative stress and relieve the injure of dilated cardiomyopathy.