[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.

CTNNAL1 promotes the structural integrity of bronchial epithelial cells through the RhoA/ROCK1 pathway.

Adhesion molecules play critical roles in maintaining the structural integrity of the airway epithelium in airways under stress. Previously, we reported that catenin alpha-like 1 (CTNNAL1) is downregulated in an asthma animal model and upregulated at the edge of human bronchial epithelial cells (HBECs) after ozone stress. In this work, we explore the potential role of CTNNAL1 in the structural adhesion of HBECs and its possible mechanism. We construct a CTNNAL1 ‒/‒ mouse model with CTNNAL1-RNAi recombinant adeno-associated virus (AAV) in the lung and a CTNNAL1-silencing cell line stably transfected with CTNNAL1-siRNA recombinant plasmids. Hematoxylin and eosin (HE) staining reveals that CTNNAL1 ‒/‒ mice have denuded epithelial cells and structural damage to the airway. Silencing of CTNNAL1 in HBECs inhibits cell proliferation and weakens extracellular matrix adhesion and intercellular adhesion, possibly through the action of the cytoskeleton. We also find that the expressions of the structural adhesion-related molecules E-cadherin, integrin ß1, and integrin ß4 are significantly decreased in ozone-treated cells than in vector control cells. In addition, our results show that the expression levels of RhoA/ROCK1 are decreased after CTNNAL1 silencing. Treatment with Y27632, a ROCK inhibitor, abolished the expressions of adhesion molecules induced by ozone in CTNNAL1-overexpressing HBECs. Overall, the findings of the present study suggest that CTNNAL1 plays a critical role in maintaining the structural integrity of the airway epithelium under ozone challenge, and is associated with epithelial cytoskeleton dynamics and the expressions of adhesion-related molecules via the RhoA/ROCK1 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.

Glycosides of Buyang Huanwu decoction inhibits pyroptosis associated with cerebral ischemia-reperfusion through Nrf2-mediated antioxidant signaling pathway both in vivo and in vitro.

BACKGROUND: Glycosides are the pharmacodynamic substances of Buyang Huanwu Decoction (BYHWD) and they exert a protective effect in the brain by inhibiting neuronal pyroptosis of cerebral ischemia-reperfusion (CIR). However, the mechanism by which glycosides regulate neuronal pyroptosis of CIR is still unclear. PURPOSE: A significant part of this study aimed to demonstrate whether glycosides have an anti-pyroptotic effect on CIR by nuclear factor erythroid 2-related factor (Nrf2)-mediated antioxidative mechanism. METHODS: Rats were used in vivo models of middle cerebral artery occlusion and reperfusion (MCAO/R). Neuroprotective effect of glycosides after Nrf2 inhibiting was observed by nerve function score, Nissl staining, Nrf2 fluorescence staining and pyroptotic proteins detection. SH-SY5Y cells were used in vitro models of oxygen-glucose deprivation/reperfusion (OGD/R). Glycosides was evaluated for their effects by measuring cell morphology, survival rate, lactate dehydrogenase (LDH) rate and expression of pyroptotic proteins. Nrf2 si-RNA 54-1 interference with lentivirus was used to create silenced Nrf2 SH-SY5Y cells (si-Nrf2-SH-SY5Y). Glycosides were evaluated on si-Con-SH-SY5Y and si-Nrf2-SH-SY5Y cells based on the expression of Nrf2 signaling pathway, pyroptotic proteins and cell damage manifestation. RESULTS: In vivo, glycosides significantly promoted the fluorescence level of nuclear Nrf2, added more Nissl bodies, reduced neurological function scores and inhibited the pyroptotic proteins level. In vitro, glycosides significantly repaired the morphological damage of cells, promoted the survival rate, reduced the LDH rate, inhibited the pyroptosis. Moreover, antioxidant activity of glycosides was enhanced via Nrf2 activation. Both Nrf2 blocking in vivo and Nrf2 silencing in vitro significantly weakened the pyroptosis inhibitory and neuroprotective effects of glycosides. CONCLUSION: These results suggested for the first time that glycosides inhibited neuronal pyroptosis by regulating the Nrf2-mediated antioxidant stress pathway, thereby exerting brain protection of CIR. As a result of this study, This study improved understanding of the pharmacodynamics and mechanism of BYHWD, as well as providing a Traditional Chinese Medicine (TCM) treatment strategy for CIR .

Daucosterol combined with umbilical cord mesenchymal stem cell-derived exosomes can alleviate liver damage in liver failure mice by regulating the IL-6/STAT3 signaling pathway.

Daucosterol is a phytosterol glycoside with hepatoprotective properties. The objective of the present study was to confirm the role of daucosterol in liver failure. Exosomes were isolated from primary mouse umbilical cord mesenchymal stem cells (UCMSCs). A liver failure mouse model was generated by injecting lipopolysaccharide/D-galactosamine. Mice were treated with exosomes alone or in combination with daucosterol (5, 10, or 20 mg/kg). Liver tissue damage was examined by hematoxylin-eosin, Masson's trichrome, and TUNEL staining. The levels of genes, proteins, and inflammatory factors were determined using real-time qPCR, western blotting, and enzyme-linked immunosorbent assay, respectively. Compared with normal mice, we noted severe damage, fibrosis, and apoptosis in the liver tissues of liver failure-induced mice. UCMSC-derived exosomes effectively alleviated hepatic damage in the mouse model. Compared with exosome treatment alone, exosomes combined with daucosterol significantly and dose-dependently reduced pathological changes in model mice. Exosome treatment alone or combined with daucosterol also markedly decreased the liver index and reduced levels of alanine aminotransferase, aspartate aminotransferase, tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 in model mice. Exosome treatment alone or combined with daucosterol suppressed mRNA expression levels of IL-6 and signal transducer and activator of transcription (STAT3) and STAT3 protein expression in model mice. Our findings revealed that treatment with daucosterol combined with UCMSC-derived exosomes was superior to exosomes alone for alleviating hepatic damage in mice with liver failure by regulating the IL-6/STAT3 signaling pathway. Accordingly, daucosterol combined with UCMSC-derived exosomes may be a prospective treatment strategy for liver failure.

[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.

ALDH2 gene rs671 G > a polymorphism and the risk of colorectal cancer: A hospital-based study.

BACKGROUND: The susceptibility to some cancers is linked to genetic factors, such as aldehyde dehydrogenase 2 (ALDH2) polymorphisms. The relationship between ALDH2 rs671 and colorectal cancer (CRC) is not clear in Hakka population. METHODS: Between October 2015 and December 2020, a total of 178 CRC patients and 261 controls were recruited. ALDH2 rs671 was genotyped in these subjects, medical records (smoking history, drinking history and blood cell parameters) were collected, and the relationship between these information and CRC was analyzed. RESULTS: The proportion of the ALDH2 rs671 G/G, G/A, and A/A genotype was 48.3%, 44.4%, and 7.3% in patients; 62.1%, 34.1%, and 3.8% in controls, respectively. The difference of ALDH2 genotypes distribution between cases and controls was statistically significant (p = 0.011). The higher percentage of smokers and alcoholics, higher level of neutrophil to lymphocyte ratio (NLR), platelet count, and platelet to lymphocyte ratio (PLR), and lower level of lymphocyte count, lymphocyte to monocyte ratio (LMR), and mean hemoglobin concentration were observed in patients. Logistic regression analysis indicated that ALDH2 rs671 G/A genotype (G/A vs. G/G) (adjusted OR 1.801, 95% CI 1.160-2.794, p = 0.009) and A/A genotype (A/A vs. G/G) (adjusted OR 2.630, 95% CI 1.041-6.645, p = 0.041) in the co-dominant model, while G/A + A/A genotypes (G/A + A/A vs. G/G) (adjusted OR 1.883, 95% CI 1.230-2.881, p = 0.004) in the dominant model were risk factors for CRC. CONCLUSIONS: Individuals carrying ALDH2 rs671 A allele (G/A, A/A genotypes) may be at increased risk of colorectal cancer.

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.

Correlation Analysis of C-terminal telopeptide of collagen type II and Interleukin-1ß for Early Diagnosis of Knee Osteoarthritis.

OBJECTIVE: To analyze the correlation between the Kellgren-Lawrence (K-L) score of knee osteoarthritis (KOA) patients with different degrees and their urine concentration of C-terminal telopeptide of collagen type II (CTX-II) and interleukin-1ß (IL-1ß), and to further evaluate the diagnostic value of CTX-II and IL-1ß during the pathological process by producing an experimental osteoarthritis (OA) model in rabbits. METHODS: From 1 January 2017 to 31 December 2018, a total of 34 subjects (7 mild, 9 moderate, 9 severe arthritis patients, and 9 healthy individuals) comprising 16 men and 18 women were included in this study. Patients were diagnosed according to the American College of Rheumatology (ACR) criteria. The urine of all subjects was collected to detect the concentration of CTX-II and IL-1ß. The rabbits in the KOA group were subjected to protease (control group with saline) injection into the articular cavity of their right knees and immobilization with gypsum. We used radiological and histological examination to identify the KOA model. ELISA was applied to investigate the concentrations of CTX-II and IL-1ß in urine and serum, and Spearman's rank correlation analysis was used to analyze the correlation. RESULTS: There was no significant difference in the mean ages and body mass index (BMI) between groups. The mean ages of mild, moderate, and severe arthritis patients and healthy individuals were 54.29 ± 5.76, 58.44 ± 6.44, 59.89 ± 6.75, and 56.67 ± 4.18 years, respectively. The mean BMI of mild, moderate, and severe arthritis patients and healthy individuals were 23.59 ± 1.56, 23.57 ± 2.06, 24.46 ± 1.64, and 23.42 ± 1.35 kg/m2 , respectively. The Kellgren-Lawrence (K-L) score was higher with the aggravation of KOA. The K-L scores of mild, moderate, and severe KOA patients were 1.14 ± 0.38, 2.56 ± 0.53, and 3.63 ± 0.52, respectively. The KOA symptoms of patients became more severe, with not only increased K-L scores but also elevated concentrations of CTX-II and IL-1ß. Moreover, there was a positive correlation between CTX-II and IL-1ß of all subjects (r = 0.974, P < 0.001), between K-L score and urine concentration of CTX-II (r = 0.900, P < 0.001), and between K-L score and IL-1ß (r = 0.813, P < 0.001) of all subjects. Both were significantly increased in KOA group rabbits at all time points after surgery. The serum concentration of CTX-II and IL-1ß was elevated as early as in the 2nd week (3.69 and 4.25 times) and reached a peak (5.41 and 7.23 times) in the 4th week after surgery. Then, until 12 weeks after surgery, the CTX-II and IL-1ß concentrations in the KOA group were slightly reduced and remained around 4.5 and 6.3 times that in the control group. Moreover, there was a positive correlation between the serum concentration of IL-1ß and CTX-II (r = 0.967, P < 0.001). CONCLUSION: CTX-II and IL-1ß, which were significantly increased during the process of KOA, can be used as biomolecular markers to provide guidelines for early diagnosis and treatment of KOA.

Five Active Components Compatibility of Astragali Radix and Angelicae Sinensis Radix Protect Hematopoietic Function Against Cyclophosphamide-Induced Injury in Mice and t-BHP-Induced Injury in HSCs.

Although the compatibility of Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) has favorable effect on promoting hematopoiesis in traditional Chinese medicine (TCM), the main active components and pharmacological mechanism are unknown. We investigated the five active components and its mechanisms in vitro and in vivo. Five active components of Astragalus glycosides (AST), Formononetin (FRM), Ferulic acid (FRA), Calycosin (CAL), and Calycosin-7-glucoside (CLG), which could be absorbed in intestinal tract, were detected in this study. The peripheral blood, hematopoietic growth factors (HGFs), and hematopoietic progenitor cells (HPCs) colony were observed to evaluate the effect of these five active components promoting hematopoiesis. Furthermore, hematopoietic stem cell (HSC) proliferation, aging, cycle, and related proteins were detected to explore the mechanism of these five components promoting HSC proliferation. i) The in vivo experiments showed that the combination of the five active components could remarkably increase the number of RBCs, WBCs, PLTs, and content of Hb in peripheral blood and the area of bone marrow hematopoietic tissue, as well as thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), and colony of CFU-GM, CFU-MK, CFU-E, and BFU-E in serum. Each of these five components promoted the recovery of RBCs and Hb, and increased TPO, CFU-MK, and CFU-E. All components except for AST increased the CFU-GM. FRA increased the number of WBCs, the area of bone marrow hematopoietic tissue, and BFU-E. FRA and AST promoted PLT recovery. FRA and CAL improved the content of GM-CSF. FRA, CAL, and CLG improved the content of EPO. ii) The in vitro experiments showed that FRA, FRM, and AST significantly promoted cell proliferation, reduced the positive rate and G0/G1 cells, and increased G2/M + S cells and the expression of cyclin D1 and CDK4 proteins in aging HSCs. Furthermore, the combination of five components had the best effect. Taken together, the five active components of AST, FRM, FRA, CAL, and CLG were the main pharmacodynamic substances of the AR-ASR compatibility, which promoted hematopoiesis. The combination of them had a synergistic effect. The mechanism of promoting hematopoiesis may be relevant to regulating cyclin-related proteins, promoting cell cycle transformation, and promoting HSC proliferation.

Assessment of the Relationship between Carotid Intima-Media Thickening and Early-Stage Diabetic Kidney Disease Coupled with Helicobacter pylori Infection.

OBJECTIVE: This study aimed to explore the associations between carotid intima-media thickness (CIMT) and early-stage diabetic kidney disease (DKD) coupled with Helicobacter pylori (H. pylori) infection in type 2 diabetic patients. METHODS: A cross-sectional study including 180 type 2 diabetic participants was conducted to explore the associations between CIMT and early-stage DKD coupled with H. pylori infection, and a stepwise multivariate regression analysis evaluated the correlations of CIMT with clinical and serologic parameters. RESULTS: The type 2 diabetic patients with early-stage DKD coupled with H. pylori infections had the highest CIMT values. Apolipoprotein B (ApoB), urine albumin/creatinine ratio (UACR), and interleukin-6 (IL-6) were independent predictors of CIMT. CONCLUSIONS: Early-stage DKD coupled with H. pylori infection may synergistically lead to significant CIMT thickening in type 2 diabetic patients. Additionally, ApoB, UACR, and IL-6 levels were important independent risk factors for increased CIMT.

[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.

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).

Synergism and mechanism of Astragaloside IV combined with Ginsenoside Rg1 against autophagic injury of PC12 cells induced by oxygen glucose deprivation/reoxygenation.

The aim of this study was to explore the effect by which the combination of Astragaloside IV (AST IV) and Ginsenoside Rg1 (Rg1) resisted autophagic injury in PC12 cells induced by oxygen glucose deprivation/reoxygenation (OGD/R). We studied the nature of the interaction between AST IV and Rg1 that inhibited autophagy through the Isobologram method, and investigated the synergistic mechanism via the PI3K I/Akt/mTOR and PI3K III/Becline-1/Bcl-2 signaling pathways. Our results showed that, based on the 50% inhibiting concentration (IC50), AST IV combined with Rg1 at a 1:1 ratio resulted in a synergistic effect, whereas the combination of the two had an antagonistic effect on autophagy at ratios of 1:2 and 2:1. Meanwhile, AST IV and Rg1 alone increased cell survival and decreased lactate dehydrogenase (LDH) leakage induced by OGD/R, reduced autophagosomes and the LC3 II positive patch, down-regulated the LC3 II/LC3 I ratio and up-regulated the p62 protein; the 1:1 combination enhanced these effects. Mechanistic study showed that Rg1 and the 1:1 combination increased the phosphorylation of PI3K I, Akt and mTOR; the effects of the combination were greater than those of the drugs alone. AST IV and the 1:1 combination suppressed the expression of PI3K III and Becline-1, and the combination elevated Bcl-2 protein expression; the effects of the combination were better than those of the drugs alone. These results suggest that after 2 h-OGD followed by reoxygenation for 24h, PC12 cells suffer excessive autophagy and damage, which are blocked by AST IV or Rg1; moreover, the combination of AST IV and Rg1 at a 1:1 ratio of their IC50 concentrations has a synergistic inhibition on autophagic injury. The synergistic mechanism may be associated with the PI3K I/Akt/mTOR and PI3K III/Becline-1/Bcl-2 signaling pathways.

Combination of total Astragalus extract and total Panax notoginseng saponins strengthened the protective effects on brain damage through improving energy metabolism and inhibiting apoptosis after cerebral ischemia-reperfusion in mice.

OBJECTIVE: To explore the effects and molecular mechanisms of the combination between total Astragalus extract (TAE) and total Panax notoginseng saponins (TPNS) against cerebral ischemia-reperfusion injury. METHODS: C57BL/6 mice were randomly divided into sham-operated group, model group, TAE (110 mg/kg) group, TPNS (115 mg/kg) group, TAE-TPNS combination group and Edaravone (4 mg/kg) group, treated for 4 days, then, cerebral ischemia-reperfusion injury was established by bilateral common carotid artery (CCA) ligation for 20 min followed by reperfusion for 1 and 24 h. RESULTS: TPNS could increase adenosine triphosphate (ATP) level, TAE and TAE-TPNS combination increased ATP, adenosine diphosphate (ADP) contents and Na+-K+-ATPase activity, and the effects of TAE-TPNS combination were stronger than those of TAE or TPNS alone after reperfusion for 1 h. After reperfusion for 24 h, TAE, TPNS and TAE-TPNS combination significantly increased neurocyte survival rate and decreased the apoptosis rate as well as down-regulated the expression of phosphorylated c-June N-terminal kinase1/2 (p-JNK1/2), cytochrome C (Cyt C), cysteine aspartic acid-specific protease (Caspase)-9 and Caspase-3. Furthermore, the effects in TAE-TPNS combination were better than those in TAE or TPNS alone. CONCLUSION: The combination of TAE 110 mg/kg and TPNS 115 mg/kg could strengthen protective effects on cerebral ischemia injury, the mechanism underlying might be related to improving jointly the early energy metabolism, and relieving the delayed apoptosis via inhibiting the mitochondrial apoptosis pathway of JNK signal transduction.

Effects of the main active components combinations of Astragalus and Panax notoginseng on energy metabolism in brain tissues after cerebral ischemia-reperfusion in mice.

BACKGROUND: Astragalus and Panax notoginseng are traditional Chinese medicines used for the treatments of cardio-cerebrovascular ischemic diseases, astragaloside IV (AST IV) and ginsenoside Rg1 (Rg1), ginsenoside Rb1 (Rb1), notoginsenoside R1 (R1) are their active components. OBJECTIVE: The purpose of this work was to investigate the effect of AST IV combined with Rg1, Rb1, R1 on energy metabolism in brain tissues after cerebral ischemia-reperfusion in mice. MATERIALS AND METHODS: C57BL/6 mice were randomly divided into 11 groups, treated for 3 days. At 1 h after the last administration, the model of cerebral ischemia-reperfusion injury was established, and brain tissues were detected. RESULTS: All drugs increased the contents of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and the level of total adenine nucleotides (TAN), the combinations increased energy charge (EC), the effects of four active components combination were better. The phosphorylation of AMP-activated protein kinaseα1/2 (p-AMPKα1/2) was increased in AST IV, R1, four active components combination, AST IV + Rg1 and AST IV + R1 groups, the increased effect of four active components combination was greater than that of the active components alone and AST IV + Rb1. All drugs increased glucose transporter 3 (GLUT3) mRNA and protein, and the increases of four active components combination were more obvious than those of the active components alone or some two active components combinations. CONCLUSION: Four active components combination of Astragalus and P. notoginseng have the potentiation on improving of energy metabolism, the mechanism underlying might be associated with promoting the activation of AMPKα1/2, enhancing the expression of GLUT3, thus mediating glucose into nerve cells, increasing the supply and intake of glucose.

Autophagy in cerebral ischemia and the effects of traditional Chinese medicine.

Autophagy is a lysosome-mediated degradation process for non-essential or damaged cellular constituents, playing an important homeostatic role in cell survival, differentiation and development to maintain homeostasis. Autophagy is involved in tumors as well as neurodegenerative, cardiovascular and cerebrovascular diseases. Recently, active compounds from traditional Chinese medicine (TCM) have been found to modulate the levels of autophagy in tumor cells, nerve cells, myocardial cells and endothelial cells. Ischemic stroke is a major cause of neurological disability and places a heavy burden on family and society. Regaining function can significantly reduce dependence and improve the quality of life of stroke survivors. In healthy cells, autophagy plays a key role in adapting to nutritional deprivation and eliminating aggregated proteins, however inappropriate activation of autophagy may lead to cell death in cerebral ischemia. This paper reviews the process and the molecular basis of autophagy, as well as its roles in cerebral ischemia and the roles of TCM in modulating its activity.

Up-regulation of CXCR4 in rat umbilical mesenchymal stem cells induced by serum from rat with acute liver failure promotes stem cells migration to injured liver tissue.

The role of C-X-C chemokine receptor type 4 (CXCR4) in umbilical mesenchymal stem cells (UMSCs) as therapy for liver disease is ill understood. The aim of the study was to evaluate rat UMSCs (rUMSCs) on CXCR4 expression and homing to injured liver tissue. rUMSCs were isolated from umbilical cords of pregnant rats. Acute liver failure (ALF) models were developed using D-galactosamine. CXCR4 expression induction by serum from rats with ALF (LFS), cytokines, growth factors, and LPS was analyzed. CXCR4 expression was analyzed by RT-PCR, western blot, and flow cytometry. rUMSCs were labeled with carboxyfluorescein and pretreated with LFS to induce CXCR4 expression and were transplanted into ALF rats. Animals were sacrificed 48 h and 1 week after transplantation. Liver-homing rUMSCs were observed under fluorescence microscopy. rUMSCs were successfully isolated, expressing CD90 and CD106, but not CD34 and CD45. mRNA and protein expressions of CXCR4 were strongly up-regulated by LFS and by the mixture of cytokines, stem cell factor, and LPS (CM). Expression of cell surface CXCR4 on rUMSCs in groups treated with LFS (42.37 ± 1.60 %) and CM (40.17 ± 1.78 %) was higher than that in the untreated control group (9.67 ± 1.06 %) (both P < 0.001). At 48 h after transplantation, more rUMSCs pretreated with LFS appeared in the portal area, and migrated to the liver parenchyma after 1 week. LFS strongly induced the surface expression of CXCR4 on rUMSCs. Increasing CXCR4 expression on rUMSCs may enhance their homing ability to injured liver tissue, and may eventually be used for treating liver diseases.

[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.

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.