BACKGROUND

Historically, traditional Chinese medicine has been widely used to treat stroke. Based on the theory of Chinese medicine and the modern pharmacological knowledge of herbal medicines, we have designed a neuroprotective formula called Post-Stroke Rehabilitation (PSR), comprising seven herbs - Astragalus membranaceus (Fisch.) Bunge, Salvia miltiorrhiza Bunge, Paeonia lactiflora Pall., Cassia obtusifolia L., Ligusticum chuanxiong Hort., Angelica sinensis (Oliv.) Diels, and Glycyrrhiza uralensis Fisch. We aim to examine the neuroprotective activity of PSR in vitro and in vivo, and to explore the underlying molecular mechanisms, to better understand its therapeutic effect and to further optimize its efficacy.

METHODS

PSR extract or vehicle was applied to primary rat neurons to examine their survival effects against N-methyl-D-aspartate (NMDA)-elicited excitotoxicity. Whole-cell patch-clamp recording was conducted to examine the NMDA-induced current in the presence of PSR. ERK- and CREB-activation were revealed by western blot analysis. Furthermore, PSR was tested for CRE promoter activation in neurons transfected with a luciferase reporter. The protective effect of PSR was then studied in the rat middle cerebral artery occlusion (MCAO) model. MCAO rats were either treated with PSR extract or vehicle, and their neurobehavioral deficit and cerebral infarct were evaluated. Statistical differences were analyzed by ANOVA or t-test.

RESULTS

PSR prominently reduced the death of cultured neurons caused by NMDA excitotoxicity in a dose-dependent manner, indicating its neuroprotective property. Furthermore, PSR significantly reduced NMDA-evoked current reversibly and activated phosphorylation of ERK and CREB with distinct time courses, with the latter's kinetics slower. PSR also triggered CRE-promoter activity as revealed by the increased expression of luciferase reporter in transfected neurons. PSR effectively reduced cerebral infarct and deficit in neurological behavior in MCAO rats when PSR decoction was administered starting either 6 days before or 6 h after onset of ischemia.

CONCLUSIONS

PSR is neuroprotective both in vitro and in vivo - it protects cultured neurons against NMDA excitotoxicity, and effectively reduces ischemic injury and neurobehavioral deficit in MCAO rats in both the pre- and post-treatment regimens. The underlying neuroprotective mechanisms may involve inhibition of NMDA receptor current and activation of ERK and CREB. This study provides important preclinical data necessary for the further development of PSR for stroke treatment.

BACKGROUND

Haemorrhagic transformation is an asymptomatic event that frequently occurs after following ischaemic stroke, particularly when pharmaceutical thrombolysis is used. However, the mechanism responsible for haemorrhagic transformation remains unknown, and therapeutics have not been identified. In this study, we administered a combination of astragalus membranaceus and ligustrazine to rats with cerebral ischaemia that had undergone thrombolysis. We analysed the effect of this combination on the attenuation of haemorrhagic transformation and the maintenance of blood-brain barrier integrity.

METHODS

A rat model of focal cerebral ischaemia was induced with autologous blood clot injections. Thrombolysis was performed via the intravenous injection of rt-PA. Astragalus membranaceus, ligustrazine or a combination of Astragalus membranaceus and ligustrazine was administered immediately after the clot injection. The cerebral infarct area, neurological deficits, blood-brain barrier integrity, and cerebral haemorrhage status were determined after 3, 6 and 24h of ischaemia. The ultrastructure of the blood-brain barrier was examined with a transmission electron microscope. The expression of tight junction proteins, including claudin-1, claudin-5, occludin, and zonula occludens-1, and matrix metallopeptidase-9 activation was further evaluated in terms of their roles in the protective effects of the combination drug on the integrity of the blood-brain barrier.

RESULTS

Ischaemia-induced Evans blue leakage and cerebral haemorrhage were markedly reduced in the combination drug-treated rats compared to the rats treated with either astragalus membranaceus or ligustrazine alone (p<0.05). The disruption of the ultrastructure of the blood-brain barrier and the neurological deficits were ameliorated by the combination treatment (p<0.05). The reductions in the expression of laudin-1, claudin-5, occludin, and ZO-1 were smaller in the rats that received the combination treatment. In addition, MMP-9 activity was suppressed in the combination-treated rats compared to the controls (p<0.05).

CONCLUSIONS

Treatment with a combination of astragalus membranaceus and ligustrazine alleviated ischaemia-induced micro-haemorrhage transformation by maintaining the integrity of the blood-brain barrier.

Astragaloside IV (AsIV) is an active saponin extracted from Astragalus membranaceus, which has shown cardioprotective effects in a number of experimental animals. In this study we investigated the molecular mechanisms by which AsIV attenuated the myocardial ischemia reperfusion (MI/R)-induced injury in vitro and in vivo by focusing on calcium-sensing receptor (CaSR) and extracellular signal-regulated kinase 1/2 (ERK1/2). Rat neonatal cardiac myocytes were subjected to a hypoxia/reoxygenation (H/R) procedure in vitro, which significantly decreased the cell viability, increased lactate dehydrogenase (LDH) release, induced cardiomyocyte apoptosis, and increased [Ca2+]i. H/R also increased the expression of CaSR and decreased ERK1/2 phosphorylation levels in H/R-exposed myocytes. Pretreatment with AsIV (60 μmol/L) significantly improved the cell viability and decreased LDH release, attenuated myocyte apoptosis, decreased [Ca2+]i and CaSR expression, and increased the ERK1/2 phosphorylation levels. The protective effects of AsIV against H/R injury were partially inhibited by co-treatment with a CaSR agonist, gadolinium chloride (GdCl3) or with a specific ERK1/2 inhibitor U0126. For in vivo studies, a rat MI/R model was established. Pre-administration of AsIV (80 mg/kg every day, ig) significantly decreased the myocardium infarct size, creatine kinase-MB (CK-MB) production, serum cardiac troponin (cTnI) levels, and cardiomyocyte apoptosis in the rats with MI/R injury. The therapeutic effects of AsIV were associated with the downregulation of CaSR expression and upregulation of ERK1/2 phosphorylation in myocardial tissues. In summary, astragaloside IV attenuates myocardial I/R injury via inhibition of CaSR/ERK1/2 and the related apoptotic signaling pathways.

Diabetic retinopathy (DR) is a severe complication of diabetes mellitus (DM) and often causes vision loss or even blindness. Vascular endothelial growth factor (VEGF) in the retina, which is mainly derived from Müller cells, is a crucial biological factor in the development of DR. Astragalin is extracted from Astragalus membranaceus and has many pharmacological properties. Studies showed that astragalin has beneficial effects on hyperglycemia. To evaluate the effect of astragalin in preventing and treating DR and determine astragalin's mechanism of action, Müller cells were collected from rat retina, cultured in vitro and identified using immunocytochemistry. They were divided into four groups: the high glucose group (20 mmol/l), the normal control group, the astragalin group (400 mg/l) and the high glucose (20 mmol/l) + astragalin (400 mg/l) group. After 3 days of treatment, immunocytochemical and reverse transcription-polymerase chain reaction (RT-PCR) analysis of VEGF was carried out. Our results demonstrated that astragalin decreased the overexpression of VEGF in Müller cells and alleviated the effects caused by high glucose. Thus, astragalin has promising application in preventing and treating DR caused by DM.

BACKGROUND

Astragalus membranaceus Bunge (Leguminosae) and Salvia miltiorrhiza Bunge (Lamiaceae) are two important Chinese herbs with a long history of extensive ethnobotanical usage in the treatment of liver-related diseases over many centuries. Presently, these two herbs are being used either as a single herbal formulation or a composite formula for the treatment of liver related conditions. In response, recent studies on these two herbs have focused on elucidating their mechanisms of action, particularly with regards to their anti-hepatocarcinogenic effects. Previously, we have reported that Compound Astragalus and Salvia miltiorrhiza extract (CASE), a synergized composite extract from Astragalus membranaceus and Salvia miltiorrhiza ameliorates liver fibrosis and hepatocellular carcinoma (HCC) by modulating the TGF-β/Smad pathway. Meanwhile, MAPK activation and MAPK-dependent linker phosphorylation of Smad2/3 and their preferential nuclear import are crucial for overall oncogenic role of TGF-β/Smad signaling in HCC. To elucidate further, we studied the effect of CASE on the MAPK pathway and how it affects MAPK-dependent regulation of TGF-β/Smad signaling using both cell and animal models of HCC.

METHODS

We used immunofluorescence and western blot techniques to monitor effect of CASE on the activation of the MAPKs (pERK, pJNK and pp38) in TGF-β1-stimulated hepatic stellate cells (HSCs), HepG2 cells and also diethylnitrosamine (DEN)-induced HCC in rats. Also phosphorylation and subcellular distribution of pSmad2/3, Smad4 and Imp7/8 in TGF-β1-stimulated HSC and HepG2 cells were monitored. The expression of pERK, pJNK, pp38 and PAI-1 gene were monitored by using western blot technique. The effect of CASE on domain-specific phosphorylation of Smad2/3 and their subcellular distribution, and the expression of Smad4 and its subcellular distribution in TGF-β1-stimulated HSCs and HepG2 cells were evaluated by using immunofluorescence technique. And the expression of Imp7/8 and their subcellular distribution were assessed by both immunofluorescence and western blot techniques, while PAI-1 gene expression was assessed by western blot

RESULTS

In vitro, CASE in a concentration-dependent manner increased the expression of pp38 but decreased the expression of pERK and pJNK; however, in vivo, CASE in a dose dependent manner decreased the expression of pERK, pJNK as well as pp38. Also, CASE concentration dependently inhibited pSmad2C/L, pSmad3L, Smad4, Imp7/8 and their nuclear import; it had no effect on pSmad3C in HepG2 cells; significantly decreased PAI-1 gene expression in both in vitro and in vivo.

CONCLUSIONS

CASE blocked MAPK activation, MAPK-dependent linker phosphorylation of Smad2/3, Smad4 expression, Imp7 expression and their nuclear import leading to significant down-regulation of PAI-1 gene expression; further highlighting the multi-target anti-HCC effect of CASE and its potential drug candidature.

Refractory nephrotic syndrome (RNS) is an immune-related kidney disease with poor clinical outcomes. Standard treatments include corticosteroids as the initial therapy and other immunosuppressants as second-line options. A substantial proportion of patients with RNS are resistant to or dependent on immunosuppressive drugs and often experience unremitting edema and proteinuria, cycles of remission and relapse, and/or serious adverse events due to long-term immunosuppression. Traditional Chinese medicine has a long history of treating complicated kidney diseases and holds great potential for providing effective treatments for RNS. This review describes the Chinese medical theories relating to the pathogenesis of RNS and discusses the strategies and treatment options using Chinese herbal medicine. Available preclinical and clinical evidence strongly supports the integration of traditional Chinese medicine and Western medicine for improving the outcome of RNS. Herbal medicine such as Astragalus membranaceus, Stephania tetrandra S. Moore, and Tripterygium wilfordii Hook F can serve as the alternative therapy when patients fail to respond to immunosuppression or as the complementary therapy to improve therapeutic efficacy and reduce side effects of immunosuppressive agents. Wuzhi capsules (Schisandra sphenanthera extract) with tacrolimus and tetrandrine with corticosteroids are two herb-drug combinations that have shown great promise and warrant further studies.

Astragalus polysaccharide (APS) is an important bioactive component extracted from Chinese herb Astragalus membranaceus. It has been widely used in treatment of cardiovascular diseases. We have previously reported that APS could inhibit isoproterenol-induced cardiac hypertrophy. The present study was designed to evaluate the protective effect of APS on vascular endothelia in cardiac hypertrophy rats induced by isoproterenol (ISO). ISO (10mg×kg(-1)) was intraperitoneally injected once daily for 2weeks to induce cardiac hypertrophy. APS (400 and 800mg×kg(-1)) was intragastrically injected once daily along with ISO. The results showed that combination with APS significantly ameliorates the endothelial dysfunction while attenuates cardiac hypertrophy induced by ISO. We found that administration with APS could attenuate the increase in number of circulating endothelial cell (CEC). APS also decreases the superoxide anion generation and the protein expression of p65 and the levels of TNF-α and IL-6; while increases the cGMP levels, an activity marker for nitric oxide (NO) in aortas. In addition, APS improves the relaxation dysfunction in isolated aortic rings and increases the protein expression of IκBα and Cu/Zn-SOD in aortas. In conclusion, our results suggested that APS had a protective effect against endothelial dysfunction in hypertrophic rats induced by ISO. The underlining mechanisms may be contributed to the anti-inflammatory effects and the improvement of the imbalance between reactive oxygen species (ROS) and NO.

BACKGROUND

Postmenopausal osteoporosis is one of the most common disorders in women after menopause, which is linked to an estrogen deficiency and characterized by an excessive loss of trabecular bone. Rubus coreanus and Astragalus membranaceus have been used for their various pharmacological properties in Asia as a traditional medicine. The present study evaluated the anti-osteoporotic effects of the optimal combination of Rubus coreanus and Astragalus membranaceus in 7:3 mixture (RAM) in ovariectomized (OVX) mice by investigating bone biomechanical properties and the serum levels of TNF-α, osteocalcin, RANKL, OPG, and RANK-RANKL signal-related osteoclast differentiation markers.

METHODS

A total of 36 mature female outbred ICR (Institute of cancer research) strain mice (7 weeks) were divided into 6 groups with 7 mice in each group as follows: (1) Sham-operated control mice (Sham) received daily oral phosphate-buffered-saline (PBS) of equal volumes through gavage. (2) OVX mice received a daily oral gavage of PBS (OVX). (3) OVX mice were treated daily with 50mg/kgb.w./day of RAM (4) with 100mg/kgb.w./day of RAM or (5) with 200mg/kgb.w./day of RAM via oral gavage. (6) OVX mice received i.p. injections of 17β-estradiol (E2) (0.1mg/kgb.w./day) three times per week for 12 weeks.

RESULTS

Micro-CT images showed that oral administration of RAM to OVX mice prevented tibial bone loss, preserved trabecular bone microarchitecture, and improved bone biomechanical properties. RAM administration also showed recovery effects on the levels of TNF-α, OPG and RANKL concentration in OVX-states. Additionally, we found that the mechanism by which RAM elicited anti-osteoporotic effects was by down-regulating the expression of TRAF6 and NFATc1 in RANKL-RANK pathway, a route of osteoclast differentiation, followed by reducing the production of osteoclast differentiation factors, calcitonin receptors and cathepsin K.

CONCLUSIONS

Our research strongly suggests that RAM can be clinically used in the prevention and treatment of postmenopausal osteoporosis.

Endothelial dysfunction is a key pathophysiological step in early stage diabetes mellitus (DM) macrovascular complications and is also crucial in the inflammatory mechanisms of macrovascular complications. However, there is currently no effective intervention to improve endothelial dysfunction associated with DM macrovascular complications. Astragaloside IV (AS‑IV), which can be extracted from the traditional Chinese medicine Astragalus membranaceus, has potential therapeutic effects on DM and its complications. The present study evaluated the effect of AS‑IV on high glucose‑induced human umbilical vein endothelial cell (HUVEC) injury and its possible mechanism. The result indicated that AS‑IV has a significant protective effect on high glucose‑induced HUVEC injury. AS‑IV could significantly promote cell proliferation, reduce apoptosis and decrease the protein and mRNA expression levels of tumor necrosis factor‑α and interleukin‑1β in HUVECs. Furthermore, AS‑IV could decrease the expression of phosphorylated c‑Jun NH2‑terminal kinase (JNK) phosphorylated apoptosis signal‑regulating kinase 1, cytochrome c, cleaved‑caspase‑9, cleaved‑caspase‑3 and the relative ratio of B‑cell lymphoma‑2 associated X protein/B‑cell lymphoma‑2 in HUVECs. In conclusion, the present study demonstrated that AS‑IV could suppress apoptosis and inflammatory reactions promoted by high glucose conditions in HUVECs by inhibiting the JNK signaling pathway. These findings suggest that AS‑IV could inhibit the process of endothelial dysfunction in diabetic macrovascular complications.

Traditional Chinese medicine (TCM) has from ancient times been applied in China for the treatment of breast cancer with its own unique theoretical system. Sanhuang decoction composed of astragalus membranaceus, prepared rhubarb, and rhizoma curcumae longae has traditionally been used for antioxidant stress, inflammatory reaction, and angiogenesis. However, the role and mechanism of Sanhuang decoction in breast cancer remains unknown. The present study demonstrated the antitumor activity of Sanhuang decoction against breast cancer xenografts in nude mice. Notably, Sanhuang decoction promoted severe necrosis and induced cell death. In addition, Sanhuang decoction obviously regulated the inflammation and oxidative stress. Despite these, Sanhuang decoction could increase the expression of Nrf2. Moreover, si-Nrf2 exhibited the opposite effects compared with the Sanhuang decoction treatment group and reversed the antibreast cancer role of Sanhuang decoction. Further, Sanhuang decoction remarkably suppressed the expression of PI3K/AKT/mTOR signaling pathway. Taken together, Sanhuang decoction was firstly evaluated to possess potent antibreast cancer effect in vivo through regulation of inflammation and oxidative stress accomplished by up-regulation of Nrf2 via PI3K/AKT/mTOR signaling pathway and Sanhuang decoction might be a powerful candidate formula for antibreast cancer.

Astragalus polysaccharides (ASP) is extracted from Astragalus, and is the main active ingredient of Astragalus membranaceus. The purpose of this study was to investigate the protective effect of ASP on rat cardiomyocytes damage induced by myocardial ischemia and reperfusion injury (MVRI) and isoprenaline(ISO) in vivo and in vitro. The model of cardiomyocytes damage was induced using MVRI in a rat in vivo and also using ISO in cell. After ASP intervention, the protective effect of ASP on cardiomyocytes was evaluated by animal experimental and cell experimental. The results show that ASP can relieve the increase of cell volume in myocardium, reduce the apoptosis of cell in myocardial tissue caused by MVRI in vivo. At the cellular level, ASP can reverse the decrease of cell activity induced by ISO, inhibit the apoptosis, and decrease the levels of intracellular reactive oxygen species. Mechanistically at the molecular level, these effects are elicited via down-regulation of the protein levels of caspase-3 and bax and up-regulation of the protein levels of bcl-2 in both in vivo and in vitro. These results demonstrate that ASP has a protective efficacy in MVRI/ISO-treated cardiomyocytes by inhibiting the apoptosis.

As iron supplement, the antioxidant activities of APS-iron (III) complex were comprehensively evaluated by 5-axe cobweb charts, which indicated the APS-iron (III) complex had a certain antioxidant activity and been weaker than that of APS. The results of immunological activity experiments indicated the stimulation index increased with APS-iron (III) complex concentration increase. When the concentration of the APS-iron (III) complex was 50 μg/mL, the lymphocytes proliferation increased by 35.7% compared with APS. APS-iron (III) complex also had better complement fixing activity than APS, 0.589 mg/mL of which achieved 50% complement fixing activities. Through the iron supplement experiments on iron-deficiency anemia mouse model, we found the APS-iron (III) complex faster increased hemoglobin concentration, SOD, CAT and faster decreased MDA to the normal level than Niferex and ferrous sulfate. Histological results revealed that the tissue sections were clear without obvious pathological changes and bone marrow had most hematopoietic cells from APS-iron (III) complex rat group, which also proved the APS-iron (III) complex had no significant side effects. Therefore, APS-iron (III) complex may be developed as a multifunctional iron supplement for clinical application.

Agroastragaloside II, a new astragaloside was isolated from the hairy root culture of Astragalus membranaceus. Its structure was established as 3-O-beta-(2'-O-acetyl)-D-xylopyranosyl-6-O-beta-D-glucopyranosyl-(24S)- 3 beta,6 alpha,16 beta,24,25-pentahydroxy-9,19-cyclolanostane on the basis of spectroscopic data. Three known astragalosides, astragaloside II, isoastragaloside I and 3-O-beta-D-xylopyranosyl-cycloastragenol were also isolated.

Carbohydrates in herbs are a relatively untapped source of new drugs and health beneficial ingredients. Their analysis has been developed as a novel aspect in quality control and herbal glycomics. In this study, glycome of Astragalus membranaceus was decoded based on optimized pressurized liquid extraction (PLE), microwave-assisted acidic hydrolysis (MAAH) and comprehensive chromatographic approaches. Twelve saccharides including sucrose, galacturonic acid, mannitol, fructose, rhamnose, ribose, arabinose, fucose, xylose, mannose, glucose and galactose were quantitatively analyzed by GC-MS and HPLC-CAD (charged aerosol detectors). Different columns, including Prevail Carbohydrate ES, XBridge Amide and CARBOSep CHO-820 CA for HPLC-CAD analysis, were compared for evaluation of oligosaccharides. The polysaccharides in water extract of Astragalus membranaceus were characterized by high performance size exclusive chromatography (HPSEC) combined with multiple angle light scattering detection (MALSD) and refractive index detection (RID). The results showed that A. membranaceus contained more than 108.5mgg(-1) free sucrose and small amounts of glucose 9.6-26.0mgg(-1) and fructose 8.7-22.9mgg(-1). While its polymeric carbohydrates were composed of glucose 71.0-162.3mgg(-1), galacturonic acid 52.0-113.4mgg(-1), arabinose 22.8-54.4mgg(-1) and small amounts of galactose, rhamnose, xylose and mannose. CARBOSep CHO-820 CA showed its potential in simultaneously analyzing oligosaccharides and uronic acid, especially only the environment-friendly water mobile phase was used. HPSEC-MALSD-RID showed that there were three different molecular weight distributions of polysaccharides in A. membranaceus and the average molecular weight were 21901.1, 2038.5, and 353.4kDa. Hierarchical clustering analysis and principal component analysis demonstrated that A. membranaceus from different regions showed variations both in free and polymeric carbohydrates, which indicated that carbohydrates should be evaluated for the proper quality control of A. membranaceus. Rha, Ara, Xyl, Man and Gal were found to be the main elements for quality evaluation of polymeric carbohydrates in A. membranaceus by factor analysis. The strategy for decoding the glycome based on chromatographic approaches including GC-MS, HPLC-CAD and HPSEC-MALSD-RID after pressurized liquid extraction and microwave-assisted hydrolysis could be applied for carbohydrates profiling in herbs and beneficial for their quality control.

Astragalus is one of the most popular Chinese herbal. Control of Astragalus quantity is most important, since that various varieties and ages largely affect bioactive metabolites and different pharmacological effects. Astragalus mongholicus and Astragalus membranaceus are both major sources of Astragalus according to the provisions in the Chinese Pharmacopoeia. Thus, a sensitive and rapid UPLC-MS/MS method for the simultaneous determination of l-Phenylalanine, Isoliquiritigenin, Liquiritigenin, Daidzein, Formononetin, Ononin, Calycosin, Calycosin-7-glucoside, Cycloastragenol, Astragaloside I, Astragaloside II, Astragaloside III and Astragaloside IV was established in this study. The detection was accomplished by MRM scanning in the positive ionization mode. Calibration curves offered linear ranges with r2>> 0.999. The method was successfully validated for the linearity, intra-day and inter day precisions, accuracy, recovery, matrix effect and stability. Then this method was successfully applied to detect the contents of 13 target flavonoids and triterpenoids metabolites in different organs and ages of A. mongholicus and A. membranaceus. Significant organs-, ages- and varieties- specificity of the 13 target metabolites were observed and discussed. The results provided basis and support for further exploration of the distribution of bioactive metabolites, namely flavonoids and triterpenoids, in different organs and ages of two Astragalus varieties. This method should be applicable to various Astragalus matrices for the quantitative analysis of the target flavonoids and triterpenoids.

Cycloastragenol (CAG) is an aglycone of astragaloside IV. It was first identified when screening Astragalus membranaceus extracts for active ingredients with antiaging properties. The present study demonstrates that CAG stimulates telomerase activity and cell proliferation in human neonatal keratinocytes. In particular, CAG promotes scratch wound closure of human neonatal keratinocyte monolayers in vitro. The distinct telomerase-activating property of CAG prompted evaluation of its potential application in the treatment of neurological disorders. Accordingly, CAG induced telomerase activity and cAMP response element binding (CREB) activation in PC12 cells and primary neurons. Blockade of CREB expression in neuronal cells by RNA interference reduced basal telomerase activity, and CAG was no longer efficacious in increasing telomerase activity. CAG treatment not only induced the expression of bcl2, a CREB-regulated gene, but also the expression of telomerase reverse transcriptase in primary cortical neurons. Interestingly, oral administration of CAG for 7 days attenuated depression-like behavior in experimental mice. In conclusion, CAG stimulates telomerase activity in human neonatal keratinocytes and rat neuronal cells, and induces CREB activation followed by tert and bcl2 expression. Furthermore, CAG may have a novel therapeutic role in depression.

Astragalus polysaccharide is an important bioactive component of Astragalus membranaceus, an herb used in traditional Chinese medicine for treating inflammatory bowel disease. The NOD-like receptor protein 3 inflammasome plays an important role in the pathogenesis of inflammatory bowel disease. However, little is known about the role of NOD-like receptor protein 3 inflammasome in Astragalus polysaccharide-treated mice with experimental colitis. For this study, we investigated the molecular mechanisms that underlie the treatment of inflammatory bowel disease by Astragalus polysaccharide. We show that Astragalus polysaccharide treatment reduces the disease activity index and histological injury scores compared to the colitis model group. Astragalus polysaccharide also effectively inhibited the expression of NOD-like receptor protein 3, apoptotic speck protein containing a c-terminal caspase recruitment domain, caspase-1, interleukin-18, and interleukin-1β, as shown by quantificational RT-PCR or the enzyme-linked immunosorbent assay. Furthermore, Astragalus polysaccharide treatments produced significant dose-dependent improvements in dextran sulfate sodium-induced experimental colitis. Our data provide the reliable evidence that Astragalus polysaccharide is able to exert a therapeutic effect in dextran sulfate sodium-induced colitis by inhibiting the activation of the NOD-like receptor protein 3 inflammasome, which acts to decrease the production of inflammatory factors such as interleukin-18 and interleukin-1β.

Astragali Radix, the root of Astragalus membranaceus Bunge, is one of the most frequently used crude drugs in Asian medicine. We developed a quantification method for 6 components (calycosin, formononetin, astragaloside I-IV) of Astragali Radix and Hwanggi-gyeji-omul-tang (HGOT) using reverse-phase high-performance liquid chromatography coupled with integrated pulsed amperometric detection (RP-HPLC-IPAD). The plants were extracted in 80% ethanol for 2h. All target components were detected with good sensitivity using sodium hydroxide (as a post-column eluent). The limit of detection (S/N=3) and limit of quantification (S/N=10) of the target components ranged from 0.10-1.00ng and from 0.30-3.00ng, respectively. The coefficients of linear regression ranged from 0.9993-1.0000, all interday and intraday precision values were <3.64%, and the average recovery ranged from 99.00-102.97% for Astragali Radix and 97.73-102.57% for HGOT. This method exhibited good selectivity, sensitivity, and reproducibility and can be used directly without any pretreatment steps. Our method will therefore be useful as a quality control measure for Astragali Radix.

Our previous studies have shown that the combination of Astragalus membranaceus and Salvia miltiorrhiza (HD) had a good antihypertensive effect, but its potential mechanism remained unclear. This study aimed to investigate the role of intestinal flora and serum metabolism induced by HD against hypertension. 16 spontaneous hypertensive rats (SHRs) were divided into HD group (5.9 g/kg) and model group (M) (normal saline), with eight Wistar-Kyoto (WKY) rats as control group (W) (normal saline). Rats were fed by gavage once a day for 28 days. The changes of intestinal flora and serum metabolism were analyzed by 16S rDNA sequencing and LC-MS/MS assay. HD decreased blood pressure steadily, improved the structure and composition of imbalance flora in SHRs, increased the abundance and diversity of flora, and decreased flora Firmicutes to Bacteroidetes (F/B) ratio. Rumen bacterium NK4A214, Clostridium sp. MC 40 increased remarkably in M group. Akkermansia, Akkermansia muciniphila, and Lactobacillus intestinalis increased significantly in HD group, which were functionally related to the significant increase of Lachnoclostridium, Faecalibaculum, and Lactobacillus reuteri in W group, which were all probiotics producing butyric acid, lactic acid, and regulating inflammation and other antihypertensive related factors. HD also changed the serum metabolic pattern of SHRs. 16 potential biomarkers related to inflammation, vasodilation, steroid hormones, oxidative stress, and etc. changed significantly, mainly enriched in arachidonic acid metabolism, tryptophan metabolism, steroid hormone biosynthesis, and glutathione metabolism. The correlation analysis demonstrated that the dominant genius and species in three groups were highly correlated with steroid hormone biosynthesis, arachidonic acid metabolism, tryptophan metabolism, and vitamin B6 metabolism. Our research indicated that HD had a good antihypertensive effect, which may be driven by the protective intestinal flora and beneficial metabolites induced by it, and the metabolites were closely related to the changes of intestinal flora. It provided new insights for the antihypertensive mechanism of HD.

OBJECTIVE

Increasing energy expenditure through adipocyte thermogenesis is generally accepted as a promising strategy to mitigate obesity and its related diseases. However, few clinically effective and safe agents are known to promote adipocyte thermogenesis. In this study, 20 traditional Chinese herbal medicines were screened to examine whether they induced adipocyte thermogenesis.

METHODS

The effects of Chinese herbal medicines or components isolated from extracts of A. membranaceus, on adipocyte thermogenesis were analysed by assessing expression of uncoupling protein 1 (UCP1) by qPCR. Eight-week-old C57BL6/J male mice were fed a high-fat diet for 8 weeks and then randomized to two groups treated with vehicle or formononetin for another 8 weeks. Glucose tolerance tests and staining of adipose tissue with haematoxylin and eosin were carried out. Whole-body oxygen consumption was measured with an open-circuit indirect calorimetry system.

RESULTS

Extracts of A. membranaceus increased expression of Ucp1 in primary cultures of mouse adipocytes. Formononetin was the only known component of A. membranaceus extracts to increase adipocyte Ucp1 expression. Diet-induced obese mice treated with formononetin gained less weight and showed higher energy expenditure than untreated mice. In addition, formononetin binds directly with PPARγ.

CONCLUSIONS

Taken together, our study demonstrates that the Chinese herbal medicine from A. membranaceus and its constituent formononetin have the potential to reduce obesity and associated metabolic disorders. Our results suggest that formononetin regulates adipocyte thermogenesis as a non-classical PPARγ agonist.

BACKGROUND

Astragaloside IV, a major component extracted from the roots of Astragalus membranaceus (AM), possesses anti-inflammatory, anti-oxidative, anti-fibrotic, anti-infarction and immunoregulatory effects. To clarify anti-stress effect of AM, anxiolytic and anti-inflammatory effects of 80% ethanol extract of AM and astragaloside IV were investigated in immobilization stress model.

METHODS

The mice were orally administered with AM (50, 200, and 500 mg/kg), astragaloside IV (5, 10, and 20 mg/kg) and buspirone, a positive drug, 1h before immobilization treated for 2h. For anxiolytic activity assay, EPM test was performed in mice. For anti-inflammatory activity assay, serum levels of corticosterone, IL-6 and TNF-α were measured using ELISA kits.

RESULTS

AM extract and astragaloside IV increased dose-dependently time spent on open arms and open arm entries in the EPM test. Anxiolytic effects of AM extract (500 mg/kg) and astragaloside IV (20 mg/kg) were comparable to those of buspirone (1 mg/kg). Their anxiolytic effects were blocked by WAY-100635 (0.5 mg/kg, i.p.), a 5-HT1A receptor antagonist (p<0.01), but not by flumazenil (3 mg/kg, i.p.) and bicuculline (0.5 mg/kg, i.p.), GABAA receptor antagonists. AM extract and astragaloside IV also reduced serum levels of corticosterone, IL-6 and TNF-α dose-dependently.

CONCLUSIONS

AM, particularly astragaloside IV, may ameliorate immobilized stress-induced anxiety and inflammation.

Ethnopharmacological relevance: Astragalus membranaceus and Codonopsis pilosula which are two Chinese medicinal herbs are often combinedly used as monarch drugs in Traditional Chinese Medicine (TCM) prescriptions to treat ulcerative colitis (UC). However, the exact mechanisms and effective constituents of the two herbs remain unclear.

Aim of the study: Polysaccharides are the main active ingredients of the two medicinal herbs and some specific polysaccharides extracted from the two medicinal herbs have been proven effective in relieving colitis. Hence, we speculated that polysaccharides of the two medicinal herbs may be the material basis for compatibility in TCM prescriptions to treat UC. In the research, total polysaccharides of A. membranaceus and C. pilosula extractum, named AERP and CERP respectively, were administrated to 2.5% dextran sulfate sodium (DSS)-induced acute colitis mice by dosing alone and in combination to test this hypothesis.

Materials and methods: 5-aminosalicylic acid (5-ASA, 100 mg/kg/d) was selected as the positive drug. The basic indexes of colitis mice including body weight, stool bleeding, stool consistency and colon lengths were recorded. In addition, tissue inflammatory factors, mucosa-associated proteins, fecal short chain fatty acids (SCFAs) and gut microbiota were also analyzed.

Results: The co-administration of AERP and CERP at specific doses could improve the clinical symptoms, reestablish the immune balance, and alleviate colonic mucosal injury in colitis mice. The unique efficacy of co-administration relied on activation of the aryl hydrocarbon receptor (AhR) and up-regulation of isovaleric acid and butyrate. In addition, the structure of intestinal flora was recovered in the co-administration group.

Conclusion: Our research revealed the efficacy after co-administration of total polysaccharides from A. membranaceus and C. pilosula on colitis which provided a theoretical basis for their compatibility in TCM prescriptions to treat UC.

Keywords: aryl hydrocarbon receptor; gut microbiota; polysaccharide; short chain fatty acids; ulcerative colitis.