[Mechanism of astragaloside â £ combined with Panax notoginseng saponins in regulating angiogenesis to treat cerebral ischemia based on network pharmacology and experimental verification].

Network pharmacology and animal and cell experiments were employed to explore the mechanism of astragaloside Ⅳ(AST Ⅳ) combined with Panax notoginseng saponins(PNS) in regulating angiogenesis to treat cerebral ischemia. The method of network pharmacology was used to predict the possible mechanisms of AST Ⅳ and PNS in treating cerebral ischemia by mediating angiogenesis. In vivo experiment: SD rats were randomized into sham, model, and AST Ⅳ(10 mg·kg~(-1)) + PNS(25 mg·kg~(-1)) groups, and the model of cerebral ischemia was established with middle cerebral artery occlusion(MCAO) method. AST Ⅳ and PNS were administered by gavage twice a day. the Longa method was employed to measure the neurological deficits. The brain tissue was stained with hematoxylin-eosin(HE) to reveal the pathological damage. Immunohistochemical assay was employed to measure the expression of von Willebrand factor(vWF), and immunofluorescence assay to measure the expression of vascular endothelial growth factor A(VEGFA). Western blot was employed to determine the protein levels of vascular endothelial growth factor receptor 2(VEGFR2), VEGFA, phosphorylated phosphatidylinositol 3-kinase(p-PI3K), and phosphorylated protein kinase B(p-AKT) in the brain tissue. In vitro experiment: the primary generation of rat brain microvascular endothelial cells(rBEMCs) was cultured and identified. The third-generation rBMECs were assigned into control, model, AST Ⅳ(50 µmol·L~(-1)) + PNS(30 µmol·L~(-1)), LY294002(PI3K/AKT signaling pathway inhibitor), 740Y-P(PI3K/AKT signaling pathway agonist), AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P groups. Oxygen glucose deprivation/re-oxygenation(OGD/R) was employed to establish the cell model of cerebral ischemia-reperfusion injury. The cell counting kit-8(CCK-8) and scratch assay were employed to examine the survival and migration of rBEMCs, respectively. Matrigel was used to evaluate the tube formation from rBEMCs. The Transwell assay was employed to examine endothelial cell permeability. Western blot was employed to determine the expression of VEGFR2, VEGFA, p-PI3K, and p-AKT in rBEMCs. The results of network pharmacology analysis showed that AST Ⅳ and PNS regulated 21 targets including VEGFA and AKT1 of angiogenesis in cerebral infarction. Most of these 21 targets were involved in the PI3K/AKT signaling pathway. The in vivo experiments showed that compared with the model group, AST Ⅳ + PNS reduced the neurological deficit score(P<0.05) and the cell damage rate in the brain tissue(P<0.05), promoted the expression of vWF and VEGFA(P<0.01) and angiogenesis, and up-regulated the expression of proteins in the PI3K/AKT pathway(P<0.05, P<0.01). The in vitro experiments showed that compared with the model group, the AST Ⅳ + PNS, 740Y-P, AST Ⅳ + PNS + LY294002, and AST Ⅳ + PNS + 740Y-P improved the survival of rBEMCs after OGD/R, enhanced the migration of rBEMCs, increased the tubes formed by rBEMCs, up-regulated the expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P<0.05, P<0.01). Compared with the LY294002 group, the AST Ⅳ + PNS + LY294002 group showed increased survival rate, migration rate, and number of tubes, up-regulated expression of proteins in the PI3K/AKT pathway, and decreased endothelial cell permeability(P<0.05,P<0.01). Compared with the AST Ⅳ + PNS and 740Y-P groups, the AST Ⅳ + PNS + 740Y-P group presented increased survival rate, migration rate, and number of tubes and up-regulated expression of proteins in the PI3K/AKT pathway, and reduced endothelial cell permeability(P<0.01). This study indicates that AST Ⅳ and PNS can promote angiogenesis after cerebral ischemia by activating the PI3K/AKT signaling pathway.

[Effects of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination on inflammatory responses in atherosclerotic mice].

The study aims to observe the effects and explore the mechanisms of Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination in the treatment of the inflammatory response of mice with atherosclerosis(AS) via the Toll-like receptor 4(TLR4)/myeloid differentiation primary response protein 88(MyD88)/nuclear factor-κB(NF-κB) signaling pathway. Male ApoE~(-/-) mice were randomly assigned into a model group, a Buyang Huanwu Decoction group, an Astragali Radix-Angelicae Sinensis Radix combination group, and an atorvastatin group, and male C57BL/6J mice of the same weeks old were used as the control group. Other groups except the control group were given high-fat diets for 12 weeks to establish the AS model, and drugs were administrated by gavage. Aortic intimal hyperplasia thickness, blood lipid level, plasma inflammatory cytokine levels, M1/M2 macrophage markers, and expression levels of proteins in TLR4/MyD88/NF-κB pathway in the vessel wall were measured to evaluate the effects of drugs on AS lesions and inflammatory responses. The results showed that the AS model was successfully established with the ApoE~(-/-) mice fed with high-fat diets. Compared with the control group, the model group showed elevated plasma total cholesterol(TC), triglyceride(TG), and low-density lipoprotein cholesterol(LDL-c) levels(P<0.05), thickened intima(P<0.01), and increased plasma tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) levels(P<0.01). Moreover, the model group showed increased expression of vascular cell adhesion molecule-1(VCAM-1) and inducible nitric oxide synthase(iNOS)(P<0.01), inhibited expression of endothelial nitric oxide synthase(eNOS) and cluster of differentiation 206(CD206)(P<0.01), and up-regulated mRNA and protein levels of TLR4, MyD88, NF-κB inhibitor alpha(IκBα), and NF-κB in the vessel wall(P<0.05). Compared with the model group, Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination lowered the plasma TC and LDL-c levels(P<0.01), alleviated the intimal hyperplasia(P<0.01), and reduced the plasma TNF-α and IL-6 levels(P<0.05). Moreover, the two interventions promoted the expression of eNOS and CD206(P<0.05), inhibited the expression of VCAM-1 and iNOS(P<0.01), and down-regulated the mRNA and protein levels of TLR4, MyD88, IκBα, and NF-κB(P<0.05) in the vessel wall. This study indicated that Buyang Huanwu Decoction and Astragali Radix-Angelicae Sinensis Radix combination could delay the progression of AS, inhibit the polarization of vascular wall macrophages toward M1 type, and attenuate vascular inflammatory response by inhibiting the activation of TLR4/MyD88/NF-κB signaling pathway in the vascular wall. Astragali Radix and Angelicae Sinensis Radix were the main pharmacological substances in Buyang Huanwu Decoction for alleviating the AS vascular inflammatory response.

[EPCs-exos combined with tanshinone â ¡_A protect vascular endothelium cells from oxidative damage via PI3K/Akt pathway].

This study aims to investigate the molecular mechanism of tanshinone Ⅱ_(A )(TaⅡ_A) combined with endothelial progenitor cells-derived exosomes(EPCs-exos) in protecting the aortic vascular endothelial cells(AVECs) from oxidative damage via the phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt) pathway. The AVECs induced by 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine(POVPC) were randomly divided into model, TaⅡ_A, EPCs-exos, and TaⅡ_A+EPCs-exos groups, and the normal cells were taken as the control group. The cell counting kit-8(CCK-8) was used to examine the cell proliferation. The lactate dehydrogenase(LDH) cytotoxicity assay kit, Matrigel assay, DCFH-DA fluorescent probe, and laser confocal microscopy were employed to examine the LDH release, tube-forming ability, cellular reactive oxygen species(ROS) level, and endothelial cell skeleton morphology, respectively. The enzyme-linked immunosorbent assay was employed to measure the expression of interleukin(IL)-1ß, IL-6, and tumor necrosis factor(TNF)-α. Real-time fluorescence quantitative PCR(qRT-PCR) and Western blot were employed to determine the mRNA and protein levels, respectively, of PI3K and Akt. Compared with the control group, the model group showed decreased cell proliferation and tube-forming ability, increased LDH release, elevated ROS level, obvious cytoskeletal disruption, increased expression of IL-1ß, IL-6, and TNF-α, and down-regulated mRNA and protein levels of PI3K and Akt. Compared with the model group, TaⅡ_A or EPCs-exos alone increased the cell proliferation and tube-forming ability, reduced LDH release, lowered the ROS level, repaired the damaged skeleton, decreased the expression of IL-1ß, IL-6, and TNF-α, and up-regulated the mRNA and protein levels of PI3K and Akt. TaⅡ_A+EPCs-exos outperformed TaⅡ_A or EPCs-exos alone in regulating the above indexes. The results demonstrated that TaⅡ_A and EPCs-exos exerted a protective effect on POVPC-induced AVECs by activating the PI3K/Akt pathway, and the combination of the two had stronger therapeutic effect.

Effect of Remote Ischemic Conditioning vs Usual Care on Neurologic Function in Patients With Acute Moderate Ischemic Stroke: The RICAMIS Randomized Clinical Trial.

Importance: Preclinical and clinical studies have suggested a neuroprotective effect of remote ischemic conditioning (RIC), which involves repeated occlusion/release cycles on bilateral upper limb arteries; however, robust evidence in patients with ischemic stroke is lacking. Objective: To assess the efficacy of RIC for acute moderate ischemic stroke. Design, Setting, and Participants: This multicenter, open-label, blinded-end point, randomized clinical trial including 1893 patients with acute moderate ischemic stroke was conducted at 55 hospitals in China from December 26, 2018, through January 19, 2021, and the date of final follow-up was April 19, 2021. Interventions: Eligible patients were randomly assigned within 48 hours after symptom onset to receive treatment with RIC (using a pneumatic electronic device and consisting of 5 cycles of cuff inflation for 5 minutes and deflation for 5 minutes to the bilateral upper limbs to 200 mm Hg) for 10 to 14 days as an adjunct to guideline-based treatment (n = 922) or guideline-based treatment alone (n = 971). Main Outcomes and Measures: The primary end point was excellent functional outcome at 90 days, defined as a modified Rankin Scale score of 0 to 1. All end points had blinded assessment and were analyzed on a full analysis set. Results: Among 1893 eligible patients with acute moderate ischemic stroke who were randomized (mean [SD] age, 65 [10.3] years; 606 women [34.1%]), 1776 (93.8%) completed the trial. The number with excellent functional outcome at 90 days was 582 (67.4%) in the RIC group and 566 (62.0%) in the control group (risk difference, 5.4% [95% CI, 1.0%-9.9%]; odds ratio, 1.27 [95% CI, 1.05-1.54]; P = .02). The proportion of patients with any adverse events was 6.8% (59/863) in the RIC group and 5.6% (51/913) in the control group. Conclusions and Relevance: Among adults with acute moderate ischemic stroke, treatment with remote ischemic conditioning compared with usual care significantly increased the likelihood of excellent neurologic function at 90 days. However, these findings require replication in another trial before concluding efficacy for this intervention. Trial Registration: ClinicalTrials.gov Identifier: NCT03740971.

The lncRNA ROR/miR-124-3p/TRAF6 axis regulated the ischaemia reperfusion injury-induced inflammatory response in human cardiac myocytes.

Myocardial ischaemia reperfusion injury (MIRI) is considered the primary cause of death in patients with cardiovascular diseases. Recently, long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been found to be involved in the pathogenesis of MIRI. However, whether lncRNA ROR and miR-124-3p play roles in MIRI and the underlying mechanism remain undetermined. HCMs were exposed to hypoxic conditions for 2 h followed by re-oxygenation (H/R) treatment. Expression of miR-124-3p and lncRNA ROR in HCMs was measured by qRT-PCR. TRAF6 expression was evaluated by qRT-PCR and western blotting. ELISA and qRT-PCR were conducted to assess the production of TNF-α, IL-6, and IL-1ß. The interaction between miR-124-3p and TRAF6, as well as between miR-124-3p and lncRNA ROR, was verified by dual-luciferase reporter assay. Cell apoptosis was detected by flow cytometry analysis. Our data revealed that miR-124-3p was significantly downregulated, while TRAF6 and lncRNA ROR were upregulated in both MIRI rat model and H/R treated HCMs. Overexpression of miR-124-3p reversed the H/R-induced cell apoptosis and upregulation of TNF-α, IL-6, and IL-1ß. Mechanistically, miR-124-3p bound and negatively regulated TRAF6 expression in HCMs. Moreover, TRAF6 overexpression significantly blocked the effects of miR-124-3p mimics on cell apoptosis and inflammatory response of HCMs, which involved the NF-κB pathway. Further analysis showed that lncRNA ROR sponged and negatively regulated miR-124-3p in HCMs. Overexpression of IL-1ß was demonstrated to promote H/R induced cell apoptosis in HCMs. In addition, overexpression of ROR further enhanced the H/R-induced inflammation and cell apoptosis through its action on miR-124-3p. The lncRNA ROR/miR-124-3p/TRAF6 axis regulated the H/R-induced cell apoptosis and inflammatory response of HCMs.

[Astragaloside IV attenuates cerebral ischemia and reperfusion injury and reduces activation of NLRP3 inflammasome and NF-κB phosphorylation in rats following a transient middle cerebral artery occlusion].

The present study was aimed to investigate the protective effect and anti-inflammation mechanism of astragaloside IV (AST-IV) on cerebral ischemia and reperfusion injury. Following the establishment of cerebral ischemia and reperfusion model in rats by modified suture method, neurological deficit scores and cerebral infarct volume were used to evaluate the pharmacological effect of AST-IV against cerebral ischemia-reperfusion injury. Western blot was used to detect the expression levels of NLRP3, pro-Caspase-1, Caspase-1, pro-IL-1ß, IL-1ß, pro-IL-18, IL-18, phosphorylated and total nuclear factor kappa B (NF-κB)/p65 protein in the brain tissue. The results showed that compared with model group, the intervention of AST-IV decreased the neurological deficit scores, reduced the cerebral infarct volume, decreased the levels of NLRP3, Caspase-1, pro-IL-1ß, IL-1ß, pro-IL-18 and IL-18, and inhibited the expression of phosphorylated NF-κB in brain tissue. The results suggest that AST-IV has a protective effect against cerebral ischemia and reperfusion injury, and its mechanism is related to inhibiting the phosphorylation of NF-κB and NLRP3 inflammasome activation.

[Pyroptosis and stroke].

Pyroptosis is a form of inflammatory programmed cell death activated by caspase-1 and caspase-4/5/11, and involves in the pathogenesis of infectious diseases and nervous system diseases. Pyroptosis is mediated by canonical inflammasome pathway and non-canonical inflammasome pathway. The canonical inflammasome pathway is activated in stroke and aggravates brain injury. Inhibition of inflammasome, caspase-1, IL-1ß and IL-18 ameliorates brain injury. These studies indicate that canonical inflammasome pathway contributes to post-stroke brain injury, therefore, pyroptosis has become a potential therapeutic target for preventing excessive cell death during stroke. We reviewed the relationship between pyroptosis and stroke to provide some perspectives on future researches in this field.

Effects of Astragalus Combined with Angelica on Bone Marrow Hematopoiesis Suppression Induced by Cyclophosphamide in Mice.

Danggui Buxue Tang (DBT), a combination of Astragalus and Angelica at a 5 : 1 ratio, mainly promotes hematopoiesis. However, in the clinic, the combination ratio of Astragalus and Angelica to treat low hematopoietic function is not an absolute 5 : 1 ratio, suggesting that the herbs may promote hematopoiesis better after being combined at a certain range of ratios. The objective of this study is to investigate the effect of different ratio combinations of Astragalus and Angelica on bone marrow hematopoiesis suppression induced by cyclophosphamide (CTX) and to probe the interaction and mechanism of Astragalus combined with Angelica in promoting hematopoiesis. Following establishment of the model, mice were administered with Astragalus (6.00 g·kg-1), Angelica (3.00 g·kg-1), and combinations of Astragalus and Angelica at different ratios, including 10 : 1 (Astragalus 9.81 g·kg-1+Angelica 0.98 g·kg-1), 5 : 1 (Astragalus 9.00 g·kg-1+Angelica 1.80 g·kg-1), 2 : 1 (Astragalus 7.71 g·kg-1+Angelica 3.08 g·kg-1), 1 : 1 (Astragalus 5.40 g·kg-1+Angelica 5.40 g·kg-1), 1 : 2.5 (Astragalus 3.08 g·kg-1+Angelica 7.71 g·kg-1), 1 : 5 (Astragalus 1.80 g·kg-1+Angelica 9.00 g·kg-1), and 1 : 10 (Astragalus 0.98 g·kg-1+Angelica 9.81 g·kg-1). Our results suggested that Astragalus mixed with Angelica synergistically promoted hematopoiesis best when the combination ratio of Astragalus and Angelica was 1 : 1, 1 : 2.5 or 1 : 5; moreover, the effect of Angelica was greater than that of Astragalus. The potential mechanisms of the combinations of Astragalus and Angelica that promote hematopoiesis include the dissolution of the effective components, promoting the synthesis and secretion of hematopoietic growth factor (HGF) and the proliferation of hematopoietic progenitor cells (HPCs).

Plasma levels of soluble receptor for advanced glycation end products in Alzheimer's disease.

BACKGROUND: A decreased plasma level of soluble form of the receptor for advanced glycation end products (sRAGE) in patients with Alzheimer's disease (AD) has been reported. However, no evidence has shown whether the sRAGE plasma level of AD patients may differentiate from other types of dementia. METHODS: Our study assessed sRAGE concentrations in the following 121 individuals in Chongqing area: 36 patients with AD, 12 with vascular dementia (VaD), 14 with mixed dementia (MD), 24 with other dementia (OD) including Parkinson's disease dementia, frontotemporal dementia, paralytic dementia and 35 cognitively normal controls. The total plasma level of sRAGE was determined using sandwich ELISA method. RESULTS: sRAGE concentration in AD is significantly decreased compared with healthy controls. However, the receiver operating characteristic curve analysis of sRAGE between the AD and the control shows a low diagnostic accuracy. CONCLUSIONS: Our results demonstrate that sRAGE may assist the diagnosis of AD from normal individuals, but cannot differentiate AD from VaD, MD or OD.

[Effects of Astragali Radix combined with Angelicae Sinensis Radix on the proliferation of hematopoietic stem cells senescence model in mice].

The aim is to study the effect and its mechanism of Astragalus Radix combined with Angelicae Sinensis Radix on the proliferation of hematopoietic stem cells(HSCs) in senescence model. After drug-containing plasma of rats was prepared via intragastric administration, HSCs of mice were cultured in vitro, and then they were divided into blank control group, model group, blank plasma group, Astragalus Radix + Angelicae Sinensis Radix 1∶1 group and 10∶1 group, Angelicae Sinensis Radix plasma group, and Astragalus Radix plasma group. HSCs senescence model was induced by using tert-butyl hydrogen peroxide(t-BHP), and intervened by drug-containing plasma. Cells senescence rate was tested by SA-ß-galactosidase staining method; cell cycle distribution was determined by flow cytometry; Cyclin D1, P21, and P53 mRNA were measured with RT-PCR, and Cyclin D1 protein expression was measured by Western blot. Results showed that after being induced by t-BHP, senescence rate of HSCs was increased; cell proliferation ability was decreased; count of G0/G1 phase cells was increased; count of G2/M+S phase cells was reduced; Cyclin D1 expression was down-regulated while P53, P21 expression was up-regulated, which were reversed by Astragalus Radix + Angelicae Sinensis Radix 1∶1 and 10∶1, single Angelicae Sinensis Radix, and single Astragalus Radix plasma. Furthermore, the above effects were most obvious in Astragalus Radix+Angelicae Sinensis Radix 1∶1 group. These results suggested that t-BHP can promote HSCs senescence and reduce cell proliferation ability. Angelicae Sinensis Radix, Astragalus Radix and their combinations can inhibit HSCs senescence, promote HSCs proliferation as well as cell cycle conversion; moreover, the effects of 1∶1 Astragalus Radix+Angelicae Sinensis Radix were strongest. The mechanisms may be related to up-regulating the expression of cell cycle positive regulator, down-regulating the expression of cell cycle negative regulator, thus promoting the cells to enter the proliferation phase from the stationary phase.

[Effect of astragaloside â £ combined with Panax notoginseng saponins on cerebral ischemia-reperfusion injury and study of pharmacokinetics in rats].

The aim is to study the effect of astragaloside Ⅳ (AST Ⅳ) combined with Panax notoginseng saponins (PNS) on cerebral ischemia-reperfusion injury, and to probe the synergistic mechanism through the pharmacokinetics of the four major components such as AST Ⅳ, ginsenoside Rg1 (Rg1), ginsenoside Rb1 (Rb1), notoginsenoside R1 (R1) in cerebral ischemia-reperfusion rats. Following the establishment of cerebral ischemia/reperfusion model in rats by modified suture method, neurological function score, cerebral infarction area and pathomorphology were used to evaluate the pharmacological effect that the combination of AST Ⅳ and PNS antagonized cerebral ischemia-reperfusion injury; the contents of AST Ⅳ, Rg1, Rb1, R1 in rat plasma of different time points were determined with ultra performance liquid chromatography tandem massspectrometry (UPLC-MS/MS), pharmacokinetic parameters were calculated and pharmacokinetics changes of the main effective components were analyzed. The results showed that AST Ⅳ, PNS alone and their combination could reduce the cerebral infarction area of rats, relieve the behavioral scores of neurologic deficit, improve the pathological changes after cerebral ischemia, the effects of the combination were better. Among AST Ⅳ, Rg1, Rb1, R1, the area under the curve (AUC) was significantly increased, the mean residence time of (MRT0-t) was delayed, the peak concentration (Cmax) was significantly raised, the apparent volume of distribution (Vz/F) was reduced, and the clearance rate in vivo was significantly slowed. It suggested that AST Ⅳ combined with PNS has synergistic enhancement on anti-cerebral ischemia/reperfusion injury, moreover, make the pharmacokinetic behavior of the main effective components change, the mechanism may be associated with prolonging the retention time of the effective components in cerebral ischemia condition, elevating the bioavailability.

[Effects of glycosides components and combinations of Buyang Huanwu decoction on vascular smooth muscle cells proliferation and related signaling pathway].

This paper was aimed to explore the effects of glycosides, the effective component of Buyang Huanwu decoction, and its main active components such as astragaloside Ⅳ, amygdalin, peoniflorin and their combinations on vascular smooth muscle cells (VSMC) proliferation, clarify the major active materials of anti-VSMC proliferation and investigate the mechanisms via the signal transduction pathway. Plasma containing drug was prepared via oral administration in rats. VSMCs of rats aorta were cultured, and then VSMC proliferation was stimulated by using platelet derived growth factor (PDGF).The plasma containing drug was added to detect the activity of cell proliferation, cell cycle and related protein expressions of signaling pathway such as extracellular signal-regulated kinase (ERK), phos-phatidylinositol-3-kinase/protein kinase B (PI3K/Akt) and Janus kinase/signal transducer and activator of transcription (JAK/STAT). After being stimulated by PDGF, the proliferation activity of VSMC was strengthened (P<0.01), G0/G1 phase cells were decreased (P<0.01), S/M phase cells were increased (P<0.01), and PcNA, cyclin D1 protein expressions related to cell cycle were up-regulated (P<0.01). Glycosides, astragaloside Ⅳ, amygdalin, peoniflorin and their combinations could inhibit the cell proliferation (P<0.05 or P<0.01) in a dose-effect relationship and time-effect relationship. They could increase G0/G1 phase cells (P<0.01), decrease S/M phase cells (P<0.01), and down-regulate the protein expressions of PCNA, cyclin D1 (P<0.01); and the effects of the combinations were greater than those of single active component (P<0.05). After VSMC proliferation was induced by PDGF, p-ERK1/2 expression was increased (P<0.01), PI3K expression was down-regulated while p-PI3K expression was up-regulated (all P<0.01), and STAT3expression was reduced while p-STAT3 expression was increased (all P<0.01). Glycosides, astragaloside Ⅳ, amygdalin, peoniflorin and the combinations of these active components could reduce p-ERK1/2 expression (P<0.05), increase PI3K expression (P<0.01), decreasep-PI3K expression (P<0.05 or P<0.01), increase STAT3 expression (P<0.01), and decrease p-STAT3 expression (P<0.05 or P<0.01). These results suggested that PDGF could induce the cell cycle conversion of VSMC, leading to VSMC proliferation. The mechanism was related to the activation of ERK, PI3K/Akt and JAK/STAT signaling pathways. Glycosides and its main active components such as astragaloside Ⅳ, amygdalin, peoniflorin and their combinations can inhibit the cell cycle conversion of VSMC, with the effect against VSMC proliferation, and the mechanisms may be associated with the inhibition of PI3K/Akt, mitogen-activated protein kinase (MAPK) and JAK/STAT signaling pathways. astragaloside Ⅳ, amygdalin and peoniflorin were the major active materials of anti-VSMC proliferation, and their combination showed enhanced effect.

Effects of borneol combined with astragaloside IV and Panax notoginseng saponins regulation of microglia polarization to promote neurogenesis after cerebral ischaemia.

OBJECTIVE: To study the effect of borneol combined with astragaloside IV and Panax notoginseng saponins (BAP) on promoting neurogenesis by regulating microglia polarization after cerebral ischaemia-reperfusion(CI/R) in rats. METHODS: A focal CI/R injury model was established. Evaluated the effects of BAP on ischaemic brain injury, on promoting neurogenesis, on inhibiting Inflammatory microenvironment and TLR4/MyD88/NFκB signalling pathway. A microglia oxygen-glucose deprivation reoxygenation (OGD/R) model was established that evaluated the effects of BAP on regulating the polarization of microglia and inflammatory microenvironment. RESULTS: BAP can inhibit the expression of TLR4, MyD88 and NFκB proteins, reduce IL-1ß and increase IL-10, reduce M1 type microglia and increase M2 microglia. The proliferation of neural stem cells increased, synaptic gap decreased, synaptic interface curvature increased, expression of SYN and PSD95 proteins increased, which improved the neurological dysfunction and reduced the volume of cerebellar infarction and nerve cell injury. CONCLUSION: BAP can reduce CI/R injury and promote neurogenesis, the effect is related to inhibition of the activation of TLR4/MyD88/NFκB, regulating the polarization of microglia from M1 type to M2 type and inhibition of inflammatory response.

Astragalus extract alleviates nerve injury after cerebral ischemia by improving energy metabolism and inhibiting apoptosis.

This aim of this study was to explore the effects and molecular mechanisms of Astragalus extract against cerebral ischemia injury through the energy metabolism and apoptosis pathways of c­Jun N-terminal kinase (JNK) signal transduction. After the bilateral common carotid artery of C57BL/6 mice was occluded for 20 min followed by 1-h reperfusion, the ATP content, total adenine nucleotides (TAN), energy charge (EC), and sodium potassium ATPase (Na(+)-K(+)­ATPase) activity were decreased markedly in brain tissues. Astragalus extract markedly increased the ATP and ADP levels, EC value, and Na(+)-K(+)-ATPase activity. Twenty-four and 48 h after reperfusion, the neurocyte survival rate decreased and apoptosis rate increased, while the expression of phosphorylated JNK1/2, cytochrome c (Cyt C), and cysteine aspartic acid-specific protease (caspase)-9 and -3 were significantly enhanced in brain tissues. Astragalus extract significantly increased neurocyte survival and decreased the apoptosis rate as well as down-regulated the expression of p-JNK1/2, Cyt C, caspase-9, and caspase-3. These results suggest that Astragalus extract has neuroprotective effects against nerve injury after cerebral ischemia-reperfusion, and the underlying mechanism may be associated with improved cellular energy metabolism, inhibition of JNK signal transduction pathway activation, and then suppression of the mitochondrial apoptosis pathway.

[Effects of the combination of active component extracts from Astragalus membranaceus and Panax notoginseng on apoptosis, reactive oxygen species and mitochondrial membrane potential of PC12 cells with oxidative injury].

OBJECTIVE: To explore the effects and mechanisms of combining astragaloside IV (the effective component of Astragalus membranaceus) with notoginsenoside R1, ginsenoside Rb1 and ginsenoside Rg1 (the effective components of Panax notoginseng) against oxidative injury in PC12 cells induced by cobalt chloride (CoCl2). METHODS: CoCl2 was used to stimulate PC12 cells to induce injury after transdifferentiation with nerve growth factor. Then the PC12 cells were divided into 10 groups and cultured with corresponding drugs. After culture, apoptotic cells were tested by using Hocchst 33258 fluorescent staining, the level of mitochondrial membrane potential (MMP) was analyzed by rhodamine 123 fluorescent staining and the content of reactive oxygen species (ROS) in PC12 cell was measured by dichlorofluorescin diacetate fluorescent staining. RESULTS: CoCl2 induced apoptosis along with the obvious decrease of MMP as well as overproduction of ROS in PC12 cells. Astragaloside IV, ginsenosides Rg1, ginsenosides Rb1 and notoginsenoside R1 had inhibition effects in different degree on PC12 cell apoptosis induced by CoCl2, reduced the overproduction of ROS and the decrease of MMP. The effects of the combination were better than those of active component alone. CONCLUSION: Active components extracted from Astragalus and Panax notoginseng can inhibit PC12 cell apoptosis induced by oxidative injury, furthermore, the effects were enhanced by combination of these components, which may be associated with jointly antagonizing the generation of ROS and raising MMP.

Borneol enhances the protective effect against cerebral ischemia/reperfusion injury by promoting the access of astragaloside IV and the components of Panax notoginseng saponins into the brain.

BACKGROUND: Astragalus and Panax notoginseng are significant traditional Chinese medicines for treating ischemic stroke, with astragaloside IV (AST IV) and Panax notoginseng saponins (PNS) being the major effective compounds, respectively. These compounds can also be used in combination. We have previously shown that AST IV and PNS have an antagonistic effect on cerebral ischemia/reperfusion (I/R) injury, and the combination of these two drugs can elevate this effect; unfortunately, AST IV and PNS cannot easily enter the brain tissues through the blood brain barrier (BBB). Previous studies have confirmed that the combination of borneol with other agents could promote the penetration of the drug components through the BBB. However, it remains unclear whether borneol can promote entry of the active components of AST IV and PNS into the brain tissues and enhance their effect against cerebral ischemia. OBJECTIVE: This study aimed to investigate the effects of a combination of borneol with AST IV and PNS against I/R injury and explore the mechanisms of borneol-promoting penetration of drug components into the BBB based on the drug transport of brain tissues. METHODS: A rat model of focal cerebral I/R injury was established, and drugs, including borneol, AST IV, and PNS, as well as their combinations were intragastrically administered. Subsequently, drug efficacy was assessed, and the condition of AST IV and PNS active components (Rg1, Rb1, R1) delivered into the brain was analyzed. Moreover, BBB permeability was determined, and the expression of related drug transporters and their genes were evaluated. RESULTS: After treatment with borneol, AST IV, PNS, AST Ⅳ+PNS, and borneol+AST Ⅳ+PNS after cerebral I/R, the neurological function deficit scores, cerebral infarct rate, and brain water content markedly decreased. The effects of the three-drug-combination were better than those of the drugs used alone and those of AST Ⅳ+PNS. Moreover, after I/R in rats, AST IV and the components of PNS (Rg1, Rb1, R1) were mainly found in the cerebral cortex and in the cerebellum, respectively, when used alone. Borneol combined with AST IV and PNS increased the contents of AST IV, Rb1, Rg1, and R1 in the cerebral cortex and in the cerebellum, thus, promoting the enrichment of active components to the cerebral cortex, especially to the affected side. In addition, following I/R, diffuse distribution of lanthanum particles in the basement membrane, intercellular and intracellular locations of rat brain tissues indicated BBB destruction and increase in permeability, which were alleviated in each drug group. The effects of borneol combined with AST IV and PNS were stronger than those of the drug single-used and those of the AST IV+PNS group. Finally, the expression of effluent transporters (ET) and their genes, including P-glycoprotein (P-gp), multidrug resistance protein (MRP)-1, MRP-2, MRP-4, and MRP-5 in brain tissues, strikingly increased after I/R. Borneol remarkedly down-regulated the protein expression of P-gp, MRP-2, and MRP-4 in the brain, whereas PNS down-regulated MRP-4 and MRP-5 protein expression. AST IV, AST IV+PNS, and bornoel+AST IV+PNS effectively decreased the expression of P-gp, MRP-2, MRP-4, and MRP-5 proteins. The effects of the three-drug combination were significantly greater than those of the drug single-used and AST IV+PNS groups. The expression of each ET gene manifested corresponding results. Meanwhile, PNS, AST IV+PNS, and bornoel+AST IV+PNS significantly inhibited the down-regulation of the uptake transporter organic anion transporting polypeptide (OATP)-2 expression, and the effect of bornoel+AST IV+PNS was stronger than that of other groups. CONCLUSION: After I/R, the brain tissues were injured, BBB permeability increased, expression of critical ET and their genes were markedly up-regulated, and the main uptake transporters were down-regulated. We propose that the combination of borneol, AST IV and PNS could enhance the effect against cerebral I/R injury and protect BBB integrity. The potential mechanism might be the delivery of AST IV and active components of PNS to the brain tissues after treatment in combination with borneol, which could be effectively promoted by down-regulating the expression of ETs and up-regulating the expression of uptake transporters in the brain tissues. This study was the first to demonstrate that borneol combined with AST IV+PNS enhanced the effect against cerebral I/R injury through promoting the entry of AST and PNS active components to the brain tissues. Thus, this study proposes an instructive role in developing effective active ingredients combination of Chinese medicine with clear ingredients and synergistic effects in terms of the characteristic of borneol.

Effect of the combination of astragaloside IV and Panax notoginseng saponins on pyroptosis and necroptosis in rat models of cerebral ischemia-reperfusion.

Pyroptosis and necroptosis are closely associated with the mechanism underlying cerebral ischemia-reperfusion (I/R) injury. The combination of astragaloside IV (AST IV) and Panax notoginseng saponins (PNS) has remarkable effects on the alleviation of cerebral I/R damage. However, whether inhibition of pyroptosis and necroptosis is the mechanism underlying the beneficial effects of this drug combination on cerebral I/R injury remains unclear. To explore the effects and mechanisms of drug treatment, middle cerebral artery occlusion was performed to induce I/R injury in rats, which was verified based on neurological deficit score (NDS), infarct volume and H&E staining. Activation of pyroptosis and necroptosis was detected by western blot analysis of associated proteins. The results of the present study demonstrated that treatment with AST IV and PNS, either alone or in combination, significantly reduced the NDS, cerebral infarct volume and cell injury rate in the cerebral cortex of rats. The treatments also improved pathological injury to the cerebral cortex and reduced the levels of proteins associated with pyroptosis and necroptosis. These effects were stronger in the combination drug group compared with groups treated with a single drug alone. The findings of the present study suggested that the combination of AST IV and PNS exhibited stronger neuroprotective effects in I/R injury than either drug alone, and that the underlying mechanism was associated with inhibition of pyroptosis and necroptosis.

Main Active Components and Cell Cycle Regulation Mechanism of Astragali Radix and Angelicae Sinensis Radix in the Treatment of Ox-LDL-Induced HUVECs Injury and Inhibition of Their Cell Cycle.

To explore the main active components and effects of cell cycle regulation mechanism of Astragali radix (AR) and Angelicae sinensis radix (ASR) on oxidative damage in vascular endothelial cells, a model of oxidative damage in human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL) treatment was developed. Based on the "knock-out/knock-in" model of the target component, cell viability, intracellular reactive oxygen species (ROS), and lactate dehydrogenase (LDH) leakage were assessed by Cell Counting Kit-8 assay, fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and colorimetric assay, respectively, to evaluate the protective effect of the active components of AR and ASR (astragaloside IV (AS IV), astragaloside I (AS I), formononetin (FRM), calycosin (CAL), calycosin-7-O-ß-D glucoside (CLG), and ferulic acid (FRA)) against oxidative damage. The cell cycle and expression of genes encoding cyclins and cyclin-dependent kinases (CDKs) were observed using flow cytometry and quantitative real-time polymerase chain reaction. The results showed that the combination of active components (ACC) significantly inhibited the decrease in cell viability as well as the increase in ROS and LDH release in HUVECs induced by ox-LDL treatment. AS IV and FRM promoted the proliferation of HUVECs but the proliferation index was decreased in the AS I and FRA groups; this inhibitory effect was counteracted by the ACC. The ACC reduced and increased the proportion of positive cells in G1 and S phases, respectively, followed by the upregulation of cyclin A (CCNA), cyclin E (CCNE), and CDK2 mRNA expression and downregulation of cyclin B (CCNB), cyclin D1 (CCND1), CDK1, CDK4, and CDK6 mRNA expression, which significantly mitigated inhibition of HUVECs proliferation induced by ox-LDL treatment. Taken together, AS IV, AS I, FRM, CAL, CLG, and FRA were the primary pharmacodynamic substances of AR and ASR that alleviated oxidative injury in HUVECs. ACC mitigated the upregulation of intracellular ROS levels and LDH release induced by ox-LDL treatment, which promoted the cell cycle procession of HUVECs by regulating the expression of genes encoding cyclins and CDKs and thus preventing oxidative damage in HUVECs.

[Thinking and practice on research of effective components of traditional Chinese medicine compound prescriptions].

By analyzing current research status and considering our own previous research results, we hold that research on active substance of Chinese herbal formula should be focused on the effect of active components according to its functions, thus determining the main effective components of traditional Chinese medicine (TCM) compound prescriptions. Study of compatibility of effective components should be carried out and compound prescriptions composed of effective components should be developed. It is a pivotal way to deepen research on active components of TCM compound prescription by further analyzing the compatibility of effective components and the relationship between functional components and active components by multilink and multitarget methods.