Smilax glabra Roxb. flavonoids protect against pathological cardiac hypertrophy by inhibiting the Raf/MEK/ERK pathway: In vivo and in vitro studies.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax glabra Roxb., the dry rhizome of Sarsaparilla, which is also known as Tu fuling (TFL) in China, is a well-known traditional CHINESE medicine that is widely used for detoxication, relieving dampness and as a diuretic. We have previously shown that the extracted TFL flavonoids (designated TFLF) possess anti-cardiac hypertrophy effects in vitro. However, the anti-cardiac hypertrophy effects of TFLF in vivo and the underlying mechanisms remain to be elucidated. AIM OF THE STUDY: To reveal the underlying therapeutic mechanism of TFLF on cardiac hypertrophy by using transverse aortic constriction (TAC) model and cellular assays in vitro. MATERIAL & METHODS: Cardiac hypertrophy was replicated by TAC surgery in rats or by isoprenaline treatment of rat H9C2 myocardial cells in vitro. Cardiac structure and function were evaluated by echocardiographic and hemodynamic examinations in vivo and histological analysis of tissues ex vivo. Biochemical kits and quantitative PCR were used to analyze markers of cardiac hypertrophy. Expression and phosphorylation of key proteins in the Raf/MEK/ERK pathway were quantified by Western blotting. We further confirmed our findings in H9C2 rat cardiomyocytes treated with isoprenaline and the ERK inhibitor in vitro. RESULTS: TFLF attenuated cardiac hypertrophy and fibrosis and improved cardiac dysfunction in TAC rats. TFLF treatment induced a strong reduction in serum NT-proBNP levels. Cardiac hypertrophy marker gene (ANP, BNP and ß-MHC) expression and the phosphorylation levels of c-Raf and ERK1/2 were decreased by TFLF treatment. TFLF also protected H9C2 cells from isoprenaline-induced hypertrophy in vitro via a similar molecular mechanism as that observed in the rat heart. Moreover, pretreatment with TRLF and the ERK inhibitor further inhibited the mRNA overexpression of hypertrophic genes in vitro. CONCLUSIONS: TFLFs may protect against pathological cardiac hypertrophy via negative regulation of the Raf/MEK/ERK pathway. Thus, TFLFs are implicated as a potential pharmacological agent for treating cardiac hypertrophy in clinical practice.

Hirsutella sinensis fungus improves cardiac function in mouse model of heart failure.

Cordyceps sinensis, including Hirsutella sinensis, is a highly valuable traditional Chinese medicine and is used to treat patients with pulmonary heart disease in clinical practice. However, the underlying mechanisms of its effects remain unclear. In this study, a mouse model of heart failure established by non-thoracic, transverse aortic constriction (TAC) was developed to determine the underlying mechanisms of therapeutic effects of Hirsutella sinensis fungus (HSF) powder. The results showed that HSF treatment remarkably ameliorated myocardial hypertrophy, collagen fiber hyperplasia, and cardiac function in mice with heart failure. Using transcriptional and epigenetic analyses, we found that the mechanism of HSF mainly involved a variety of signaling pathways related to myocardial fibrosis and determined that HSF could reduce the levels of TGF-ß1 proteins in heart tissue, as well as type I and III collagen levels. These data suggest that HSF alleviates heart failure, inhibits irreversible ventricular remodeling, and improves cardiac function through the regulation of myocardial fibrosis-related signaling pathways, which can provide novel opportunities to improve heart failure therapy.

The Target MicroRNAs and Potential Underlying Mechanisms of Yiqi-Bushen-Tiaozhi Recipe against-Non-Alcoholic Steatohepatitis.

MicroRNAs (miRNAs) have emerged as potential therapeutic targets for non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH). Traditional Chineses Medicine (TCM) plays an important role in the prevention or treatment of NAFLD/NASH. However, miRNA targets of TCM against NASH still remain largely unknown. Here, we showed that Yiqi-Bushen-Tiaozhi (YBT) recipe effectively attenuated diet-induced NASH in C57BL/6 mice. To identify the miRNA targets of YBT and understand the potential underlying mechanisms, we performed network pharmacology using miRNA and mRNA deep sequencing data combined with Ingenuity Pathway Analysis (IPA). Mmu-let-7a-5p, mmu-let-7b-5p, mmu-let-7g-3p and mmu-miR-106b-3p were screened as the main targets of YBT. Our results suggested that YBT might alleviate NASH by regulating the expression of these miRNAs that potentially modulate inflammation/immunity and oxidative stress. This study provides useful information for guiding future studies on the mechanism of YBT against NASH by regulating miRNAs.

Effect of Hirsutella sinensis Fungus on the Hypothalamic-Pituitary-Adrenal Axis in Lewis Rats with Kidney-Yang Deficiency Syndrome.

Kidney-yang deficiency syndrome (KYDS) is a classic syndrome in traditional Chinese medicine, which is mainly caused by damage to the hypothalamic-pituitary-adrenal (HPA) axis. Hirsutella sinensis fungus (HSF), an artificial substitute of Cordyceps sinensis, has been widely used in TCM. However, the effects and the possible mechanism of HSF on the HPA axis and corresponding KYDS have not yet been investigated. In this study, Lewis rats were used as a spontaneous KYDS model. HSF was intragastrically administered to the Lewis rats at two doses: low dose (1 g/kg) and high dose (2 g/kg). Body weight, temperature, and behavioral tests including grip strength, open field, and Morris water maze (MWM) tests were used to evaluate the KYDS symptoms. Enzyme-linked immunosorbent assay was used to detect the level of circulating adrenocortisol (ACTH), corticosterone (CORT), corticotropin releasing hormone (CRH), cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP). In addition, mRNA expression of tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 10 (IL-10), CRH, glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) was detected by quantitative real-time polymerase chain reaction (Q-PCR). The Lewis rats were indicated to have KYDS symptoms and HSF treatment ameliorated these symptoms via enhancement of the HPA axis function, which was evidenced by the increased levels of CRH, ACTH, and CORT in serum and 17-OHCS in urine. HSF also significantly improved the expression of TNF-α, IFN-γ, and IL-2, secreted by Th1 cells, which might accelerate the activation of the immune system related to the HPA axis function. Thus, we conclude that HSF can alleviate KYDS symptoms in Lewis rats by regulating the HPA axis through accelerated immune system activation.

Transcriptome profiling reveals multiple pathways responsible for the beneficial metabolic effects of Smilax glabra flavonoids in mouse 3T3-L1 adipocytes.

Smilax glabra Roxb. (SG) is a well-known traditional Chinese medicine that has been extensively used as both food and folk medicine in many countries. Although many beneficial health effects of SG and its primary components have been reported, their action on adipocyte function remains unknown. In the present study, we investigated the effects of the total flavonoids from Smilax glabra Roxb. (SGF) on lipid accumulation in mouse 3T3-L1 adipocytes and further elucidated its potential mechanism using RNA-Seq transcriptome technique. Our results showed that SGF exposure significantly decreased the lipid droplet size and the levels of cellular free fatty acids, while triglyceride accumulation was not affected by SGF. Transcriptome analysis revealed that SGF induced the expression of genes involved in triglyceride storage, fatty acid ß-oxidation and mitochondrial biogenesis. Furthermore, we also observed an increased cellular ATP level and mitochondrial mass after SGF exposure, indicating that SGF enhanced mitochondrial function. The other relevant transcriptional changes appeared to be involved in AMPK/PGC-1α signaling, inflammatory response, as well as PI3K/AKT and calcium signaling pathways, which might contribute to the beneficial metabolic effects of SGF on adipocyte function. The results of Western blotting confirmed that SGF could increase the phosphorylation of AMPK while decrease the phosphorylation of AKT in adipocytes. Altogether, our results provided novel information about the molecular mechanism responsible for the effects of SGF on fat storage in adipocytes and highlights the potential metabolic benefits of SGF on human obesity and its related chronic diseases.

Total glucosides of peony improve ovalbumin-induced allergic asthma by inhibiting mast cell degranulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonia lactiflora Pall. (peony) is a medicinal plant used in the Xiaoqinglong decoction, a commonly prescribed traditional Chinese medicine for asthma. The main active ingredients of peony roots-described as the total glucosides of peony (TGP)-have anti-inflammatory, immunomodulatory, and protective effects on endothelial cells, and they are known to improve rheumatoid arthritis. This study explored the underlying mechanism of TGP activity in the treatment of allergic asthma. MATERIALS AND METHODS: Allergic asthma was induced in BALB/c mice by administering injections of ovalbumin (OVA) mixed with aluminum hydroxide gel and inhaling nebulized OVA. The OVA-sensitized mice were treated with TGP by oral gavage, and the potentially anti-asthmatic treatment effect was studied by testing airway hyperresponsiveness, classifying and counting of leukocytes, performing cytokine assays, and analyzing the lung histopathology. The ß-hexosaminidase activity was assayed as a biomarker to evaluate the effect of TGP on mast cell degranulation. The mechanism of TGP was explored by monitoring the Ca2+ influx level in mast cells (RBL-2H3) using a Ca2+ fluorescent probe technique. RESULTS: In mice with OVA-induced allergic asthma, TGP reduced airway hyperresponsiveness and improved lung tissue pathology, which included a decrease in inflammatory cell infiltration and collagen deposition. TGP also significantly lowered BALF leukocyte, eosinophil, and neutrophil counts, along with chemokines and cytokines, such as eotaxin, TNF-α, IL-4, and MIP-1α, in serum and lungs of OVA-challenged mice. These effects were further confirmed with the decrease of ß-hexosaminidase release and the inhibition of Ca2+ influx in mast cell degranulation. CONCLUSIONS: Our findings suggest that TGP improved OVA-induced allergic asthma in mice mainly by suppressing Ca2+ influx-dependent mast cell degranulation.

Total flavonoids from Smilax glabra Roxb blocks epithelial-mesenchymal transition and inhibits renal interstitial fibrosis by targeting miR-21/PTEN signaling.

BACKGROUND: Smilax glabra Roxb, a traditional Chinese herb, has been widely used in folk medicine. The current study was performed to investigate the protective effect of S. glabra Roxb extract, pure total flavonoids from Smilax glabra Roxb (PTFS), on renal interstitial fibrosis (RIF) and its underlying mechanism. METHODS: First, a surgical model of unilateral ureteral obstruction was established in rats to induce RIF. Then, rats were grouped and treated with PTFS at different concentration. Second, HK-2 cells underwent an epithelial-mesenchymal transition (EMT) by the addition of transforming growth factor-ß1 (TGF-ß1). Additionally, HK-2 cells after inducing for EMT were transfected with microRNA-21 (miR-21) mimic or inhibitor. These HK-2 cells were grouped and treated with PTFS at different concentration. Finally, real-time polymerase chain reaction and Western blot analysis were performed to detect the expression of possible signaling factor involved in RIF in renal tissues or HK-2 cells after PTFS treatment. RESULTS: In vivo and in vitro experiments indicated that PTFS treatment could decrease the expression of α-smooth muscle actin (α-SMA; mesenchymal marker) and increase the expression of E-cadherin (epithelial marker) in both messenger RNA and protein level. Moreover, PTFS also attenuated the expression of TGF-ß1/Smad signaling in both renal tissues and HK-2 cells that underwent EMT. Overexpression or inhibition of miR-21 in HK-2 cells activated or blocked the PI3K/Akt signaling via targeting phosphatase and tension homolog (PTEN), and then promoted or suppressed the progress of TGF-ß1-induced EMT by regulating the expression of α-SMA and E-cadherin. Furthermore, PTFS treatment inhibited TGF-ß1-induced EMT progress by blocking miR-21/PTEN/PI3K/Akt signaling. CONCLUSION: PTFS has strong anti-EMT and antifibrosis effects both in vitro and in vivo. The mechanism underlying these effects may be related to inhibition of TGF-ß1/Smad, and their downstream miR-21/PTEN signaling, leading to blocks of EMT process during RIF.

Effect of Total Flavone of Haw Leaves on Nuclear Factor Erythroid-2 Related Factor and Other Related Factors in Nonalcoholic Steatohepatitis Rats.

OBJECTIVE: To investigate the effect of total flavone of haw leaves (TFHL) on the expression of nuclear factor erythroid-2 related factor (Nrf2) and other related factors in nonalcoholic steatohepatitis (NASH) rats induced by high-fat diet and then to further discuss the mechanism of TFHL's prevention against NASH. METHODS: High-fat diet was fed to 40 rats to establish the NASH model. Then model rats were intragastrically administrated with 40, 80, 160 mg/(kg•day) TFHL, respectively. The pathological changes of liver tissues in NASH rats were detected by oil red O and hematoxylin-eosin (HE) stainings. The expression of Nrf2 in rat liver was examined through immunohistochemistry. The level of 8-iso-prostaglandin F2α in serum was detected through enzyme linked immunosorbent assay (ELISA). The mRNA and protein levels of Nrf2 and other related factors in liver tissue were measured by real-time reverse transcriptionpolymerase chain reaction and western blot. RESULTS: Lipid deposition, hepatic steatosis, focal necrosis in lobular inflammation and ballooning degeneration were emerged in livers of NASH rats. The 8-iso-prostaglandin F2α in the serum of NASH rats increased significantly compared with the control group (P<0.05). The mRNA of Nrf2, hemeoxyenase1 (HO-1) and the mRNA and protein levels of quinine oxidoreductase (NQO1) in NASH rats liver tissue showed a striking increase, while the mRNA levels of Keap1, r-glutamylcysteine synthethase (rGCS) and glutathione S-transferase (GST) were significantly decreased compared with the control group (P<0.05). After TFHL treatment, 8-iso-prostaglandin F2α level in serum significantly decreased, and Nrf2 mRNA and protein levels in hepatocytes nucleus enhanced compared with the model group (P<0.05 or 0.01). Meanwhile the Keap1 mRNA, the mRNA and protein levels of HO-1, NQO1 antibody, rGCS antibody, GST increased after TFHL treatment (P<0.05 or 0.01). CONCLUSIONS: Nrf2 and other related factors were involved in development of NASH, and they also served as an important part in its occurrence. By regulating expression of Nrf2 and other related factors, TFHL may play a role in antioxidative stress and prevention of NASH.

[Protective effect of astragalus saponin extracts on kidneys of diabetic rats].

To study the protective effect of astragalus saponin extracts (AS) on kidneys of diabetic rats. Totally 32 diabetic rats induced by streptozotocin (STZ) were divided into AS high and low dose groups, the positive control group and the model group (DM group) and orally administered with 50 mg x- kg(-1) x d(-1) AS 200, 25 mg x kg(-1) x d(-1) valsartan, 10 mL x kg(-1) x d(1) physiological saline, respectively. Another 8 healthy rats were collected in the normal control group (NC group, physiological saline 10 mL x kg(-1). d(-1)). All rats were treated for consecutively 6 weeks. After the administration, the body weight was measured every week, the concentration of blood glucose was monitored on week 2, 4 and 6. The total urine and total urinary protein (U-TP) in 24 h were measured by the metabolic cage method on week 6; At the end of week 6, blood samples were collected from hearts to detect blood urea nitrogen (BUN), serum creatinine (Scr), uric acid (UA) , total cholesterol (CH) triglyceride (TG) by biochemical methods. Kidneys were collect to calculate the kidney hypertrophy index and observe the pathological sections. The laboratory results show that in the DM group, the blood glucose, metabolic cost in 24 h, kidney hypertrophy index, U-TP, BUN, Scr, UA, TG were significantly higher than that in the NC group (P < 0.01, P < 0.05) , with significant pathological changes; After the intervention with AS, the metabolic value in 24 h, kidney hypertrophy index, U-TP, BUN, Scr, UA, TG were significantly lower in the high dose group (P < 0.01, P < 0.05), and the kidney hypertrophy index, BUN, Scr, UA, TG in the low dose group were also significantly lower (P < 0.05), with slight reduction in renal pathological changes in both groups. In conclusion, Astragalus saponin extracts have a certain protective effect on kidneys of diabetic rats.

Medicinal effect and its JP2/RyR2-based mechanism of Smilax glabra flavonoids on angiotensin II-induced hypertrophy model of cardiomyocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizome and root of Smilax glabra Roxb (Liliaceae family) is a widely used traditional Chinese medicine (TCM) named Tu-fu-ling (TFL) for cardiac disease therapy. The TFL flavonoids (TFLF) has been extracted and proven to possess the anti-cardiac hypertrophy effect in our previous reports. Such effect could be mediated by the modulation of intracellular Ca(2+) flux in myocardial cells, in which junctophilin-2 (JP2) and ryanodine receptor 2 (RyR2) play an important role. However, its mechanism of the anti-cardiac hypertrophy effect remains unclarified. MATERIALS AND METHODS: 2µmol/L Ang II was applied to induce hypertrophy model of rat primary cardiomyocytes. After treatment of TFLF at 0.25, 0.5 and 1.0mg/ml, the cell size was microscopic measured, and the protein and mRNA expressions of JP2 and RyR2 in cardiomyocytes were estimated by immunofluorescence imaging, ELISA and real-time PCR assay. RESULTS: Obvious hypertrophy of cardiomyocytes was induced by Ang II but reversed by TFLF from 0.5 to 1.0mg/ml. The protein and mRNA expressions of JP2 and RyR2 in cardiomyocytes were also inhibited by Ang II but restored by TFLF at its dose range. Such effect of TFLF was exerted at a dose dependent manner, which was even better than that of verapamil. CONCLUSIONS: Our findings may evidence the correlation between JP2/RyR2 and myocardiac hypertrophy, and indicate the JP2/RyR2-mediated anti-hypertrophy mechanism of TFLF for the first time. It deserves to be developed as a promising TCM candidate of new drug for myocardial hypertrophy treatment.

Qingyihuaji Formula Inhibits Pancreatic Cancer and Prolongs Survival by Downregulating Hes-1 and Hey-1.

The dire prognosis of pancreatic cancer has not markedly improved during past decades. The present study was carried out to explore the effect of Qingyihuaji formula (QYHJ) on inhibiting pancreatic cancer and prolonging survival in related Notch signaling pathway. Proliferation of pancreatic cancer cells (SW1990 and PANC-1) was detected by MTT assay at 24, 48, and 72 h with exposure to various concentrations (0.08-50 mg/mL) of QYHJ water extract. Pancreatic tumor models of nude mice were divided into three groups randomly (control, QYHJ, and gemcitabine). mRNA and protein expression of Notch target genes (Hes-1, Hey-1, Hey-2, and Hey-L) in dissected tumor tissue were detected. Results showed that proliferation of SW1990 cells and PANC-1 cells was inhibited by QYHJ water extract in a dose-dependent and time-dependent manner. QYHJ effectively inhibited tumor growth and prolonged survival time in nude mice. Expression of both Hes-1 and Hey-1 was decreased significantly in QYHJ group, suggesting that Hes-1 and Hey-1 in Notch signaling pathway might be potential targets for QYHJ treatment. This research could help explain the clinical effectiveness of QYHJ and may provide advanced pancreatic cancer patients with a new therapeutic option.

Modulation effect of Smilax glabra flavonoids on ryanodine receptor mediated intracellular Ca2+ release in cardiomyoblast cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Smilax glabra rhizome, a plant material from Liliaceae family, is a widely used traditional Chinese medicine for anti-cardiac hypertrophy treatment. We have previously found that Smilax glabra flavonoids (SGF) exerted such anti-cardiac hypertrophy activity. However, the mechanism of this activity of SGF has not been clarified yet. MATERIALS AND METHODS: This study was aimed to investigate the inhibitory role of SGF on intracellular Ca(2+) release in rat cardiomyoblast cells (H9C2). Intracellular Ca(2+) release was determined by Ca(2+) indicator fluorescence (fluo 4-AM) in H9C2 cell line. RESULTS: SGF at concentrations of 0.25, 0.5, 1.0mg/ml significantly inhibited the phenylephrine or angiotensin II induced intracellular Ca(2+) release in a dose-dependent manner. Furthermore, SGF could also inhibit ryanodine receptor (RyR) agonist caffeine induced Ca(2+) release and phenylephrine (PE)-induced Ca(2+) release under the condition in which inositol trisphosphate (IP3) receptors were blocked with 2-Aminoethoxydiphenyl borate (2-APB). Nevertheless, SGF had no impact on PE-induced Ca(2+) release under the condition in which RyRs were blocked with tetracaine. CONCLUSIONS: Our results suggest that the protective effects of SGF are mediated via targeting inhibition of RyR mediated intracellular Ca(2+) release.

[Study on treatment effect and mechanism of Hirsutella sinensis mycelium in idiopathic pulmonary fibrosis in rats].

OBJECTIVE: To study and discuss the effect and mechanism of Hirsutella sinensis mycelium (HSM) on idiopathic pulmonary fibrosis in rats. METHOD: Forty Wistar rats were divided into five groups: the normal control group, the model control group, the high-dose group (1.0 g x kg(-1) HSM), the low-dose group (0.5 g x kg(-1) HSM), and the positive control group (10 mg x kg(-1) hydrocortisone). In addition to rats in the normal control group, the pulmonary fibrosis model was established by injecting 5 mg x kg(-1) bleomycin into rat tracheas for consecutively 28 days, in order to observe their lung function, lung tissue hydroxyproline, cytokines and pathology. RESULT: After rats were administered with HSM, 0.5 g x kg(-1) and 1.0 g x kg(-1) HSM could significantly decrease lung index and hydroxyproline content (P<0.01), while notably improving pulmonary function, alveolus inflammation and fibrosis degree (P<0.05, P<0.01); 1.0 g x kg(-1) HSM could decrease significantly protein expressions of TNF-alpha, IL-1beta and TGF-beta1 in lung tissues, while increasing significantly protein expressions of IFN-gamma (P<0.05). CONCLUSION: HSM have better effect in treating idiopathic pulmonary fibrosis in rats. Its treatment effect and mechanism are related to the regulation of TNF-alpha, IL-1beta and TGF-beta1 and IFN-gamma imbalance.