Use of network pharmacology to analyze compound reserpine and triamterene tablets in the treatment of hypertension / 药学学报

Compound reserpine and triamterene tablets (CRTT), a compound antihypertensive drug developed by Chinese scientists, is still widely used in clinical practice. However, the mechanisms by which CRTT treats hypertension remain to be fully understood. This study used network pharmacology to analyze CRTT's antihypertensive mechanisms with in vitro experiments. The targets of the four chemical components of CRTT were collected from the Swiss Target Prediction database; 1 828 protein targets related to hypertension were collected from the Therapeutic Target Database (TTD) and Online Mendelian Inheritance in Man (OMIM) database. The CRTT-hypertension network model was constructed using a search tool for recurring instances of neighbouring genes (STRING). Gene ontology (GO) and pathway enrichment analysis of targets of interest was conducted with the Metascape database. In the in vitro study, human umbilical vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) were treated with 1 μmol·L-1 angiotensin Ⅱ (AngⅡ) and CRTT was administered at concentrations of 0.01, 0.1, and 1 μmol·L-1. Changes in the phosphatidylinositol-3-kinase/protein serine threonine kinase/endothelial nitric oxide synthase (PI3K/Akt/eNOS) pathway in HUVEC and the cyclic guanosine monophosphate/cGMP-dependent protein kinase (cGMP/PKG) pathway in VSMC were determined by Western blot. Network pharmacological analysis revealed that the antihypertensive effect of CRTT is closely associated with biological pathways such as vascular tone regulation, adrenergic receptor activation, protein kinase activity and signaling pathways such as the cGMP/PKG signaling pathway, vascular smooth muscle contraction, neuroactive ligand-receptor interaction, adrenergic signaling in cardiomyocytes and calcium signaling pathways. The in vitro study confirmed that CRTT increased the levels of phosphorylated phosphatidylinositol-3-kinase (p-PI3K), phosphorylated protein serine threonine kinase (p-Akt), phosphorylated endothelial nitric oxide synthase (p-eNOS) in HUVEC and the levels of eNOS, phosphorylated vasodilator-stimulated phosphoprotein (p-VASP), and PKG in VSMC through multiple targets and pathways. These results suggest that the activation of PI3K/Akt/eNOS pathway and endothelial-dependent NO/cGMP signaling may be involved in the CRTT-mediated hypotensive effect.

Gastrodin improves hippocampal neurogenesis by NO-cGMP-PKG signaling pathway in cerebral ischemic mice / 中国中药杂志

This paper was aimed to investigate the effect of gastrodin( GAS) on hippocampal neurogenesis after cerebral was chemic and to explore its mechanism of action related to NO. The cerebral ischemia model of C57 BL/6 mice was established by bilateral common carotid artery occlusion. The pathological changes in hippocampal CA1 region and the cognitive function of mice were assessed by HE staining and Morris water maze test,respectively. The count of Brd U/Neu N positive cells in dentate gyrus was detected by immunofluorescence assay. The NOS activity and the NO content were determined by colorimetric and nitrate reduction methods,respectively.The level of c GMP was measured by ELISA kit,and the PKG protein expression was tested by Western blot. On postoperative day 8,the hippocampal CA1 pyramidal neurons of mice showed irregular structure,with obvious nuclear pyknosis,loose cell arrangement and obvious decrease in the number of neurons. On postoperative day 29,the spatial learning ability and memory were decreased. These results indicated cerebral ischemia in mice. Meanwhile,the Brd U/Neu N positive cells were increased significantly in ischemic mice,indicating that neurogenesis occurred in hippocampus after cerebral ischemia. Treatment with different doses of gastrodin( 50 and 100 mg·kg-1) significantly ameliorated the pathological damages in the CA1 region,improved the ability of learning and memory,and promoted hippocampal neurogenesis. At the same time,both the NOS activity and the NO concentration were decreased in model group,but the c GMP level was increased,and the PKG protein expression was up-regulated. Gastrodin administration activated the NOS activity,promoted NO production,further increased c GMP level and up-regulated PKG protein expression. These results suggested that gastrodin can promote hippocampal neurogenesis after cerebral ischemia and improve cognitive function in mice,which may be related to the activation of NO-cGMP-PKG signaling pathway.

Possible mechanisms involved in the vasorelaxant effect produced by clobenzorex in aortic segments of rats

Clobenzorex is a metabolic precursor of amphetamine indicated for the treatment of obesity. Amphetamines have been involved with cardiovascular side effects such as hypertension and pulmonary arterial hypertension. The aim of the present study was to investigate whether the direct application of 10-9-10-5 M clobenzorex on isolated phenylephrine-precontracted rat aortic rings produces vascular effects, and if so, what mechanisms may be involved. Clobenzorex produced an immediate concentration-dependent vasorelaxant effect at the higher concentrations (10-7.5-10-5 M). The present outcome was not modified by 10-6 M atropine (an antagonist of muscarinic acetylcholine receptors), 3.1×10-7 M glibenclamide (an ATP-sensitive K+ channel blocker), 10-3 M 4-aminopyridine (4-AP; a voltage-activated K+ channel blocker), 10-5 M indomethacin (a prostaglandin synthesis inhibitor), 10-5 M clotrimazole (a cytochrome P450 inhibitor) or 10-5 M cycloheximide (a general protein synthesis inhibitor). Contrarily, the clobenzorex-induced vasorelaxation was significantly attenuated (P<0.05) by 10-5 M L-NAME (a direct inhibitor of nitric oxide synthase), 10-7 M ODQ (an inhibitor of nitric oxide-sensitive guanylyl cyclase), 10-6 M KT 5823 (an inhibitor of protein kinase G), 10-2 M TEA (a Ca2+-activated K+ channel blocker and non-specific voltage-activated K+ channel blocker) and 10-7 M apamin plus 10-7 M charybdotoxin (blockers of small- and large-conductance Ca2+-activated K+ channels, respectively), and was blocked by 8×10-2 M potassium (a high concentration) and removal of the vascular endothelium. These results suggest that the direct vasorelaxant effect by clobenzorex on phenylephrine-precontracted rat aortic rings involved stimulation of the NO/cGMP/PKG/Ca2+-activated K+ channel pathway.

Pharmacological study of the mechanisms involved in the vasodilator effect produced by the acute application of triiodothyronine to rat aortic rings

A relationship between thyroid hormones and the cardiovascular system has been well established in the literature. The present in vitro study aimed to investigate the mechanisms involved in the vasodilator effect produced by the acute application of 10-8–10-4 M triiodothyronine (T3) to isolated rat aortic rings. Thoracic aortic rings from 80 adult male Wistar rats were isolated and mounted in tissue chambers filled with Krebs-Henseleit bicarbonate buffer in order to analyze the influence of endothelial tissue, inhibitors and blockers on the vascular effect produced by T3. T3 induced a vasorelaxant response in phenylephrine-precontracted rat aortic rings at higher concentrations (10-4.5–10-4.0 M). This outcome was unaffected by 3.1×10-7 M glibenclamide, 10-3 M 4-aminopyridine (4-AP), 10-5 M indomethacin, or 10-5 M cycloheximide. Contrarily, vasorelaxant responses to T3 were significantly (P<0.05) attenuated by endothelium removal or the application of 10-6 M atropine, 10-5 M L-NG-nitroarginine methyl ester (L-NAME), 10-7 M 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10-6 M (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i](1,6)benzodiazocine-10-carboxylic acid, methyl ester KT 5823, 10-2 M tetraethylammonium (TEA), or 10-7 M apamin plus 10-7 M charybdotoxin. The results suggest the involvement of endothelial mechanisms in the vasodilator effect produced by the acute in vitro application of T3 to rat aortic rings. Possible mechanisms include the stimulation of muscarinic receptors, activation of the NO-cGMP-PKG pathway, and opening of Ca2+-activated K+ channels.

Oxidative Stress-Activated NHE1 Is Involved in High Glucose-Induced Apoptosis in Renal Tubular Epithelial Cells

PURPOSE: Diabetic nephropathy (DN) is a prevalent chronic microvascular complication of diabetes mellitus involving disturbances in electrolytes and the acid-base balance caused by a disorder of glucose metabolism. NHE1 is a Na+/H+ exchanger responsible for keeping intracellular pH (pHi) balance and cell growth. Our study aimed to investigate roles of NHE1 in high glucose (HG)-induced apoptosis in renal tubular epithelial cells. MATERIALS AND METHODS: Renal epithelial tubular cell line HK-2 was cultured in medium containing 5 mM or 30 mM glucose. Then, cell apoptosis, oxidative stress, NHE1 expression, and pHi were evaluated. NHE1 siRNA and inhibitor were used to evaluate its role in cell apoptosis. RESULTS: HG significantly increased cell apoptosis and the production of reactive oxygen species (ROS) and 8-OHdG (p<0.05). Meanwhile, we found that HG induced the expression of NHE1 and increased the pHi from 7.0 to 7.6 after 48 h of incubation. However, inhibiting NHE1 using its specific siRNA or antagonist DMA markedly reduced cell apoptosis stimulated by HG. In addition, suppressing cellular oxidative stress using antioxidants, such as glutathione and N-acetyl cysteine, significantly reduced the production of ROS, accompanied by a decrease in NHE1. We also found that activated cyclic GMP-Dependent Protein Kinase Type I (PKG) signaling promoted the production of ROS, which contributed to the regulation of NHE1 functions. CONCLUSION: Our study indicated that HG activates PKG signaling and elevates the production of ROS, which was responsible for the induction of NHE1 expression and dysfunction, as well as subsequent cell apoptosis, in renal tubular epithelial cells.

Effect of PKG on the secretion of inflammatory cytokines in THP-1 macrophage-derived foam cells / 实用医学杂志

Objective To investigate the effect of PKG on the secretion of IL-6, IL-10, and TNF-αin THP-1 macrophage-derived foam cells. Methods THP-1 monocytes were induced to construct macrophages by treating with 160 nmol/L TPA. Then the macrophages were further treated with 50 mg/L ox-LDL to become foam cells. Four groups were set in this study, including the macrophage group, the foam cell group, the group of foam cell treated with PKG agonist 8-Br-cGMP, and the group of foam cell treated with PKG inhibitor KT-5823. The morphology of THP-1 cells, macrophages and foam cells were observed under microscope. The cellular lipid accumulation was detected by oil red ostaining. The secretion of IL-6, IL-10, and TNF-α into the supernatant was detected by ELISA assay. Results The foam cell was obtained after macrophage incubated with ox-LDL for 48 hours. The secretion of IL-6 and TNF-α increased significantly from the foam cells than that from the macrophages (P0.05). After incubation with 8-Br-cGMP, the secretion of IL-6 and TNF-α from the macrophage-derived foam cells decreased significantly (P 0.05). Conclusions PKG may enhance the expression of anti-inflammatory cytokine IL-10, and inhibit the expression of inflammatory cytokine IL-6 and TNF-α, contributing to prevent the development of inflammation. PKG might have a potential anti-atherosclerosis effect.

Construction and identification of recombinant adenovirus containing wild-type human PKG I α gene / 第二军医大学学报

Objective: To clone human PKG I α gene and construct a recombinant adenovirus vector containing wild-type PKG I α. Methods: RT-PCR was used to amplify the full-length PKG gene from human pulmonary arterial smooth muscle. After T/A cloning, PKG I α cDNA was cloned into shuttle plasmid pAdTrack-CMV to construct pAdTrack-PKG I α. The plasmid was linearized by Pme I and transformed into BJ5183 E. coli, where the plasmid was recombined with pAdEasy-1 by homologous recombination, The recombinants were then transfected into Ad293 cells by Lipofectamine2000 for packaging the adenovirus; the recombinant adenovirus was traced by monitoring GFP expression under fluorescence microscope to determine the titer. Results: PKG I α was successfully amplified from human pulmonary arterial smooth muscle by RT-RCR. After 3 cycles of amplification, the titer of adenovirus containing wild-type PKG I α reached the indicated level. Conclusion: We have successfully constructed PKG I α gene and constructed the PKG I α recombinant adenovirus, which provides a foundation for the study of PKG Iα function and its role in hypoxia pulmonary vessel remodeling.

Role of PKG-L-type calcium channels in the antinociceptive effect of intrathecal sildenafil

Sildenafil increases the cyclic guanosine monophosphate (cGMP) by inhibition of a phosphodiesterase 5, thereby leading to an antinociceptive effect. The increased cGMP may exert the effect on an L-type calcium channel through the activation of protein kinase G (PKG). The purpose of this study was to examine the possible involvement of a PKG-L-type calcium channel on the effect of sildenafil at the spinal level. Catheters were inserted into the intrathecal space of male SD rats. Pain was induced by applying 50 microliter of a 5% formalin solution to the hindpaw. The sildenafil-induced effect was examined after an intrathecal pretreatment of a PKG inhibitor (KT 5823), or a L-type calcium channel activator (FPL 64176). Intrathecal sildenafil produced an antinociceptive effect during phase 1 (0~10 min interval) and phase 2 (10~60 min interval) in the formalin test. Intrathecal KT 5823 and FPL 64176 attenuated the antinociceptive effect of sildenafil during both phases. Sildenafil is effective against both acute pain and the facilitated pain state at the spinal level. In addition, the inhibition of an L-type calcium channel by activation of the PKG may contribute to the antinocieptive mechanism of sildenafil in the spinal cord.

Proliferation of vascular smooth muscle cells regulated by NO/PKG mediated via Ca~(2+)/calcineurin signaling pathway / 基础医学与临床

Objective To study the regulation of VSMC proliferation by NO/PKG via modulating intracellular Ca~(2+)/calcineurin(CaN) signaling pathway.Methods Primary VSMCs from rat aorta were used as the experimental model.CaN protein and its activity were assayed using immunoblotting and free inorganic phosphate content analysis respectively.Growth and viability of cells were determined by MTT assay and acridine orange and ethidium bromide staining.Results The addition of SNAP and Sp-8-pCPT-cGMPS decrease absorbance of cells stimulated by phenylephrine(PE),whereas the addition of Rp-8-pCPT-cGMPS increases it.In SMCs p retreated with Ver,absorbance of cells stimulated by PE decreased and was further inhibited by the additional treatment of SNAP and Sp-8-pCPT-cGMPS.In SMCs pretreated with CsA,absorbance of cells stimulated by PE decrease,but it can not be further altered by the additional treatment of SNAP,Sp-8-pCPT-cGMPS and Rp-8-pCPT-cGMPS.Moreover,expression and activities of CaN induced by PE is inhibited by SNAP and Sp-8-pCPT-cGMPS.Conclusion NO/PKG inhibits the proliferation of vascular SMCs without decreasing cell survival rate,which is mediated via intracellular Ca~(2+)/CaN signaling pathway.

Characteristic of NO-cGMP signal pathway in regulation of thoracic aortic relaxation in thyroxine-induced hypertensive rats / 中国药理学通报

Aim To explore the characteristic of NO-cGMP signal pathway in the regulation of thoracic aortic relaxation in thyroxine-induced hypertensive rats.Methods Hyperthyroidism was induced by administering Lthyroxine(T4,0.5 mg?kg~-1,sc)daily for 16 days.Sham-treated euthyroid control rats received only vehicle saline for 16 days.SNAP,an NO donor,was used to define the differential relaxation in the thoracic aorta from euthyroid and hyperthyroid rats.To determine the mechanisms involved changes in NO-cGMP signal pathway in the regulation of aortic relaxation from hyperthyroid rats,BAY 41-2272(BAY)was used to activate soluble guanylate cyclase(sGC),ODQ was used to inhibit sGC,and 8-Br-cGMP was used to acti vate protein kinase G(PKG),respectively.Results Thyroid hormone excess for 16 days showed characteristic changes in body weight,heart rate and systolic blood pressure in rats.The body weight was significantly decreased,while heart rate,pulse pressure and systolic blood pressure were increased in T4-treated rats.SNAP caused relaxation in the aorta in both euthyroid and hyperthyroid rats.However,SNAP-induced aortic relaxation was significantly attennuated in hyperthyroid rats than in euthyroid rats.In the presence of ODQ,SNAP-induced aortic relaxation was blocked in both euthyroid and hyperthyroid rats.BAY and 8-BrcGMP-induced aortic relaxation was significantly attennuated in hyperthyroid rats than in euthyroid rats.Conclusion These data suggest that the attenuated effect of NO-cGMP signal pathway is involved in the regulation of aortic relaxation in the pathophysiology of hyperthyroidism,which may be related to the downregulation of sGC and PKG functions.