Astragaloside IV attenuates myocardial ischemia/reperfusion injury in rats via inhibition of calcium-sensing receptor-mediated apoptotic signaling pathways.

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.

[Effect of Combined Intervention of Electroacupuncture and Astragaloside IV on Myocardial Hypertrophy and TGF-ß 1/Smad Signaling in Rats with Myocardial Fibrosis].

OBJECTIVE: To observe the effect of combined intervention of electroacupuncture (EA) and astragaloside IV(ASIV) on cardiac hypertrophy and transforming growth factor ß 1 (TGF-ß 1)/Smad signaling in isoproterenol (ISO) induced cardiac hypertrophy rats, so as to investigate its underlying mechanisms in improving myocardial fibrosis. METHODS: A total of 50 SD rats were randomly divided into 5 groups: normal control, model (ISO), Propranolol (PRO)，ASIV and EA＋ASIV groups (n＝10 in each group). The myocardial fibrosis model was established by intraperitoneal injection (i.p.) of ISO (10 mg·kg－1·d－1), once daily for 30 days. Rats of the control group were given normal saline (i.p.), those of the PRO group given with PRO (40 mg·kg－1·d－1, gavage), and those of the ASIV and EA＋ASIV groups were treated by gavage of ASIV (40 mg·kg－1·d－1), once daily for 30 days. EA (20 Hz, 6 V) was applied to bilateral "Neiguan" (PC 6) for 10 min, once every day for 30 d. The heart mass index (HMI, whole heart weight/body weight) and left ventricular (LV) mass index (LVMI, weight of the LV/body weight) were calculated to assess the state of cardiac hypertrophy. The enzyme linked immunosorbent assay (ELISA) was used to determine the levels of procollagen I carboxy-terminal propeptide (PICP，a marker of extracellular matrix remodeling) and carboxyterminal telopeptide of type I collagen (ICTP, a metabolite of type I collagen) in serum, and Western blot was used to test protein contents of TGF- ß 1, Smad 2 / 3, Smad 4, Smad 7 in the left ventricle tissue of the heart. RESULTS: After modeling, the HMI and LVMI, serum PICP and ICTP contents and the expression levels of myocardial TGF-ß 1, Smad 2/3 and Smad 4 proteins were significantly increased in the model (ISO) group (P<0.05), suggesting a deposition of collagen and cardiac hypertrophy, and were considerably decreased in PRO, ASIV and EA＋ASIV groups after the intervention (P<0.05). The expression level of myocardial Smad 7 protein was significantly lower in the model group than in the normal control group (P<0.05), and significantly up-regulated in PRO, ASIV and EA＋ASIV groups (P<0.05). Sirius Red staining of the left ventricular myocardium showed a dense deposition of collagen and a severer myocardial fibrosis in the model group, and a relatively lighter fibrosis in the PRO, ASIV and EA＋ASIV groups. The therapeutic effects of EA＋ASIV were comparable to those of PRO, and were significantly superior to those of ASIV in down-regulating HMI, serum ICTP, and myocardial Smad 2/3 and Smad 4 expression and up-regulating Smad 7 protein (P<0.05). There were no significant differences among the PRO, ASIV and EA＋ASIV groups in LVMI, PICP and TGF-ß 1 levels, and between the PRO and EA＋ ASIV groups in HMI, ICTP, Smad 2/3, Smad 4 and Smad 7 levels (P> 0.05). CONCLUSIONS: EA stimulation of PC 6 combined with ASIV can relieve cardiac hypertrophy and myocardial fibrosis in rats, which may be associated with its effects in regulating myocardial TGF-ß 1/Smad signaling pathway.

Potential Signaling Pathways Involved in the Clinical Application of Oxymatrine.

Oxymatrine, an alkaloid component extracted from the roots of Sophora species, has been shown to have antiinflammatory, antifibrosis, and antitumor effects and the ability to protect against myocardial damage, etc. The potential signaling pathways involved in the clinical application of oxymatrine might include the TGF-ß/Smad, toll-like receptor 4/nuclear factor kappa-light-chain-enhancer of activated B cells, toll-like receptor9/TRAF6, Janus kinase/signal transduction and activator of transcription, phosphatidylinositol-3 kinase/Akt, delta-opioid receptor-arrestinl-Bcl-2, CD40, epidermal growth factor receptor, nuclear factor erythroid-2-related factor 2/heme oxygenase-1 signaling pathways, and dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine metabolism pathway. In this review, we summarize the recent investigations of the signaling pathways related to oxymatrine to provide clues and references for further studies on its clinical application. Copyright © 2016 John Wiley & Sons, Ltd.

Zhuanggu Jianxi Decoction () limits interleukin-1 ß-induced degeneration chondrocytes via the caveolin-p38 MAPK signal pathway.

OBJECTIVE: To evaluate the effect of Zhuanggu Jianxi Decoction (, ZGJXD) on interleukin-1 ß (IL-1 ß)-induced degeneration of chondrocytes (CDs) as well as the activation of caveolin-p38 mitogen-activated protein kinase (MAPK) signal pathway, investigating the possible molecular mechanism that ZGJXD treats osteoarthritis. METHODS: Serum pharmacology was applied in the present study, where ZGJXD was orally administrated to New Zealand rabbits and then ZGJXD containing serum (ZGJXD-S) was collected for following in vitro experiments. CDs were isolated aseptically from New Zealand rabbits and then cultured in vitro. Upon IL-1 ß stimulation, the degeneration of CDs was verified by inverted microscope, toluidine blue stain and type II collagen immunocytochemistry. After IL-1 ß-stimulated CDs were intervened with blank control serum, ZGJXD-S, together with or without SB203580 (a specific inhibitor of p38 MAPK) for 48 h, caveolin-1 protein expression and the phosphorylation level of p38 were determined by Western blotting, and the mRNA expression of IL-1 ß, tumor necrosis factor α (TNF-α), matrix metalloproteinase 3 (MMP-3) and MMP-13 were examined by real-time polymerase chain reaction. RESULTS: IL-1 ß stimulation induced degeneration of CDs, increased caveolin-1 expression and p38 phosphorylation, up-regulated the mRNA level of IL-1 ß, TNF-α, MMP-3 and MMP-13. However, the IL-1 ß-induced activation of caveolin-p38 signaling and alteration in the expression of p38 downstream target genes were suppressed by ZGJXD-S and/or SB203580 in CDs. CONCLUSION: ZGJXD can prevent CDs degeneration via inhibition of caveolin-p38 MAPK signal pathway, which might be one of the mechanisms that ZGJXD treats osteoarthritis.

[Effect of naringin of Drynaria Rhizome, a Chinese medical component of Zhuanggu Jianxi Recipe containing serum on caveolin-p38MAPK signal pathway in IL-1ß induced rabbit degenerated chondrocytes].

OBJECTIVE: To observe the effect of naringin of Drynaria Rhizome, a Chinese medical component of Zhuanggu Jianxi Recipe (ZJR) containing serum on caveolin-p38MAPK signal factors (such as caveolin-1, p-p38, p-ATF-2, IL-1ß, and TNF-α) in IL-1ß induced rabbit degenerated chondrocytes, and further to explore its mechanism for protecting articular cartilages. METHODS: Naringin of Drynaria Rhizome was obtained and analyzed by HPLC-TOF/MS. Four weeks old New Zealand rabbits were killed and their bilateral knee joints were isolated aseptically. CDs were isolated and then cultured in vitro. The second generation of CDs were used for later experiment. The effect of naringin on CDs proliferation was detected by MTT assay. The effect of naringin on the expression of IL-1ß-induced collagen II in CDs was detected by immunohistochemical method. The effect of naringin on caveolin-1, p-p38, and p-ATF-2 protein in IL-1ß-induced CDs was detected by Western blot. The effect of naringin on mRNA expression of IL-1ß and TNF-α in IL-1ß-induced CDs was detected by RT-PCR. RESULTS: The appearance time of naringin in flow graphs of naringin standard solution and ZJR containing serum was 23.5 min, and the molecular weight ranged between 581.0 and 581.5 m/z. Naringin could promote the proliferation of CDs, and inhibit the effect of IL-1ß on collagen II in CDs. Compared with the model group, naringin could reduce the expression of caveolin-1, p-p38, p-ATF-2, IL-1ß, and TNF-α in IL-1ß induced CDs (P < 0.05), which was approximate to the level of the normal group. CONCLUSIONS: Naringin could not only promote the proliferation of CDs, but also protect IL-1ß-induced CDs. Its mechanism might be associated with decreasing the expression of caveolin-1, p-p38, and p-ATF-2 proteins, inhibiting caveolin-p38MAPK signal pathway, and further reducing mRNA expression of IL-1ß and TNF-α in the downstream of caveolin-p38MAPK signal pathway.

Astragaloside IV attenuates inflammatory cytokines by inhibiting TLR4/NF-кB signaling pathway in isoproterenol-induced myocardial hypertrophy.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragaloside IV (As IV) is one of the main effective components isolated from the traditional Chinese medical herb Astragalus membranaceus. The protective effect of Astragalus membranaceus on myocardial hypertrophy has been extensively proved. To test the hypothesis that Astragaloside IV can ameliorate the myocardial hypertrophy and inflammatory effect induced by ß-adrenergic hyperactivity, we carried out in vivo and in vitro experiments. MATERIAL AND METHODS: In in vivo study, the isoproterenol (Iso) (5 mg kg(-1) d(-1)) was used as a model of myocardial hypertrophy by intraperitoneal injection. SD rats were randomly assigned to following six groups: A: the control; B: Iso group; C: Iso plus As IV 20 mg kg(-1) d(-1); D: Iso plus As IV 40 mg kg(-1) d(-1); E: Iso plus As IV 80 mg kg(-1) d(-1); F: Iso plus Propranolol 40 mg kg(-1) d(-1). In in vitro study, cultured neonatal rat cardiomyocytes were pretreated with As IV (3, 10, 30 µ mol L(-1)), Propranolol (2 µ mol L(-1)) and BAY11-7082 (5 µ mol L(-1)) for 30 min, and then incubated with Iso (10 µ mol L(-1)) for 48 h. For the rats in each group, the heart mass index (HMI) and the left ventricular mass index (LVMI) were measured. To measure the transverse diameter of left ventricular myocardial cells (TDM), the hematoxylin-eosin (HE) staining method was applied. In addition, the volume and the total protein content of cardiomyocytes were measured, the mRNA expression of ANP and TLR4 were quantified by RT-PCR, the protein expression of TLR4, IκBα and p65 were quantified by Western blot, and the level of TNF-α and IL-6 were measured by ELISA. RESULTS: In vivo: Comparing the Iso group to the control, the HMI, LVMI, TDM were significantly increased; the protein expression of TLR4 and p65 were increased, while the IκBα were decreased; the expression of ANP, TLR4 mRNA, and TNF-α, IL-6 in serum were significantly increased. These changes could be partly prevented by As IV and Pro. In vitro: the over-expression of the cell size, total protein content could remarkably down-regulated by As IV and Pro, and the results of RT-PCR, Western blot and ELISA were similar to those of in vivo. CONCLUSIONS: The results of these studies indicate that Astragaloside IV has good protective effect on myocardial hypertrophy induced by isoproterenol. More specifically, the cardioprotection is related to inhibiting the TLR4/NF-кB signaling pathway and the attenuating inflammatory effect.