Mingjun Zhu

TRPV1 agonism inhibits endothelial cell inflammation via activation of eNOS/NO pathway

BACKGROUND AND AIMS Transient receptor potential vanilloid type 1 channel (TRPV1) is found to be expressed in endothelial cells (ECs) and activate endothelial nitric oxide synthase (eNOS). Recent studies implicate TRPV1 in attenuating inflammatory responses. However, the mechanisms underlying the beneficial effects remain unclear. In this study, we investigated whether TRPV1 suppresses inflammatory responses of ECs via eNOS/NO pathway. METHODS Human umbilical vein endothelial cells (HUVECs) and renal microvascular endothelial cells (MVECs) isolated from deoxycorticosterone (DOCA)-salt hypertensive mice were cultured in the presence of capsaicin (CAP, a specific TRPV1 agonist) with or without the specific inhibitor of TRPV1, NOS, or Ca2+-dependent phosphatidylinositol 3-kinase (PI3K)/Akt pathway, before lipopolysaccharide (LPS) stimulation. NO metabolites, protein expression, and inflammatory molecules were evaluated by Griess assay and immune assay-based multiplex analysis, respectively. Monocyte adhesion was determined by measuring the fluorescently labeled human monocytes attached to LPS-stimulated ECs. RESULTS In HUVECs, treatment with CAP increased NO production, and CAP-induced NO production was accompanied by increased eNOSser1177 phosphorylation. Additionally, CAP attenuated LPS-induced cytokine and chemokine production, adhesion molecule expression, activation of NF-κB, and monocyte adhesion in HUVECs, and these effects were abrogated by the inhibition of TRPV1, NOS, or Ca2+-dependent PI3K/Akt pathway. Moreover, these protective actions of TRPV1 were also observed in renal MVECs isolated from DOCA-salt hypertensive mice. CONCLUSIONS Our results indicate that TRPV1 activation suppresses the inflammatory response of ECs via the activation of Ca2+/PI3K/Akt/eNOS/NO pathway, the protective effects are also documented in ECs derived from salt-sensitive hypertensive mice.

Role of the monocyte chemoattractant protein-1/C-C chemokine receptor 2 signaling pathway in transient receptor potential vanilloid type 1 ablation-induced renal injury in salt-sensitive hypertension

Our recent studies indicate that the transient receptor potential vanilloid type 1 (TRPV1) channel may act as a potential regulator of monocyte/macrophage recruitment to reduce renal injury in salt-sensitive hypertension. This study tests the hypothesis that deletion of TRPV1 exaggerates salt-sensitive hypertension-induced renal injury due to enhanced inflammatory responses via monocyte chemoattractant protein-1 (MCP-1)/C-C chemokine receptor 2 (CCR2)-dependent pathways. Wild type (WT) and TRPV1-null mutant (TRPV1−/−) mice were subjected to uninephrectomy and deoxycorticosterone acetate (DOCA)-salt treatment for four weeks with or without the selective CCR2 antagonist, RS504393. DOCA-salt treatment increased systolic blood pressure (SBP) to the same degree in both strains, but increased urinary excretion of albumin and 8-isoprostane and decreased creatinine clearance with greater magnitude in TRPV1−/− mice compared to WT mice. DOCA-salt treatment also caused renal glomerulosclerosis, tubulointerstitial injury, collagen deposition, monocyte/macrophage infiltration, proinflammatory cytokine and chemokine production, and NF-κB activation in greater degree in TRPV1−/− mice compared to WT mice. Blockade of the CCR2 with RS504393 (4 mg/kg/day) had no effect on SBP in DOCA-salt-treated WT or TRPV1−/− mice compared to their respective controls. However, treatment with RS504393 ameliorated renal dysfunction and morphological damage, and prevented the increase in monocyte/macrophage infiltration, cytokine/chemokine production, and NF-κB activity in both DOCA-salt hypertensive strains with a greater effect in DOCA-salt-treated TRPV1−/− mice compared to DOCA-salt-treated WT mice. No differences in CCR2 protein expression in kidney were found between DOCA-salt-treated WT and TRPV1−/− mice with or without RS504393 treatment. Our studies for the first time indicate that deletion of TRPV1 aggravated renal injury in salt-sensitive hypertension via enhancing MCP-1/CCR2 signaling-dependent inflammatory responses.

Jia-Shen decoction-medicated serum inhibits angiotensin-II induced cardiac fibroblast proliferation via the TGF-β1/Smad signaling pathway

Jia-Shen decoction (JSD) is a traditional Chinese medicine, which is used widely to treat chronic heart failure. However, the underlying mechanism remains to be elucidated. The present study aimed to investigate the mechanism underlying the effects of JSD on cardiac fibroblast (CF) proliferation and differentiation. The CFs were obtained from the hearts of neonatal (1-3-day old) Sprague-Dawley rats and treated with JSD-medicated serum (JSDS) with or without angiotensin II (Ang II). Cell proliferation was assessed using Cell Counting Kit-8 reagent. In addition, the mRNA expression levels of transforming growth factor-β1 (TGF-β1) and phosphorylated small mothers against decapentaplegic (p-Smad)2/3 and their protein expression levels were analyzed. CF proliferation was significantly increased in the Ang II-treated group, compared with the control group (P<0.05). The expression levels of collagen, α-smooth muscle actin, TGF-β1 and p-Smad2/3 were also increased in the Ang II-treated group (P<0.05). Following JSDS treatment, the increased levels of collagen and cell proliferation were inhibited, and the increased expression levels of p-Smad2 and p-Smad3 were also inhibited (P<0.05). These data suggested that JSDS may inhibit CF proliferation via attenuating the TGF-β1/Smad signaling pathway.

GW24-e2125 PROTECTIVE EFFECTS OF A PRESCRIPTION OF JIASHEN ON MYOCARDIAL INFARCTION VIA INHIBITION OF INFLAMMATION IN RATS

Objectives To determine the role of inflammation in myocardial protection induced by a prescription of Jiashen (PJS) in the early period of myocardial infarction (MI) in rats. Methods MI was induced by the ligation of left anterior descending coronary artery in Sprague-Dawley rats. The rats were divided into five groups: sham-operated group; MI + vehicle group; PJS-3g (3 g/kg/day) group; PJS-6g (6g/kg/day) group and losartan (10 mg/kg/day) group. Infarct size (IS) was determined by Evans blue and 2,3,5-Triphenyltetrazolium chloride (TTC) 3 days after MI; the left ventricular structure and function were measured by echocardiography performed 1 week after MI; and myocardial inflammatory mediators including tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1) were measured by ELISA. Results Compared with the MI + vehicle group, treatment with PJS at the dose of 6 g/kg/day reduced myocardial IS (P < 0.05), left ventricular end diastolic diameter (LVEDD) and left ventricular end systolic diameter (LVESD) (P < 0.01), and enhanced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (P < 0.01). Compared with the MI + vehicle group, administration of PJS dose-dependently attenuated the increased levels of TNF-α, IL-1β and MCP-1 in ischaemic myocardium (P < 0.01). PJS at the dose of 6 g/kg/day had equally effectiveness with losartan. Conclusions Our studies showed that consistent with losartan-induced cardioprotection, PJS administered after MI reduced myocaidial IS and improved cardiac function that was associated with the decreased inflammatory mediators. The data indicate that PJS exert its cardioprotection possibly via inhibiting inflammatory response.

GW24-e3612 Effects of prescription of Jiashen on ventricular remodelling and infiltration of monocyte/macrophages in the early period after myocardial infarction in rats

Objectives This study was designed to test the hypothesis that PJS modulates inflammatory processes to prevent cardiac functional deterioration and reduce ventricular remodelling after MI. Methods Male Sprague-Dawley rats (9-10 weeks) were subjected to sham-MI or MI by ligating the left anterior descending coronary artery for 1 and 4 weeks. The rats were divided into five groups: sham, MI, PJS (3 g/kg/day), PJS (6 g/kg/day), and losartan (an AT1 antagonist, 10 mg/kg/day). The vehicle, PJS, or losartan was given by oral gavage once a day after MI. Cardiac functions were determined by echocardiographic measurements at 1 and 4 weeks after MI. Fibrinogen at the site of infarction and in ischaemic myocardium were determined by Masson staining. Results of quantitative analysis were used to detect the level of hydroxyproline. Monocyte/macrophages expression was detected by immunohistochemistry staining and quantitative analysis. Results 1) The echocardiographic measurement showed that Both LVEDd and LVESd in the PJS-6 and Losartan groups were significantly shorter than in the MI group at both week 1 and week 4 post-MI. Both FS and EF were well-maintained in the PJS-6 and Losartan groups than in the MI group at both week 1 and week 4 post-MI. 2) Masson trichrome staining of cardiac sections: At 1 and 4 weeks after myocardial infarction, fibrinogen proliferation was much more obvious in MI group than in sham operation group.a detectable reduction of fibrinogen with PJS-6 and Losartan groups at the site of infarction was not found but was reduced at ischaemic sites. Results of quantitative analysis shows: compared to the MI group, the PJS-6 and losartan groups significantly decrease at both the infarction and ischaemia areas the level of fibrinogen. 3) Immunohistochemical results of monocyte/macrophages shows: At 1 and 4 weeks after myocardial infarction, monocyte/macrophages was much more obvious in MI group than in sham operation group compared to the MI group, the PJS-6 and losartan groups significantly decrease at both the infarction and ischaemia areas of monocyte/macrophages. Conclusions Our studies demonstrate that the PJS improves cardiac function, inhibits cardiac remodelling and suppresses infiltration of monocyte/macrophages after myocardial infarction. The data indicate that PJS inhibits its ventricular remodelling possibly via inhibiting infiltration of monocyte/macrophages. The results suggest that PJS may have a promising potential for the prevention and treatment of MI.

GW24-e2192 NK-1 Receptor-mediated Renal Injury during DOCA-salt Hypertension

Objectives Salt-sensitive hypertension is a leading risk factor contributing to the development of chronic renal disease. We found that high-salt intake activated sensory nerves, leading to release of sensory neuropeptides including substance P (SP). It is well established that the neurokinin-1 receptor (NK-1R) is activated preferentially by SP. This study was designed to determine whether activation of NK-1R contributes to the development of renal injury during DOCA-salt hypertension. Methods We induced salt-sensitive hypertension by uninephrectomy and deoxycorticosterone (DOCA)-salt in C57BL/6 mice with or without selective NK1 antagonists. Results DOCA-salt treatment for 5 weeks increased mean arterial pressure (MAP) determined by the telemetry system (144 ± 3 vs. 108 ± 4 mmHg, P < 0.01) and renal hypertrophy (116.3 ± 2.7 vs. 75.7 ± 1.4 mg/10 g of BW, P < 0.01) compared with sham mice. We also found that urinary 8-isoprostane and albumin excretion were increased in DOCA-salt mice compared with sham mice (1.76 ± 0.16 vs. 0.49 ± 0.10 ng/24 h; 41.1 ± 3.5 vs. 5.8 ± 0.6 μg/24 h, P < 0.05). Periodic acid-Schiff and Masson’s trichrome staining showed that DOCA-salt treatment caused obvious glomerulosclerosis and tubulointerstitial injury in the renal cortex (0.62 ± 0.06 vs. 0.08 ± 0.01; 3.00 ± 0.30 vs. 0.21 ± 0.10, P < 0.05), and tubulointerstitial fibrosis compared with the sham mice. Consistent with the results, renal collagen level determined using hydroxyproline assay was higher in DOCA-salt treated mice compared with the sham mice (26.8 ± 2.4 vs. 12.2 ± 1.3 μg/mg dry tissue, P < 0.05). In addition, F4/80-staining showed that interstitial monocyte/macrophage infiltration were greater in DOCA-salt mice compared with sham mice (61 ± 4 vs. 9 ± 2 cells/mm2, P < 0.05). Blockade of the NK-1R with RP-67580 (8 mg/kg/day, ip) or L-733,060 (20 mg/kg/day, ip) had no effect on MAP and renal hypertrophy. However, the NK-1R antagonists attenuated renal morphological injury, interstitial monocyte/macrophage infiltration, and the increase in renal collagen, and reduced the increase in urinary 8-isoprostane and albumin excretion in DOCA-salt treated mice (P < 0.05). Conclusions Our study showed that blockade of NK-1R with RP-67580 or L-733,060 attenuated renal injury induced by DOCA-salt hypertension independently of their effects on blood pressure. The results suggest that activation of NK-1R, possibly by SP, may contribute to renal injury during DOCA-salt hypertension. This work was supported by the National Natural Science Foundation of China (No. 81170243).