Influence of electroacupuncture on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway in spontaneously hypertensive rats / 中西医结合学报

OBJECTIVE@#To investigate the influence of electroacupuncture (EA) on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathway in spontaneously hypertensive rats (SHRs).@*METHODS@#Eight Wistar-Kyoto rats were used as the healthy blood pressure (BP) control (normal group), and 32 SHRs were randomized into model group, EA group, EA plus ghrelin group (EA + G group), and EA plus PF04628935 group (a potent ghrelin receptor blocker; EA + P group) using a random number table. Rats in the normal group and model group did not receive treatment, but were immobilized for 20 min per day, 5 times a week, for 4 continuous weeks. SHRs in the EA group, EA + G group and EA + P group were immobilized and given EA treatment in 20 min sessions, 5 times per week, for 4 weeks. Additionally, 1 h before EA, SHRs in the EA + G group and EA + P group were intraperitoneally injected with ghrelin or PF04628935, respectively, for 4 weeks. The tail-cuff method was used to measure BP. After the 4-week intervention, the rats were sacrificed by cervical dislocation, and pathological morphology of the abdominal aorta was observed using hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ghrelin, nitric oxide (NO), endothelin-1 (ET-1) and thromboxane A2 (TXA2) in the serum. Isolated thoracic aortic ring experiment was performed to evaluate vasorelaxation. Western blot was used to measure the expression of PI3K, Akt, phosphorylated Akt (p-Akt) and eNOS proteins in the abdominal aorta. Further, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to measure the relative levels of mRNA expression for PI3K, Akt and eNOS in the abdominal aorta.@*RESULTS@#EA significantly reduced the systolic BP (SBP) and diastolic BP (DBP) (P < 0.05). HE staining showed that EA improved the morphology of the vascular endothelium to some extent. Results of ELISA indicated that higher concentrations of ghrelin and NO, and lower concentrations of ET-1 and TXA2 were presented in the EA group (P < 0.05). The isolated thoracic aortic ring experiment demonstrated that the vasodilation capacity of the thoracic aorta increased in the EA group. Results of Western blot and qRT-PCR showed that EA increased the abundance of PI3K, p-Akt/Akt and eNOS proteins, as well as expression levels of PI3K, Akt and eNOS mRNAs (P < 0.05). In the EA + G group, SBP and DBP decreased (P < 0.05), ghrelin concentrations increased (P < 0.05), and the concentrations of ET-1 and TXA2 decreased (P < 0.05), relative to the EA group. In addition, the levels of PI3K and eNOS proteins, the p-Akt/Akt ratio, and the expression of PI3K, Akt and eNOS mRNAs increased significantly in the EA + G group (P < 0.05), while PF04628935 reversed these effects.@*CONCLUSION@#EA effectively reduced BP and protected the vascular endothelium, and these effects may be linked to promoting the release of ghrelin and activation of the PI3K/Akt/eNOS signaling pathway.

Screening of mRNA expression differences of PI3K/Akt/eNOS in the aortic tissue of rats exposed to sodium fluoride based on mRNA sequencing / 中华地方病学杂志

Objective:To explore the effect of fluoride exposure on the gene expression of phosphatidylinositol-3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) in rat aortic tissue, and to provide a theoretical basis for studying the mechanism of cardiovascular injury caused by endemic fluorosis.Methods:A total of 40 male Wistar rats were divided into 4 groups (10 rats in each group) via the random number table method according to body weight (80 - 100 g), namely control group (drinking distilled water), low-dose, medium-dose and high-dose groups [drinking distilled water containing 50, 100 and 150 mg/L sodium fluoride (NaF), respectively]. The rats were free to drink and eat. After feeding for 90 days, rats were sacrificed and the aortic tissue was taken. Three aortic tissue samples from the control group and the high-dose group were taken for mRNA sequencing, the differential genes were screened, and the differential genes were analyzed by GO function enrichment analysis and KEGG function enrichment analysis. At the same time, the mRNA expression levels of PI3K, Akt and eNOS in the aortic tissue of rats in each group were determined by real-time fluorescence quantitative PCR.Results:Compared with control group, there were 756 differential genes in high-dose group, including 654 up-regulated genes and 102 down-regulated genes. These differential genes were mainly related to biological processes such as muscle contraction, muscle regulation, muscle tissue development, striated muscle cell development, muscle cell differentiation, blood circulation regulation and striated muscle tissue development. They were mainly enriched in cyclic guanosine phosphate (cGMP-PKG) signaling pathway, relaxin signaling pathway and PI3K/Akt signaling pathway, etc. Compared with control group, the mRNA expression levels of PI3K and eNOS in aortic tissue of rats in low-dose, medium-dose and high-dose groups were significantly reduced ( P < 0.05); the mRNA expression level of Akt in low-dose group was significantly increased ( P < 0.05). Conclusion:Fluoride exposure has certain effects on the function and gene expression of rat aortic tissue, and PI3K/Akt/eNOS signaling pathway may play an important role in the process of fluoride induced aortic tissue injury in rats.

Effects of PI3K/Akt-eNOS signaling pathway on total flavones in Clematis filamentosa post-conditioning alleviated of myocardial ischemia-reperfusion injury in rats / 中国中药杂志

The aim of this paper was to study the effect of total flavones of Clematis filamentosa Dunn(TFCD) post-conditioning against myocardial ischemia-reperfusion injury (MIRI) and the role of PI3K/Akt-eNOS signaling pathway. Forty male SD rats were divided randomly into five groups: Sham group, model group (I/R), TFCD post-conditioning group (TFCD), TFCD post-condition-ing+LY294002 (a PI3K/Akt signaling pathway inhibitor) group (TFCD+LY), and LY294002 group (LY). At the end of reperfusion, hemodynamic parameters were recorded, morphology changes of myocardial tissue were evaluated by using HE staining, and myocardial infarct size were observed, blood samples were obtained to determine plasma activation of lactate dehydrogenase (LDH), creatine kinase (CK) nitric oxide (NO), endothelial nitric oxide synthase (eNOS), superoxide dismutase (SOD), maleic dialdehyde (MDA) and glutathione peroxidase (GSH-Px). The expressions of Akt, p-Akt, eNOS and p-eNOS proteins were assessed by using Western blot, and eNOS and inducible nitric oxide synthase (iNOS) mRNA was measured by RT-PCR. The results showed that, compared with the model group, TFCD post-conditioning remarkably improved hemodynamics function and myocardial structure, reduced myocardial infarct size and enhanced the contents of NO, eNOS, SOD and GSH-Px, and decreased the contents of LDH, CK and MDA, increased the levels of phosphorylation of Akt and eNOS protein expression, eNOS and iNOS mRNA expression significantly(P＜0.05 or P＜0.01). These effects were inhibited by LY294002, a blocker of PI3K/Akt signaling pathway. The above experiments indicated that TFCD post-conditioning could significantly reduce MIRI in rats, the mechanism of which may be associated with increasing antioxidation, scavenging oxygen free radicals, regulating NO generation and activating PI3K/Akt-eNOS signaling pathway.

An integrative pharmacological-based study on network mechanism of Xinkeshu Tablets intervention in atherosclerosis / 中草药

Objective: To explore the mechanism of the intervention of Xinkeshu Tablets (XKST) on atherosclerosis (AS) and provide reference for the secondary development and clinical application of XKST. Methods: The integrated pharmacology platform was used to predict the key targets and pathways of the intervention of XKST on AS and its molecular mechanism was also explored. Results: In the integrative analysis of heterogeneous network of “TCM-component-target-pathway”, 80 relevant effective ingredients were found, including B4GALT4, B4GALT2, PRKCD, GCK, GNB1, and other key targets; Endocrine system, thyroid hormone signaling pathway, nervous system, estrogen signaling pathway, and chemokine signaling pathway were key pathways related with its anti-atherosclerosis. Conclusion: According to the analysis and prediction of the enrichment information, the effect of XKST on common regulating PI3K/Akt/eNOS and Raf/MEK/ERK signaling pathway and protecting vascular endothelial cells is first prompted, thus achieving the intervention in AS.

Lycium barbarum polysaccharides attenuate oxidative stress injury of hu-man endothelium-like EA. hy926 cells induced by hydrogen peroxide / 中国病理生理杂志

AIM:To study the effect of Lycium barbarum polysaccharides (LBP) on oxidative stress injury of human endothelium-like EA. Hy926 cells induced by hydrogen peroxide (H2O2). METHODS:The EA. Hy926 cell model of oxidative stress injury was established by H2O2 treatment. The EA. Hy926 cells were divided into 5 groups:control group, damage (H2O2 at 50 mmol/L) group, LBP (100 mg/L) group, anti-damage groups (LBP at 50 mg/L, 100 mg/L or 200 mg/L+50 mol/L H2O2), and LY294002 (20 μmol/L) group. The effect of LBP at different concentrations on the cell viability of EA. Hy926 cells was measured by CCK-8 assay, and the optimum concentration of LBP was screened out. The apoptotic of EA. Hy926 cells was analyzed by flow cytometry. Acridine orange/ethidium bromide ( AO/EB) staining was used to observe the morphological characteristics of the apoptotic cells. The cell migration ability was detected by scratch method. The levels of nitric oxide (NO) and vascular endothelial growth factor (VEGF) in the cell culture medium were examined. The protein levels of cleaved caspase-3, Bax, Bcl-2, endothelial NO synthase (eNOS), p-eNOS and p-Akt were determined by Western blot. RESULTS:LBP at concentration of 100 mg/L significantly attenuated the injury of EA. Hy926 cells induced by H2O2, as indicated by improved cell viability ( P <0.05 ) and decreased apoptosis ( P <0.05). Pretreatment with LBP elevated the levels of NO and VEGF (P<0.05), and promoted the migration ability of EA. Hy926 cells. LBP also increased the Bcl-2/Bax ratio, down-regulated the protein level of cleaved caspase-3, and up-regulated the protein levels of eNOS and p-eNOS. The protective effect of LBP were abolished by pretreatment of the EA. Hy926 cells with the inhibitor of PI3K (P<0.05). As a result, the protein level of p-Akt was down-regulated, and the level of NO was also significantly reduced. CONCLUSION:LBP has protective effect on H2O2-induced EA. Hy926 cells by attenuating apoptosis of the cells. The mechanism is closely related to the activation of PI3K/Akt/eNOS signaling pathway.

Role of PI3K/Akt/eNOS signaling pathway in inhibitory effects of puerarin on ox-LDL-induced TF expression in vascular endothelial cells / 中国病理生理杂志

AIM: To explore the role of phosphatidylinositiol 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathways in the inhibitory effects of puerarin on oxidized low-density lipoprotein (ox-LDL)-induced tissue factor (TF) expression in vascular endothelial cells.METHODS: The mRNA expression of TF was detected by real-time fluorescent quantitative PCR.The protein levels of TF and Akt was determined by Western blot.The content of the nitric oxide (NO) was measured by nitrate reduction method.RESULTS: Compared with control group, incubating endothelial cells with ox-LDL significantly induced TF expression at mRNA and protein levels and the dephosphorylation of Akt protein, and decreased NO production.Incubation of the endothelial cells with puerarin for 1 h and then treatment of the cells with ox-LDL decreased the TF expression at mRNA and protein levels, increased Akt protein phosphorylation and intracellular NO content.Co-incubation of the endothelial cells with PI3K inhibitor LY294002 and puerarin for 1 h and then treatment of the cells with ox-LDL augmented the TF expression at mRNA and protein levels and the Akt protein dephosphorylation, and decreased NO production.Co-incubation of the endothelial cells with eNOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and puerarin significantly decreased the inhibitory effect of puerarin on ox-LDL-induced TF expression at mRNA and protein levels in the endothelial cells, and reduced Akt protein phosphorylation and NO production.CONCLUSION: Puerarin inhibits ox-LDL-induced TF expression at mRNA and protein levels in the human umbilical vein endothelial cells via activation of PI3K/Akt/eNOS signaling pathway.

Effects of miRNA-29a on monocrotaline-induced pulmonary hypertension in rats / 中国病理生理杂志

AIM: To detect the expression of microRNA (miRNA)-29a in pulmonary arteries of monocrotaline (MCT)-induced pulmonary hypertensive rats, and to investigate the effects of miRNA-29a on pulmonary hypertension.METHODS: MCT-induced pulmonary hypertensive model was established in Wistar rats.The expression of miRNA-29a in the lung tissue was determined by qPCR.miRNA-29a was overexpressed in the pulmonary hypertension rats by tail vein injection of miRNA-29a-mimic.Pulmonary artery pressure (PAP) and systemic arterial pressure (SAP) were measured.The morphological changes of the pulmonary arteries were observed by HE staining.The protein levels of p-Akt and p-eNOS were detected by Western blot.RESULTS: The mRNA expression of miRNA-29a was significantly decreased in the pulmonary arteries of MCT-induced pulmonary hypertensive rats.Furthermore, after overexpression of miRNA-29a, PAP was remarkably reduced, while SAP remained unchanged.In addition, the increased thickness of tunica media, the remodeling of pulmonary arteries and the decreased protein levels of p-Akt and p-eNOS in the pulmonary hypertensive rats were dramati-cally changed after miRNA-29a overexpression.CONCLUSION: Overexpression of miRNA-29a ameliorates pulmonary hypertension in rats.These effects may be associated with the activation of PI3K/Akt-eNOS signaling pathway.