CTRP3 attenuates post-infarct cardiac fibrosis by targeting Smad3 activation and inhibiting myofibroblast differentiation.

UNLABELLED: C1q/tumor necrosis factor-related protein-3 (CTRP3) is a novel adipokine with modulation effects on metabolism, inflammation, and cardiovascular system. This study aimed to investigate the effect of CTRP3 on cardiac fibrosis and its underlying mechanism. The myocardial expression of CTRP3 was significantly decreased after myocardial infarction (MI). Adenovirus-delivered CTRP3 supplement attenuated myocardial hypertrophy, improved cardiac function, inhibited interstitial fibrosis, and decreased the number of myofibroblasts post-MI. In cultured adult rat cardiac fibroblasts (CFs), CTRP3 attenuated cell proliferation; migration; and the expression of connective tissue growth factor, collagen I, and collagen III induced by transforming growth factor (TGF)-ß1. Moreover, CTRP3 inhibited whereas CTRP3 small interfering RNA (siRNA) facilitated the expression of α-SMA and profibrotic molecules induced by TGF-ß1. CTRP3 also attenuated TGF-ß1-induced Smad3 phosphorylation, nuclear translocation, and interaction with p300. CTRP3 increased the phosphorylation of AMP-activated protein kinase (AMPK) and Akt in both rat hearts and CFs. Adenine 9-ß-D-arabinofuranoside (AraA), an AMPK inhibitor, abolished the protective effect of CTRP3 against TGF-ß1-induced profibrotic response and Smad3 activation. Taken together, CTRP3 attenuates cardiac fibrosis by inhibiting myofibroblast differentiation and the subsequent extracellular matrix production. AMPK is required for the anti-fibrotic effect of CTRP3 through targeting Smad3 activation and inhibiting myofibroblast differentiation. KEY MESSAGE: CTRP3 alleviates cardiac fibrosis in a rat post-MI model and in cardiac fibroblasts. CTRP3 inhibits fibroblast-to-myofibroblast differentiation. CTRP3 exerts anti-fibrotic effect through targeting Smad3 activation. AMPK mediates the anti-fibrotic effect of CTRP3 by inhibition of Smad3 activation.

C1q/tumor necrosis factor-related protein-6 attenuates post-infarct cardiac fibrosis by targeting RhoA/MRTF-A pathway and inhibiting myofibroblast differentiation.

C1q/tumor necrosis factor-related protein-6 (CTRP6) is a newly identified adiponectin paralog with modulation effects on metabolism and inflammation. However, the cardiovascular function of CTRP6 remains unknown. This study aimed to determine its role in cardiac fibrosis and explore the possible mechanism. Myocardial infarction (MI) was induced by left anterior descending coronary artery ligation in rats. CTRP6 was mainly expressed in the cytoplasm of adult rat cardiomyocytes and significantly decreased in the border and infarct zones post-MI. Adenovirus-mediated CTRP6 delivery improved cardiac function, attenuated cardiac hypertrophy, alleviated cardiac fibrosis, and inhibited myofibroblast differentiation as well as the expression of collagen I, collagen III, and connective tissue growth factor post-MI. In cultured adult rat cardiac fibroblasts (CFs), exogenous or cardiomyocyte-secreted CTRP6 inhibited, whereas knockdown of CTRP6 facilitated transforming growth factor-ß1 (TGF-ß1)-induced expression of α-smooth muscle actin, smooth muscle 22α, and profibrotic molecules. CTRP6 had no effect on CFs proliferation but attenuated CFs migration induced by TGF-ß1. CTRP6 increased the phosphorylation of AMP-activated protein kinase (AMPK) and Akt in CFs and post-MI hearts. Pretreatment with adenine 9-ß-D-arabinofuranoside (AraA), an AMPK inhibitor, or LY294002, a phosphatidylinositol-3-kinase (PI3 K) inhibitor, abolished the protective effect of CTRP6 on TGF-ß1-induced profibrotic response. Furthermore, CTRP6 had no effect on TGF-ß1-induced Smad3 phosphorylation and nuclear translocation, whereas significantly decreased TGF-ß1-induced RhoA activation and myocardin-related transcription factor-A (MRTF-A) nuclear translocation, and these effects were blocked by AMPK or Akt inhibition. In conclusion, CTRP6 attenuates cardiac fibrosis via inhibiting myofibroblast differentiation. AMPK and Akt activation are responsible for the CTRP6-mediated anti-fibrotic effect by targeting RhoA/MRTF-A pathway.

Angiotensin II reduces cardiac AdipoR1 expression through AT1 receptor/ROS/ERK1/2/c-Myc pathway.

Adiponectin, an abundant adipose tissue-derived protein, exerts protective effect against cardiovascular disease. Adiponectin receptors (AdipoR1 and AdipoR2) mediate the beneficial effects of adiponectin on the cardiovascular system. However, the alteration of AdipoRs in cardiac remodeling is not fully elucidated. Here, we investigated the effect of angiotensin II (AngII) on cardiac AdipoRs expression and explored the possible molecular mechanism. AngII infusion into rats induced cardiac hypertrophy, reduced AdipoR1 but not AdipoR2 expression, and attenuated the phosphorylations of adenosine monophosphate-activated protein kinase and acetyl coenzyme A carboxylase, and those effects were all reversed by losartan, an AngII type 1 (AT1) receptor blocker. AngII reduced expression of AdipoR1 mRNA and protein in cultured neonatal rat cardiomyocytes, which was abolished by losartan, but not by PD123319, an AT2 receptor antagonist. The antioxidants including reactive oxygen species (ROS) scavenger NAC, NADPH oxidase inhibitor apocynin, Nox2 inhibitor peptide gp91 ds-tat, and mitochondrial electron transport chain complex I inhibitor rotenone attenuated AngII-induced production of ROS and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. AngII-reduced AdipoR1 expression was reversed by pretreatment with NAC, apocynin, gp91 ds-tat, rotenone, and an ERK1/2 inhibitor PD98059. Chromatin immunoprecipitation assay demonstrated that AngII provoked the recruitment of c-Myc onto the promoter region of AdipoR1, which was attenuated by PD98059. Moreover, AngII-induced DNA binding activity of c-Myc was inhibited by losartan, NAC, apocynin, gp91 ds-tat, rotenone, and PD98059. c-Myc small interfering RNA abolished the inhibitory effect of AngII on AdipoR1 expression. Our results suggest that AngII inhibits cardiac AdipoR1 expression in vivo and in vitro and AT1 receptor/ROS/ERK1/2/c-Myc pathway is required for the downregulation of AdipoR1 induced by AngII.

Ischemic preconditioning reduces transplanted submandibular gland injury.

BACKGROUND: Ischemic preconditioning (IPC) can reduce ischemia/reperfusion (I/R) injury in multiple organs and species. However, the effect of IPC on transplanted submandibular glands remains unknown. We explored the protection of IPC in transplanted submandibular glands in the rabbit and the underlying mechanism. METHODS: IPC was performed by clamping the lingual artery for 10 min, with 10 min of reperfusion before transplantation. Male rabbits were randomly divided into control, transplantation, and IPC groups (n = 6 each). Saliva secretion, oxidative stress, pro-inflammatory cytokine levels, and apoptosis-related protein levels were determined at 1, 12, and 24 h after reperfusion. RESULTS: Salivary flow was significantly increased at 12 h and decreased at 24 h in the transplanted glands. IPC treatment prevented the reduced saliva secretion at 24 h after reperfusion (P < 0.01). The mRNA levels of tumor necrosis factor-α, interleukin-1ß, and reactive oxygen species, as well as malondialodehyde (MDA) and myeloperoxidase activity, were significantly increased and superoxide dismutase activity was decreased in the transplanted glands. However, these changes were all attenuated with IPC treatment (all P < 0.05). Also, acinar cell apoptosis and Bax protein expression were decreased and Bcl-2 protein expression was increased in the IPC-treated glands at 1 and 12 h after reperfusion (all P < 0.05). CONCLUSIONS: IPC protects the secretory function of transplanted submandibular gland in the rabbit by reducing the inflammatory response, attenuating oxidative stress, and an anti-apoptosis process.

Insulin decreases myocardial adiponectin receptor 1 expression via PI3K/Akt and FoxO1 pathway.

AIMS: Adiponectin is considered an important adipokine protecting against diabetes, atherosclerosis, and cardiovascular disease. Because adiponectin receptors (AdipoRs) are critical components in the adiponectin signalling cascade, we investigated the effect of insulin on the expression of myocardial AdipoRs and explored the possible molecular mechanism. METHODS AND RESULTS: The hyperinsulinaemia rat model was induced by infusion of insulin (1 U/day) for 28 days: serum and myocardial adiponectin levels were increased, and skeletal muscle and myocardial AdipoR1 expression and AMP-activated protein kinase (AMPK) phosphorylation were decreased. In primary cultured neonatal rat ventricular myocytes (NRVMs), insulin decreased AdipoR1 but not AdipoR2 expression and AMPK phosphorylation; high glucose had no affect on AdipoRs expression. Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was increased in insulin-treated hearts and in NRVMs. P13K inhibitor LY294002 and Akt1/2 kinase inhibitor but not the ERK1/2 kinase (MEK) inhibitors PD98059 and U0126 blocked the insulin-induced reduction in AdipoR1 expression and AMPK phosphorylation. Insulin induced forkhead/winged helix box gene group O-1 (FoxO1) phosphorylation and translocation from the nucleus to the cytosol, and this was blocked by LY294002. FoxO1 small interfering RNA reduced AdipoR1 expression and AMPK phosphorylation. In electrophoretic mobility shift assay and chromatin immunoprecipitation, FoxO1 bound to the putative site from -167 to -157 bp of the AdipoR1 promoter both in vitro and in living cells; insulin suppressed this binding, which was blocked by LY294002. CONCLUSION: Insulin inhibits myocardial AdipoR1 expression via PI3K/Akt and FoxO1 pathways, and FoxO1 mediates AdipoR1 transcription by binding to its promoter directly.

[Effect of parasympathectomy on the salivary secretion of submandibular gland in rats].

OBJECTIVE: To investigate the effect of parasympathectomy on secretion of submandibular glands and the feasibility of treatment for xerostomia in rats. METHODS: Twenty Sprague-Dawley male rats weighing 200 - 300 g were randomly divided into the experimental group (n = 12), in which the right chorda-lingual nerve was cut, and the control group (n = 8). The secretion of submandibular gland was measured for 5 min by Schirmer test for both groups. RESULTS: The stimulated saliva flow rate decreased on 1st, 12th and 24th week after denervation in the right operated submandibular glands (P < 0.05). No difference in secretion was found between the left non-operated glands and the control group. Comparing with the left non-operated gland and the control gland, the saliva flow rate at rest in the right operated submandibular gland increased on the 1st, 12th and 24th week (P < 0.05). CONCLUSIONS: After parasympathectomy of rat submandibular glands, the saliva flow rate at rest increased in the denervated gland, which suggests that parasympathectomy of submandibular gland might be used as a therapy for xerostomia.

[Effects of baicalin on pulmonary functions of acute respiratory distress syndrome induced by oleic acid in rats].

OBJECTIVE: To investigate the effect of baicalin on pulmonary functions and its mechanism during the development of acute respiratory distress syndrome (ARDS) induced by oleic acid (OA) in rats. METHODS: Rats were randomized into 5 groups: control, ARDS (OA induction, 0.12 mg/kg), baicalin-treated group (150 mg/kg), baicalin-treated group (300 mg/kg) and baicalin-treated group (450 mg/kg). The blood samples and lung tissue were collected at 10 min, 1, 2 and 6 h after OA injection. The lung concentration of myeloperoxidase (MPO) was detected by an ELISA (enzyme-linked immunosorbent assay) kit. Meanwhile, blood gas analysis and pulmonary pathological examination were also performed. RESULTS: The level of arterial oxygen partial pressure and oxygenation index decreased (P < 0.01 vs. control) and oxygenation index (190 mm Hg, 1 mm Hg = 0.133 kPa) reached the diagnostic standard of ARDS at 2 h in ARDS group. In baicalin-treated group (150 mg/kg and 300 mg/kg), the level of arterial oxygen partial pressure and oxygenation index increased versus the ARDS group. In baicalin-treated group (450 mg/kg), the level of arterial oxygen partial pressure was undifferentiated at 1, 2 and 6 h (P > 0.05) and decreased at 10 min (46.8 mm Hg, P < 0.05) versus the ARDS group. The level of MPO increased in baicalin-treated (300 mg/kg) and ARDS groups. Compared with the ARDS group, the level of MPO decreased significantly in baicalin-treated group (300 mg/kg) at 10 min, 1 and 2 h. Meanwhile, the pulmonary pathological damage improved in baicalin-treated group (300 mg/kg). CONCLUSION: An appropriate dose of baicalin may improve hypoxemia of ARDS induced by OA in rats. It may be due to the inhibition of MPO activity.

[Effects of low-G preconditioning on contractive ability of left ventricle and contents of endothelin and prostacycline in myocardium of rats after repeated +10 Gz stress].

OBJECTIVE: To further test the protective effect of low-G pre-conditioning on +Gz stress induced cardiac damage. METHOD: Forty-two male Wistar rats were randomly divided into 3 groups (n=14 for each group): group A (+1 Gz group), group B (+10 Gz group) and group C (low-G pre-conditioning group). The rats in group B were exposed to five +10 Gz plateaus of 30 s with 1 min intervals at +1 Gz, 3 times a week, for 3 weeks; while group A were only submitted to +1 Gz for 5 min. The rats in group C were exposed to +2 Gz for 5 min about 1 h prior to +10 Gz stress. All the animals were decapitated in the next day after the last centrifuge run and function of the isolated working hearts and contents of endothelin and prostacycline in the myocardium were observed. RESULT: Compared with the control, a significant decrease of left ventricular systolic pressure (LVSP) and increase of myocardial contents of endothelin in rats were observed after repeated +10 Gz exposure; but low-G pre-conditioning could remarkably elevate the LVSP and reduce the myocardial endothelin level in rats after repeated +10 Gz stress. CONCLUSION: Low-G pre-conditioning has a significant protective effect on contractive ability of the left ventricle and secretion function of the vascular endothelium in rat heart after high G stress.