The Role And Mechanism Of ’Unblocking Collaterals’ Therapy On Target Organ Damages In Essential Hypertension

BackgroundHypertensive heart disease is one of cardiac complications due to persistent and chronic hypertension, which is characterized that left ventricular diastolic dysfunction and hypertrophy at the early stage and ventricular systolic dysfunction and heart fail-ure at last. It is commonly accepted that the hypertrophy and phenotypic transition of cardiac myocytes lead to compensatory ventricular hypertrophy and the systolic dys-function of these myocytes result in heart failure. But recent preclinic studies shows that the abnormal deposition and turnover of extracellular matrix is the vital factor of basic pathophysiological mechanism responsive for target organ injury in hyperten-sion. Cardiac fibroblasts(CFs) being transactivated to myofibroblasts and extracel-lular matrix depositing in the cardiac interstitium, which is known as cardiac fibrosis, is the important characteristic of ventricular remodeling. In the patients with essential hypertension cardiac fibrosis is closely associated with the ventricular diastolic dys-function in the early stage and structural adverse remodeling and functional failure in the subsequent stage. Simply controlling blood pressure could not completely im-prove cardiac fibrosis. Therefore, effective prevention and therapy of cardiac fibrosis play an important role in the prognostic improvement of hypertension and hyperten-sive heart disease.CFs is the initial and vital effector cell in the cardiac fibrotic response, which preside over the homeostasis of extracellular matrix. The activation of cardiac fibro-Part Ⅰ Role and Mechanism of Tongxinluo on Cardiac Fibrosis Related to Essential Hypertension (Invitro Experiment) blasts is an early event of hypertensive heart disease, and also represents the pivotal event response for cardiac remodeling and dysfunction. Smad3/CTGF signaling criti-cally controls the activation and functions of CFs. Meanwhile the cardiac microvas-cular endothelial cells(CMEC) is the earliest cell type which is influenced by the he-modynamic and neurohumoral changes of hypertension. The abnormal function of CMECs could promote the activation of CFs and the microvessle rarefaction is an important feature of essential hypertension, which further aggra-vates the proceeding of cardiac adverse remodeling and dysfunction. eNOS/NO sig-naling plays an important role in the protection of endothelium and angiogenesis. Therefore, strategies about inhibiting the activation of CFs and protecting the function of CMECs by regulating the signaling pathway involved, are important to the therapy of hypertension.microRNAs (miR) is a kind of highly conserved and endogenous non-coding single-stranded RNA. miR could result in the degradation of mRNA of target genes or inhibition of their translation, which negatively controls the expression of target genes. In many animal models of cardiovascular diseases, the expression profiling of miR changes significantly. miR could participated in the process of cardiac fibrosis and could promote or inhibit the adverse development of cardiovascular disease. miR-133a is extensively expressed in main cell types of heart. In the physiological state, miR-133a regulate the development of cardiac morphology. In the pathological state,miR-133a directly regulates many signaling pathways related to ventricular hy-pertrophy, cardiac fibrosis and angiogenesis. miR-133a plays an protective role in the cardiac diseases. The expression of miR-133a is down-regulated during hypertension. Therefore, it is an promising therapeutic target of cardiac fibrosis in hypertension.Cardiac fibrosis related to hypertension is the complication during the slowly progressive course of hypertension, which accord with the viewpoint "persistent ill-ness entering collaterals" in traditional Chinese medicine. The pathogenic factors dis-turbs the function that cardiac collaterals preside over the interaction and transfor-mation between Ying-nutrient and Wei-defense. Therefore, the cardiac collaterals are blocked and their structure and function are injured by qi stagnation, blood stasis and secondary pathological products such as phlegm and toxic metabolites, which result in the abnormal accumulation in the heart. As a result, the strategy unblocking collat-erals unblocking collaterals with subsequent convergence and substance-qi transformation is suitable for the therapy of cardiac fibrosis related to hypertension. Tongxinluo is one of representative prescription according to the "unblocking collat-erals unblocking collaterals with subsequent convergence and substance-qi transformation" theory. Fundamental researches showed that Tongxinluo protect against cardiac fibrosis in spontaneously hypertensive rats, but the mechanisms need to be further researched. Therefore we observe CFs and CMECs in the vitro experi-ments and SHR in the vivo experiments and study the effect and mechanism of Tongxinluo on cardiac fibrosis related to hypertension.Objectives1. To investigate the effects and mechanisms of Tongxinluo on AngⅡ-induced transformation and functional activation of CFs;2. To investigate the effects and mechanisms of Tongxinluo on AngⅡ-induced CMEV functional injury and on the microenvirmental changes;3. To investigate the effects and mechanisms of Tongxinluo and the role of miR-133a.Methods1.Isolation, culture and identification of primary cellsCMECs and CFs were isolated from neonatal rat ventricular tissues by en-zyme digestion and differential time attachment. CMECs and CFs were separately identified by Immunofluorescence analysis of Dil-ac-LDL and FSP-1 antibody.2.Screening the appropriate exposure concentration and time of TongxinluoCell viability was measured by cell counting kit(CCK)-8 in order to screen the the appropriate exposure concentration and time of Tongxinluo.3.Groups of CFs and CMEC.Cells were divided into the control group, Ang Ⅱ group and treatment groups. After miR-133a transfection, cells were divided into blank group, control group, treatment group, and negative control group. After 24 h serum starving, cells were given treatment with or without Tongxinluo and/or Ang Ⅱ for 24 h.4. Transfection of miR-133a inhibitior.miR-133a inhibitors were were transfected into CFs or CMECs with Lipofec-tamine 2000 according to the manufactures’instruction. Fluores-cence immunostain and qRT-PCR were used to qualify the transfection efficiency of miR-133a inhibitors.5.CFs function assayCFs migration assays were performed in Transwell chambers. Collagen synthesis of CFs was measured by 3H-hydropoline incorporation assay. The collagen I and IL-6 secretory function of CFs were measured by ELISA assays.6. CMEC function of invitro tube formationMatrigel invitro tube formation assay was used to observe CMEC function of invitro tube formation.7.CMEC secretion of pro-inflammatory and pro-fibrotic factorsThe IL-6 and CTGF secretion of CMECs in the supernatant were measured by ELISA assays.8. Real-time RT-PCRTotal RNA was extracted from CMECs and CFs. In the experiment, the expres-sion of miR-133a in CMECs and CFs were analyzed.9. Western blottingProteins were extracted from CMECs and CFs. The protein expression of a-SMA, smad3,p-smad3,CTGF,eNOS,p-eNOS and caveolin-1 was analyzed in our experiment.10. Statistical analysisAll statistical analyses involved use of SPSS 18.0. Data are reported as mean± standard deviation. Comparisons between two groups involved in student’s t-test and comparisons among group involved one-way ANOVA. Probabilities of 0.05 or less were considered to be statistically significant.Results1 Tongxinluo inhibited Ang II-induced CF phenotypic transformation(1) Screening the appropriate exposure concentration and time of TongxinluoThe appropriate concentration of Tongxinluo in the experiment is within 400 μg/mL and these is the minimal effect on cell viability within 24 h.(2)Tongxinluo inhibited AngⅡ-induced CF expression of alpha-SMACompared with the control group, Ang Ⅱ up-regulated the expression of al-pha-SMA protein; in comparison with the Ang II group, Tongxinluo inhibited the ex-pression of alpha-SMA protein.(3)Tongxinluo inhibited Ang II-induced functinonal activities of CFsCompared with the control group, Ang II enhanced CF migration and 3H-hydropoline incorporation;in comparison with the Ang II group, Tongxinluo in-hibited CF migration and 3H-hydropoline incorporation.Compared with the control group, Ang II enhanced CF secretion of IL-6 and collagen I; in comparison with the Ang II group, Tongxinluo inhibited CF secretion of IL-6 and collagen I.(4)Tongxinluo protectsed against Ang Ⅱ-inhibited expression of miR-133a in CFs.Compared with the control group, AngⅡ down-regulated expression of miR-133a in CFs;in comparison with the AngⅡ group, Tongxinluo increased the expression of miR-133a.(5) Modulation of miR-133a expression participates in the effects of Tongxinluo on AngⅡ-induced expression of alpha-SMA in CFs.Compared with the negative control group in which CFs were transfected with scrambled siRNA, the inhibitory effects of Tongxinluo on AngⅡ-induced CF activa-tion disappeared in CFs transfected with miR-133a inhibitors.(6) Modulation of miR-133a expression participates in the effects of Tongxinluo on AngⅡ-induced activation of smad3/CTGF signaling.Compared with the negative control group in which CFs were transfected with scrambled siRNA, the inhibitory effects of Tongxinluo on AngⅡ-induced activation of smad3/CTGF signaling disappeared in CFs transfected with miR-133a inhibitors.2 Tongxinluo improved Ang Ⅱ-induced injury and functional activation of CMECs(1) Screening the appropriate exposure concentration and time of TongxinluoThe appropriate concentration of Tongxinluo in the experiment is within 400 μg/mL and these is the minimal effect on cell viability within 24 h.(2) Tongxinluo prevented CMECs from AngⅡ-induced injury of invitro tube formationCompared with the control group, AngⅡ induced the injury of invitro tube for-mation in CMEC; in comparison with the AngⅡ group, Tongxinluo prevented CMEC from injury of invitro tube formation.(3) Tongxinluo regulated AngⅡ-induced changes in the level of NO and in the secretion of IL-6 and CTGF in CMECs.Compared with the control group, Ang Ⅱ decreased significantly NO content and increased the secretion of CTGF and IL-6 in CMECs; in comparison with the AngⅡ group, Tongxinluo increased NO content and decreased the secretion of CTGF and IL-6.(4)Tongxinluo protected against AngⅡ-inhibited expression of miR-133a in CMECs.Compared with the control group, Ang Ⅱ down-regulated expression of miR-133a in CMECs; in comparison with the Ang Ⅱ group, Tongxinluo increased the expression of miR-133a.(5) Modulation of miR-133a expression participates in the effects of Tongxinluo on AngⅡ-induced injury of invitro tube formation in CMECs.Compared with the negative control group in which CMECs were transfected with scrambled siRNA, the protective effects of Tongxinluo on AngⅡ-induced injury of invitro tube formation disappeared in CMECs transfected with miR-133a inhibi-tors.(6) Modulation of miR-133a expression participates in the effects of Tongxinluo on AngⅡ-induced changes of caveolin-1/eNOS signaling.Compared with the negative control group in which CMECs were transfected with scrambled siRNA, the protective effects of Tongxinluo on Angll-induced changes of caveolin-1/eNOS signaling disappeared in CMECs transfected with miR-133a inhibitors.Conclusion1. Tongxinluo inhibits AngⅡ-induced activation and functional activities of CFs.miR-133a is a vital factor regulating AngⅡ-induced CF activation. Tongxinluo increases the expression of miR-133a and inhibits the activation of smad3/CTGF sig-naling, which directly related to the anti-fibrotic effects of Tongxinluo.2. Tongxinluo protects against AngⅡ-induced injury and functional activation of invitro tube formation in CMECs. miR-133a is a vital factor regulating AngⅡ-induced injury. Tongxinluo increases the expression of miR-133a, regulated the changes of caveolin-1/eNOS signaling and inhibits the secretion of inflammatory factor in CMECs.3. Tongxinluo inhibited the functional activation of CMECs, improved the ab-nomal metabolism and secretion and therefore modulated the microenvironment which promoted the activation of CFs and fibrogenesis.BackgroundHypertensive heart diseases are the main complication of heart.. The ventricular hypertrophy is commonly regarded as the most important pathological changes, which is the independent risk factor of heart failure. Despite the therapeutic strategies of hy-pertension and ventricular protection, the morbidity of heart failure in patients with hypertension still keep high level. Recently preclinic and clinic researches shows the desposition and abnomal turnover of extracellular matrix protein result in cardiac fi-brosis which may be the vital factor of pathophysiology of cardiac structural and functional damages. The biopsy of human heart in patients with hypertension shows the extensive fibrosis and maladjustment of collagen types. In the patients with essen-tial hypertension cardiac fibrosis is closely associated with the ventricular diastolic dysfunction in the early stage and structural adverse remodeling and functional failure in the subsequent stage. Therefore the control of cardiac fibrosis play an important role in protecting against the hypertensive heart diseases.Recently the important roles of cardiac microvessels and microvascular endothe-lial cells are valued. Histopathological study showed cardiac fibrosis and angiogenic response co-exist frequently, and perivascular fibrosis often occurs in the ventricles with overload pressure; these changes also suggested that the myocardial fibrosis is often related to the changes of cardiac microvessels. Angiogenesis is the compensato-ry mechanism of stress response in the heart. Pathological stimuli in hypertension such as abnormal hemodynamic changes, ischemia and hypoxia, chronic neu-Paper Ⅱ Role and Mechanism of Tongxinluo on Cardiac Fibrosis Related to Essential Hypertension (Invivo Experiment) ral-humoral hyperactivation, regulate microvascular growth in the heart, in order to keep enough oxygen and nutrients to the growing demand of hypertrophic ventricles. More experiments suggest that in the heart, brain, kidney and other organs of adult spontaneously hypertensive rat are often characterized as microvascular rarefaction. In the last stage of the course which myocardial fibrosis accompanied with deteriora-tive cardiac function significantly, angiogenesis resulting from compensatory re-sponse to pathological stimuli is often insufficient. Therefore, the hypertrophy of the cardiomyocytes increased demand of oxygen and energy, microvascular angiogenesis quantity is insufficient; the transformation of the microvascular network accompanied with microvascular damage or degradation, so the microvascular structure and func-tion is not perfect. At the same time, the injury caused by microvascular endothelial activation, abnormal metabolism and paracrine and endocrine function, lead to cardiac inflammation and oxidative stress, and thus promote formation of cardiac microenvi-ronment which is in favor of fibrogenesis. Angiogenesis does not significantly im-prove cardiac blood and oxygen supply, the outcome is to further aggravate the heart of the functional and structural remodeling of the heart to heart failure. It is speculated that the microvascular rarefaction may be one of the important characteristics of hy-pertension, and it can be used as an important indicator of cardiac function.Therefore, it is of great significance to improve cardiac microvascular angiogenesis and improve microenvironmental disturbance, which is important for reducing myocardial fibrosis and protecting the heart function.microRNAs (miR) is a kind of highly conserved and endogenous non-coding single-stranded RNA. miR could result in the degradation of mRNA of target genes or inhibition of their translation, which negatively controls the expression of target genes. In many animal models of cardiovascular diseases, the expression profiling of miR changes significantly. miR could participated in the process of cardiac fibrosis and could promote or inhibit the adverse development of cardiovascular disease. miR-133a is extensively expressed in main cell types of heart. In the physiological state, miR-133a regulate the development of cardiac morphology. In the pathological state,miR-133a directly regulates many signaling pathways related to ventricular hy- pertrophy, cardiac fibrosis and angiogenesis. miR-133a plays an protective role in the cardiac diseases. The expression of miR-133a is down-regulated during hypertension. Therefore, it is an promising therapeutic target of cardiac fibrosis in hypertension.Cardiac fibrosis related to hypertension is the complication during the slowly progressive course of hypertension, which accord with the viewpoint "persistent ill-ness entering collaterals" in traditional Chinese medicine. The pathogenic factors disturbs the function that cardiac collaterals preside over the interaction and transfor-mation between Ying-nutrient and Wei-defense. Therefore, the cardiac collaterals are blocked and their structure and function are injured by qi stagnation, blood stasis and secondary pathological products such as phlegm and toxic metabolites, which re-sult in the abnormal accumulation in the heart. As a result, the strategy unblocking collaterals unblocking collaterals with subsequent convergence and sub-stance-qi transformation is suitable for the therapy of cardiac fibrosis related to hy-pertension. Tongxinluo is one of representative prescription according to the "un-blocking collaterals unblocking collaterals with subsequent convergence and substance-qi transformation" theory. Fundamental researches showed that Tongxinluo protect against cardiac fibrosis in spontaneously hypertensive rats, but the mechanisms need to be further researched.Objectives1. To investigate the effects and mechanisms of Tongxinluo on cardiac fibrosis related to essential hypertension and cardiac microenvironment related to fibrogene-sis;2. To investigate the effects and mechanisms of Tongxinluo on cardiac fibrosis in SHRs and roles of miR-133a.Methods1.Animal protocol40 eight-week-old male SHRs were randomLy assigned to 4 groups(n=10 per group):untreated SHR and TXL.Eight eight-week-old male Wistar Kyoto rats(WKY;Experimental Animal Center of Shandong University, Jinan, China)were used as the control group. The TXL groups were fed intragastrically with 0.4 g-kg-l-d-1(TXL-L). TXL ultrafine powder dissolved in saline for 12 weeks. The WKY and untreated SHR groups were fed intragastrically with equal volumes of sa-line once daily for 12 weeks.2.Measurement of blood pressure and cardiac function.Systolic blood pressure was measured by tail cuff method. Cardiac diameter and function was measured by use of the Vevo 770 imaging system.2D echocardiography, M-mode echocardiography, pulsed-wave Doppler echocardiography and tissue Dop-pler imaging were used to evaluate cardiac function.3. Histology and immunohistochemistryTissue was paraffin-embedded and sectioned (4μm) for Masson’s trichome staining to examine heart extracellular matrix deposition. Immunohistochemistry was used to determine the levels of CD31 in myocardial tissues. Cardiac microvascular density was determined by CD31 positive staining.4. Real-time RT-PCRTotal RNA was extracted from cardiac tissues. In the experiment, the expression of miR-133a in cardiac tissues was analyzed.5. Western blottingProteins were extracted from cardiac tissues. The protein expression of collagen I, smad3,p-smad3,CTGF,eNOS,p-eNOS and VEGF-A was analyzed in our experiment.6. Measurement of myocardial NO content and the concentration of IL-6 and TNFa.Myocardial NO content was measured by Nitric Oxide Colorimetric Assay Kit and myocardial IL-6 and TNFa were measured by ELISA kit.7. Statistical analysisAll statistical analyses involved use of SPSS 18.0. Data are reported as mean± standard deviation. Comparisons between two groups involved in student’s t-test and comparisons among group involved one-way ANOVA. Probabilities of 0.05 or less were considered to be statistically significant.Results1. Tongxinluo decreased systolic blood pressure and cardiac diastolic function in SHRsCompared with the untreated group, systolic blood pressure of SHRs was de-creased significantly in the TXL-treated groups and meanwhile cardiac diastolic func-tion was improved significantly. 2. Tongxinluo decreased cardiac collagen volume fraction and collagen I expres-sion in SHRsCompared with the untreated group, cardiac collagen volume fraction and colla-gen I expression of SHRs was decreased significantly in the TXL-treated groups.3. Tongxinluo increased cardiac microvascular density in SHRsCompared with the untreated group, cardiac microvascular density of SHRs was increased significantly in the TXL-treated groups.4. Tongxinluo decreased cardiac expression of IL-6 and TNFa in SHRsCompared with the untreated group, cardiac IL-6 and TNFa expression of SHRs was decreased significantly in the TXL-treated groups.5. Tongxinluo increased cardiac expression of miR-133a in SHRsCompared with the untreated group, cardiac expression of miR-133a of SHRs was increased significantly in the TXL-treated groups.6. Tongxinluo decreased the phosphorylation of smad3 and the expression of CTGF in SHR hearts.Compared with the untreated group, the phosphorylation of smad3 and the ex-pression of CTGF of SHR heart was decreased significantly in the TXL-treated groups.7. Tongxinluo promoted the phosphorylation of eNOS and decreased the expres-sion of caveolin-1 in SHR hearts.Compared with the untreated group, the expression of caveolin-1 in SHR hearts was increased significantly in the TXL-treated groups. Compared with the untreated group, the phosphorylation of eNOS in SHR hearts was decreased significantly in the TXL-treated groups.8. Tongxinluo increased the content of NO in SHR hearts.Compared with the untreated group, the content of NO in SHR hearts was de-creased significantly in the TXL-treated groups.Conclusion1.Tongxinluo protects against cardiac fibrosis in SHRs; and improves cardiac microvascular rarefaction and inflammation in cardiac microvironment that is favor of fibrogenesis.2.The protective mechanisms of action involves up-regulation of miR-133a,inhibition of pro-fibrotic smad3/CTGF signaling and modulation of cardi-ovascular protective caveolin-1/eNOS.3. The’unblocking collaterals’therapeutical stratege is suit for cardiac fibrosis related to hypertension.Paper III Role and Mechanism of Tongxinluo in HypertensiveBackgroundIn clinic practice, the hypertension-induced renal injury is an important factor in the pathogenesis of end-stage nephropathy and the need for dialysis. Renal injury re-lated to hypertension is characterized by glomerular and tubulointerstitial damages, which eventually lead to renal dysfunction.Gradually increasing blood pressure and activation of the ren-in-angiotensin-aldosterone system are pro-oxidant and pro-inflammatory effects, and the initial factors contributing to renal damage. Oxidative stress plays a critical role in the pathological development of renal injury related to hypertension. Reactive oxygen species (ROS) generated during oxidative stress influence nearly all types of intrinsic kidney cells. In hypertensive kidney damage, oxidative stress determines podocyte apoptosis and generation of segmental glomerulosclerosis, thereby influencing glo-merular permeability. Furthermore, oxidative stress also promotes the accumulation of myofibroblasts via epithelial-mesenchymal transition of proximal tubular and mesangial cells in the kidney, resulting in remodeling of the extracellular matrix of the tubulointerstitium. In addition, oxidative stress and inflammatory responses act synergistically in the pathogenesis of renal injury. Therefore, antioxidant therapy is an important aspect of the therapeutic strategy for hypertensive kidney damage.Among the various signaling pathways activated in response to oxidative stress, the forkhead box O1(FoxO1) transcription factor plays an important role in protecting cells. Under normal and pathological conditions,FoxO1 regulates the expression of specific antioxidant enzymes to protect cells against oxidative stress.FoxO1 also in-hibits epithelial-mesenchymal transition of mesangial cells and secretion of extracel-lular matrix(ECM) protein. Furthermore, post-translational modifications control the function of FoxO1 protein.Tongxinluo (TXL)superfine powder, a traditional Chinese medicinal prescription, has been used clinically for 2 decades to treat a wide range of cardiovascular diseases including angina pectoris and hypertension. Experimental evidence has shown the pleiotropic effects of TXL in animals as well as its antioxidant, anti-inflammatory, and antifibrotic effects in subjects with cardiac and renal injury. However, the thera-peutic effects of TXL in hypertensive kidney damage have not been investigated.Objectives1.To investigate the effects of Tongxinluo on hypertensive kidney structural and functional injury in SHRs;2.To investigate the effects of Tongxinluo on oxidative stress injury and an-ti-oxidant activities in SHR kidneys;3.To investigate whether the mechanisms of Tongxinluo on hypertensive kidney injury are involved in the activation and post-transcription of FoxO1.Methods1.Animal protocolTwenty eight-week-old male SHRs were randomLy assigned to 4 groups(n=10 per group):untreated SHR and TXL.Eight eight-week-old male Wistar Kyoto rats(WKY;Experimental Animal Center of Shandong University, Jinan, China)were used as the control group. The TXL groups were fed intragastrically with 0.4 g-kg-1·d-1. TXL ultrafine powder dissolved in saline for 16 weeks. The WKY and untreated SHR groups were fed intragastrically with equal volumes of saline once daily for 16 weeks.2. Measurement of blood pressureSystolic blood pressure was measured by tail cuff method.3.Measurements of renal functional parametersUrinary albumin was quantified using ELISA.Urinary and serum creatinine were quantified using a Quantichrom Creatinine Assay kit and creatinine clearance was calculated from these data and normalized to kidney weight as an index of glomerular filtration rate.4. Histology and immunohistochemistryTissue was paraffin-embedded and sectioned (4μm) for PSA staining to examine kidney injury and calculate GSI. Immunohistochemistry was used to determine the levels of a-SMA,desmin,fibronectin,collagen IV and CD68 in kidney tissues.5.Measurement of markers of oxidative stress injury.NOX activity, MDA and protein carbonyl were measured as the markers of oxi-dative stress injury.6.Measurement of anti-oxidant activities.The SOD and catalase activities were measured as the anti-oxidant activities in the kidney.7. Real-time RT-PCRTotal RNA was extracted from kidney tissues. In the experiment, the expression of p47 and p67 phox, SOD,catalase,IL-6 and TNF-a were analyzed.8. Western blottingProteins were extracted from kidney tissues. The protein expression of FoxO1,phospho-FoxO1, ERK1/2 phospho-ERK1/2, P38, phospho-P38, PI3K, phos-pho-PI3K, Akt, phospho-Akt, AMPK, phospho-AMPK, SIRT1, TGFβ1, SMAD3, phospho-SMAD3 was analyzed in our experiment.9. Statistical analysisAll statistical analyses involved use of SPSS 18.0. Data are reported as mean± standard deviation. Comparisons between two groups involved in student’s t-test and comparisons among group involved one-way ANOVA. Probabilities of 0.05 or less were considered to be statistically significant.Results1.Tongxinluo decreases systolic blood pressure in SHRsCompared with the untreated SHR, systolic blood pressure of SHRs was de-creased significantly in the treatment group.2. TXL attenuated renal functional injury in SHRs.Compared with the untreated SHR, TXL treatment significantly decreased the urine albumin excretion rate and increased creatinine clearance.3. TXL inhibited oxidative stress injury in SHR kidneys.Compared with the untreated SHR, treatment with TXL significantly decreased the levels of MDA and protein carbonyl in the SHR kidneys and inhibited the expres-sion of NOX subunits p47phox and p67phox, as well as NOX activities.4.Tongxinluo enhanced the anti-oxidant activities in SHR kidneys.Compared with the untreated SHR, treatment with TXL significantly increased the gene expression and activities of SOD and catalase in SHR kidney.5. TXL promoted post-transcription and activation of FoxO1 in SHR kidneys.Compared with untreated SHR, Tongxinluo attenuated the phosphorylation of ERK1/2 and P38 and did not alter the the phosphorylation of AMPK,PI3K and AKT and the expression of SIRT1. Compared with untreated SHR, Tongxinluo significant-ly inhibited the phosphorylation of FoxO1 and promoted its nuclear retention.6. TXL prevented glomerular injury in SHR kidneysCompared with untreated SHR, Tongxinluo attenuated GSI, the expression of desmin, interstitial fibrosis. Compared with untreated SHR, Tongxinluo inhibited the expression of TGF and the phosphorylation of Smad3.7. TXL inhibited inflammatory responses in SHR kidneys.Compared with untreated SHR, CD68 immunopositivity and the expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were decreased in treated SHRs.Conclusion1.Tongxinluo improves structural and functional damage in the hypertensive kidney injury;2.Tongxinluo attenuates oxidative stress and inflammation and enhances the an-ti-oxidant activities;3.Tongxinluo promotes the anti-oxidant signaling FoxO1 by modulating its post-transcription and activation.