DR1-CSE/H2S pathway upregulates autophagy and inhibits H9C2 cells damage induced by high glucose.

In the cardiovascular system, long-term high glucose (HG) can lead to cardiomyocyte damage. Hydrogen sulfide (H2S) reduces cell autophagy in cardiomyocytes. Dopamine 1 receptors (DR1), a specific binding receptor for dopamine, which has a significant regulatory effect on cardiomyocytes. However, it is unclear whether DR1 inhibits HG-induced cardiomyocyte damage by regulating endogenous H2S production and the level of cell autophagy. The present data indicated that the expression of DR1 and cystathionine-γ-lyase (CSE, a key enzyme for endogenous H2S production) and H2S content were significantly reduced in HG-induced cardiomyocytes, which was reversed by SKF38393 (an agonist of DR1). NaHS (an exogenous H2S donor) only increased H2S content and the expression of CSE with no effect on DR1 expression. HG reduced cell viability, the expression of Bcl-2 and Beclin1, the production of autophagosomes and LC3 II/I ratio and increased the cell apoptotic ratio, the expression of cleaved caspase-3, cleaved caspase-9, cytochrome c, P62, and p-mTOR/t-mTOR ratio. SKF38393 and NaHS reversed the effects of HG. PPG (an inhibitor of CSE) and 3MA (an inhibitor of autophagy) abolished the beneficial effect of SKF38393. In addition, AICAR (an agonist of AMPK) and Rapamycin (an inhibitor of mTOR) increased the production of autophagosomes but decreased the p-mTOR/t-mTOR ratio, which was similar to the effects of SKF38393 and 3MA. Our findings suggest that DR1 reduces the HG-induced cardiomyocyte damage via up-regulating the CSE/H2S pathway, which increases cell autophagy by inhibiting the activation of mTOR.

Exogenous hydrogen sulfide inhibits the senescence of cardiomyocytes through modulating mitophagy in rats.

Hydrogen sulfide (H2S), a gaseous molecule, has been shown to be involved in the regulation of body pathophysiological processes. Aging is related to structural and functional alterations within the heart. There is evidence that diminished mitophagy accelerates the aging process. Studies in recent years have revealed that plasma levels of H2S in humans and old rats decrease with age, and H2S acts as a cytoprotective mediator in the aging process. However, it is unclear whether H2S can delay the senescence of cardiomyocytes by regulating mitophagy. Our present results showed that exogenous H2S inhibited mitochondrial damage, oxidative stress and cell apoptosis, and enhanced mitophagy through upregulating the SIRT1-PINK1-parkin pathway in myocardial tissues of aged rats and cultured aged cardiomyocytes. Furthermore, the effect of exogenous H2S on the above indicators was the same as that of SRT1720 (a SIRT1 agonist) and kinetin (a PINK1 activator). Our findings suggest that exogenous H2S inhibits the senescence of cardiomyocytes by increasing mitophagy via upregulation of the SIRT1-PINK1-parkin pathway in rats.

DR1 Activation Inhibits the Proliferation of Vascular Smooth Muscle Cells through Increasing Endogenous H2S in Diabetes.

Tissue ischemia and hypoxia caused by the abnormal proliferation of smooth muscle cells (SMCs) in the diabetic state is an important pathological basis for diabetic microangiopathy. Studies in recent years have shown that the chronic complications of diabetes are related to the decrease of endogenous hydrogen sulfide (H2S) in diabetic patients, and it has been proven that H2S can inhibit the proliferation of vascular SMCs (VSMCs). Our study showed that the endogenous H2S content and the expression of cystathionine gamma-lyase (CSE), which is the key enzyme of H2S production, were decreased in arterial SMCs of diabetic mice. The expression of PCNA and Cyclin D1 was increased, and the expression of p21 was decreased in the diabetic state. After administration of dopamine 1-like receptors (DR1) agonist SKF38393 and exogenous H2S donor NaHS, the expression of CSE was increased and the change in proliferation-related proteins caused by diabetes was reversed. It was further verified by cell experiments that SKF38393 activated calmodulin (CaM) by increasing the intracellular calcium ([Ca2+]i) concentration, which activated the CSE/H2S pathway, enhancing the H2S content in vivo. We also found that SKF38393 and NaHS inhibited insulin-like growth factor-1 (IGF-1)/IGF-1R and heparin-binding EGF-like growth factor (HB-EGF)/EGFR, as well as their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways. Taken together, our results suggest that DR1 activation up-regulates the CSE/H2S system by increasing Ca2+-CaM binding, which inhibits the IGF-1/IGF-1R and HB-EGF/EGFR pathways, thereby decreasing their downstream PI3K/Akt, JAK2/STAT3 and ERK1/2 pathways to achieve the effect of inhibiting HG-induced VSMCs proliferation.

Dopamine 1 receptors inhibit apoptosis via activating CSE/H2 S pathway in high glucose-induced vascular endothelial cells.

High glucose (HG)-induced dysfunction of vascular endothelial cells plays a crucial role in the development of diabetic vascular complications. Inhibition of cystathionine γ-synthase/hydrogen sulfide (CSE/H2 S) pathway is one of the causes of vascular endothelial cell injury induced by HG. Dopamine D1 receptors (DR1) are widely expressed and regulate important physiological functions in the vascular system. However, the effect of DR1 inhibition on HG-induced vascular endothelial apoptosis by regulating the CSE/H2 S pathway is unclear. Therefore, we aimed to determine if DR1 can regulate the CSE/H2 S pathway and regulate the effect of DR1 on HG-induced apoptosis in human umbilical vein endothelial cells. In this study, we found that HG treatment significantly decreased the expression of DR1 and CSE and the endogenous content of H2 S; DR1 agonist SKF 38393 reversed these effects, while sodium hydrosulfide (NaHS) only increased CSE expression and the endogenous H2 S production and had no effect on DR1 expression. Meanwhile, HG significantly increased the intracellular calcium concentration ([Ca2+ ]i ), and SKF 38393 further increased HG-induced [Ca2+ ]i . In addition, HG increased the lactate dehydrogenase activity, malondialdehyde and reactive oxygen species contents, apoptotic rate, the expression of cleaved caspase-3, caspase-9, and cytochrome c, and the activity of phosphorylated-inhibitor of nuclear factor-kappaBα (NF-κBα) (p-IκBα) and phosphorylated-NF-κB (p-NF-κB), and reduced cell viability, superoxide dismutase activity, and Bcl-2 expressions. SKF 38393 and NaHS markedly reversed the effect of HG. The effect of SKF 38393 was similar to N-acetyl- l-cysteine (an inhibitor of oxidative stress) or pyrrolidinedithiocarbamate ammonium (an NF-kB inhibitor). Taken together, DR1 upregulates the CSE/H2 S pathway by increasing the [Ca2+ ]i , which inhibits HG-induced apoptosis via downregulating NF-κB/IκBα pathway in vascular endothelial cells.

SKF38393 prevents high glucose (HG)-induced endothelial dysfunction by inhibiting the effects of HG on cystathionine γ-lyase/hydrogen sulfide activity and via a RhoA/ROCK1 pathway.

BACKGROUND: Endothelial dysfunction plays a crucial role in diabetic vascular complications. A decrease in hydrogen sulfide (H2S) levels is increasingly becoming a vital factor contributing to high glucose (HG)-induced endothelial dysfunction. Dopamine D1-like receptors (DR1) activation has important physiological functions in the cardiovascular system. H2S decreases the dysfunction of vascular endothelial cells. However, no studies have reported whether DR1 protects the function of vascular endothelial cells by regulating H2S levels. AIM: The present study aimed to determine whether DR1 regulates the levels of endogenous H2S, which exerts protective effects against HG-induced injury of human umbilical vein endothelial cells (HUVECs) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing kinase 1 (ROCK1) signalling. METHODS: HUVECs were exposed to HG (30 mM) or normal glucose (5.5 mM) after different treatments. Cell viability, proliferation and migration were measured by Cell Counting Kit-8, EdU cell proliferation assay, transwell assay and wound healing assay, respectively. H2S probe (7-Azido-4-Methylcoumarin) was used to detect levels of H2S. The intracellular calcium concentration ([Ca2+]i) were measured using Fluo-4 AM. The protein expressions were quantified by Western blot. RESULTS: We found that HG decreased the expression of DR1 and cystathionine γ-lyase (CSE) and H2S production. The DR1 agonist SKF38393 significantly increased DR1 and CSE expression and H2S production, whereas NaHS (a H2S donor) only increased CSE expression and H2S production but had no effect on DR1 expression. Meanwhile, SKF38393 further increased the [Ca2+]i induced by HG. In addition, HG reduced cell viability and the expression of Cyclin D1 and proliferating cell nuclear antigen and increased the expression of p21C⁢i⁢p/W⁢A⁢F-1, collagen I, collagen III, matrix metalloproteinase 9, osteopontin and α-smooth muscle actin and the activity of phosphorylated RhoA and ROCK1. SKF38393 and NaHS reversed these effects of HG. PPG (a CSE inhibitor) abolished the beneficial effect of SKF38393. These effects of SKF38393 were similar to those of Y-27632 (a ROCK inhibitor). CONCLUSION: Taken together, our results suggest that DR1 activation upregulates the CSE/H2S pathway by increasing the [Ca2+]i, which protects endothelial cells from HG-induced injury by inhibiting the RhoA/ROCK1 pathway.

DR1 activation promotes vascular smooth muscle cell apoptosis via up-regulation of CSE/H2 S pathway in diabetic mice.

The important role of hydrogen sulfide (H2 S) as a novel gasotransmitter in inhibiting proliferation and promoting apoptosis of vascular smooth muscle cells (VSMCs) has been widely recognized. The dopamine D1 receptor (DR1), a G protein coupled receptor, inhibits atherosclerosis by suppressing VSMC proliferation. However, whether DR1 contributes to VSMC apoptosis via the induction of endogenous H2 S in diabetic mice is unclear. Here, we found that hyperglycemia decreased the expressions of DR1 and cystathionine-γ-lyase (CSE, a key enzyme for endogenous H2 S production) and reduced endogenous H2 S generation in mouse arteries and cultured VSMCs. DR1 agonist SKF38393 increased DR1 and CSE expressions and stimulated endogenous H2 S generation. Sodium hydrosulfide (NaHS, a H2 S donor) increased CSE expressions and H2 S generation but had no effect on DR1 expression. In addition, high glucose (HG) increased VSMC apoptosis, up-regulated IGF-1-IGF-1R and HB-EGF-EGFR, and stimulated ERK1/2 and PI3K-Akt pathways. Overexpression of DR1, the addition of SKF38393 or supply of NaHS further promoted VSMC apoptosis and down-regulated the above pathways. Knock out of CSE or the addition of the CSE inhibitor poly propylene glycol diminished the effect of SKF38393. Moreover, calmodulin (CaM) interacted with CSE in VSMCs; HG increased intracellular Ca2+ concentration and induced CaM expression, further strengthened the interaction of CaM with CSE in VSMCs, which were further enhanced by SKF38393. CaM inhibitor W-7, inositol 1,4,5-trisphosphate (IP3 ) inhibitor 2-APB, or ryanodine receptor inhibitor tetracaine abolished the stimulatory effect of SKF38393 on CaM expression and intracellular Ca2+ concentration. Taken together, these results suggest that DR1 up-regulates CSE/H2 S signaling by inducing the Ca2+ -CaM pathway followed by down-regulations of IGF-1-IGF-1R and HB-EGF-EGFR and their downstream ERK1/2 and PI3K-Akt, finally promoting the apoptosis of VSMCs in diabetic mice.

The DR1­CSE/H2S system inhibits renal fibrosis by downregulating the ERK1/2 signaling pathway in diabetic mice.

Glomerular mesangial cell (MC) proliferation and extracellular matrix deposition are the main pathological changes in diabetic nephropathy. Hydrogen sulfide (H2S) inhibits the proliferation of MCs. Dopamine 1 receptors (DR1) are expressed in MCs and serve important physiological roles. However, it is unclear whether DR1 activation inhibits MC proliferation by increasing endogenous H2S. The present study found that the production of H2S and the expression of DR1 and cystathionine­Î³­lyase (CSE) were decreased in the renal tissues of diabetic mice and high glucose (HG)­induced MCs. SKF38393 (a DR1 agonist) increased the production of H2S and the expression of DR1 and CSE and NaHS (an exogenous H2S donor) only increased H2S production and CSE expression but not DR1 expression. HG increased the thickness of the glomerular basement membrane, cell viability and proliferation, the expression of cyclin D1, PCNA, collagen 1 and α­smooth muscle actin and the activity of phosphorylated ERK1/2 and decreased the expression of P21 and MMP9. SKF38393 and NaHS reversed the effects of HG. PPG (a CSE inhibitor) abolished the beneficial effects of SKF38393. The beneficial effects of SKF38393 were similar to those of PD98059 (an ERK1/2 inhibitor). Taken together, the findings suggested that the DR1­CSE/H2S pathway activation attenuated diabetic MC proliferation and extracellular matrix deposition by downregulating the ERK1/2 signaling pathway.

[Effects of exogenous H2S on hepatic fibrosis in diabetic mice and its mechanism].

OBJECTIVE: To investigate the effects of exogenous hydrogen sulfide (H2S) on the hepatic fibrosis in diabetic mice and its mechanism. METHODS: Twenty-four C57 male mice (weight 22±2 g) were randomly divided into three groups (n=8): ① Normal control group (Control): Mice were intraperitoneally injected equal amount of normal saline, the injection time was the same as that of the experimental groups; ② Diabetes model groups (HG): Streptozotocin (STZ) was injected intraperitoneally once according to body weight (150 mg/kg) to establish diabetes model; ③ NaHS treatment groups (HG + NaHS): Mice were intraperitoneally injected with NaHS (100 µmol/L·kg·d) once a day for 12 consecutive weeks. The hepatocyte injury was detected by HE staining; the hepatic fibrosis was observed through Masson staining; the protein expressions of cystathionine - ß - synthetase (CBS), collagen-I (CoL-I), collagen-III (CoL-III) and matrix metalloproteinase-9 (MMP-9) were detected by Western blot. RESULTS: Compared with the control group, the damage and fibrosis of hepatocyte were significantly aggravated, the expression of CBS proteins was decreased (P＜0.01), and the expression levels of CoL-I, CoL-III and MMP-9 proteins were increased (P＜0.01) in the diabetic model group. Compared with the diabetic model group, the damage and fibrosis of hepatocyte were significantly lightened, the expression of CBS proteins was obviously increased (P＜0.01), and the expression levels of CoL-I, CoL-III and MMP-9 proteins were markedly decreased (P＜ 0.01). CONCLUSION: H2S inhibits the hepatic fibrosis in diabetic mice, and its mechanism is related to the decrease of collagen and matrix metalloproteinase-9.

Reversal of Multidrug Resistance in Cancer by Multi-Functional Flavonoids.

Multidrug resistance (MDR) resulting from different defensive mechanisms in cancer is one of the major obstacles of clinical treatment. To circumvent MDR many reversal agents have been developed, but most of them fail in clinical trials due to severely adverse effects. Recently, certain natural products have been reported to overcome MDR, including flavonoids which are abundant in plants, foods, and herbs. The structure of flavonoids can be abbreviated as C6-C3-C6 (C for carbon), and further categorized into flavonoids, iso-flavonoids and neo-flavonoids, according to their structural backbones. Flavonoids possess multiple bioactivities, and a growing body of research has indicated that both flavonoids and iso-flavonoids can either kill or re-sensitize conventional chemotherapeutics to resistant cancer cells. Here, we summarize the research and discuss the underlying mechanisms, concluding that these flavonoids do not function as specific regulators of target proteins, but rather as multi-functional agents that negatively regulate the key factors contributing to MDR.

The functional expression of extracellular calcium-sensing receptor in rat pulmonary artery smooth muscle cells.

BACKGROUND: The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown. METHODS: The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca2+]i) was detected by a laser-scanning confocal microscope. RESULTS: The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca2+]o (extracellular calcium concentration) or Gd3+ (an agonist of CaSR) induced an increase of [Ca2+]i and PAs constriction in a concentration-dependent manner. In addition, the above-mentioned effects of Ca2+ and Gd3+ were inhibited by U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP3 receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase). CONCLUSIONS: CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca2+]i through G-PLC-IP3 pathway.

The calcium-sensing receptor mediates hypoxia-induced proliferation of rat pulmonary artery smooth muscle cells through MEK1/ERK1,2 and PI3K pathways.

Activation of the calcium-sensing receptor (CaSR) leads to an increase of intracellular calcium concentration and alteration of cellular activities. High level of intracellular calcium is involved in hypoxia-induced proliferation of pulmonary arterial smooth muscle cells (PASMCs). However, whether the CaSR is expressed in PAMSCs and is related to the hypoxia-induced proliferation of PASMCs is unclear. In this study, the expression and distribution of CaSRs were detected by RT-PCR, western blotting and immunofluorescence; the intracellular concentration of free calcium ([Ca(2+) ](i) ) was determined by confocal laser scanning microscopy; cell proliferation was tested using an MTT and BrdU incorporation assay; cell cycle analysis was carried out using a flow cytometric assay; and the expression of proliferating cell nuclear antigen (PCNA), extracellular signal-regulated protein kinase 1,2 (ERK1,2) and AKT were analysed by western blotting. We observed that both CaSR mRNA and protein were expressed in rat PASMCs. Lowering of oxygen from 21% to 2.5% led to increased [Ca(2+) ](i) and CaSR expression. This condition of hypoxia also stimulated PASMCs proliferation accompanying with increased phosphorylation of ERK1,2 and AKT. GdCl(3) (an agonist of CaSR) or NPS2390 (an antagonist of CaSR) amplified or weakened the effect of hypoxia, respectively. PD98059 (a MEK1 inhibitor) or LY294002 (a PI3K inhibitors) decreased the up-regulation of PCNA expression and the increase of the cell proliferation index induced by hypoxia and GdCl(3) in PASMCs. Our results suggest that CaSR is expressed in rat PASMCs, and that CaSR activation through MEK1/ERK1,2 and PI3 kinase pathways is involved in hypoxia-induced proliferation of PASMCs.