调经促孕方对胚胎着床障碍性不孕症小鼠子宫内膜容受性及AREG/EGFR/HIF-1α信号通路的影响

目的观察调经促孕方对胚胎着床障碍(EID)性不孕症小鼠子宫内膜容受性及AREG/EGFR/HIF-1α信号通路的影响,探讨其改善子宫内膜容受性的可能机制。方法80只昆明小鼠于妊娠第1日随机分为正常组、模型组、黄体酮组和调经促孕方组,每组20只,黄体酮组和调经促孕方组分别予相应药物灌胃。妊娠第4日,除正常组外,其余各组小鼠皮下注射米非司酮溶液(2.3 mg/kg)建立EID性不孕症模型,正常组皮下注射等体积丙二醇。妊娠第5日,每组随机取10只小鼠,剖腹取子宫,HE染色、Masson染色观察子宫内膜组织形态,扫描电镜观察子宫内膜胞饮突形态及数量,ELISA检测子宫内膜组织双调蛋白(AREG)含量,Western blot和免疫荧光检测子宫内膜组织表皮生长因子受体(EGFR)、p-EGFR、缺氧诱导因子(HIF)-1α表达。其余小鼠灌胃至妊娠第8日取材,记录平均胚胎着床点数。结果与正常组比较,模型组小鼠平均胚胎着床点数显著减少(P<0.01),子宫内膜腺体发育不良,胶原纤维增加,血管及胞饮突数量减少,子宫内膜组织AREG含量显著降低(P<0.01),p-EGFR、HIF-1α蛋白表达显著降低(P<0.05);与模型组比较,调经促孕方组小鼠平均胚胎着床点数显著增加(P<0.05),子宫内膜胶原纤维增生减少,间质内腺体、血管及胞饮突数量均明显增加,胞饮突发育饱满、均匀,子宫内膜组织AREG含量显著升高(P<0.01),p-EGFR、HIF-1α蛋白表达显著升高(P<0.05,P<0.01),免疫荧光检测结果与Western blot一致。结论调经促孕方能促进子宫内膜腺体和血管发育,增加胞饮突数量,改善EID性不孕症小鼠子宫内膜容受性,利于胚胎黏附及植入,从而提高妊娠率,其机制可能与激活AREG/EGFR/HIF-1α信号通路,改善子宫内膜血流灌注,恢复子宫内膜相对缺氧环境有关。

Timosaponin AⅢ induces drug-metabolizing enzymes by activating constitutive androstane receptor (CAR) via dephosphorylation of the EGFR signaling pathway

The current study aimed to assess the effect of timosaponin AⅢ(T-AⅢ)on drug-metabolizing enzymes during anticancer therapy.The in vivo experiments were conducted on nude and ICR mice.Following a 24-day administration of T-AⅢ,the nude mice exhibited an induction of CYP2B10,MDR1,and CYP3A11 expression in the liver tissues.In the ICR mice,the expression levels of CYP2B10 and MDR1 increased after a three-day T-AⅢ administration.The in vitro assessments with HepG2 cells revealed that T-AⅢ induced the expression of CYP2B6,MDR1,and CYP3A4,along with constitutive androstane receptor(CAR)activation.Treatment with CAR siRNA reversed the T-AⅢ-induced increases in CYP2B6 and CYP3A4 expression.Furthermore,other CAR target genes also showed a significant increase in the expression.The up-regulation of murine CAR was observed in the liver tissues of both nude and ICR mice.Subsequent findings demonstrated that T-AⅢ activated CAR by inhibiting ERK1/2 phosphorylation,with this effect being partially reversed by the ERK activator t-BHQ.Inhibition of the ERK1/2 signaling pathway was also observed in vivo.Additionally,T-AⅢ inhibited the phosphorylation of EGFR at Tyr1173 and Tyr845,and suppressed EGF-induced phosphorylation of EGFR,ERK,and CAR.In the nude mice,T-AⅢ also inhibited EGFR phosphorylation.These results collectively indicate that T-AⅢ is a novel CAR activator through inhibition of the EGFR pathway.

Solanine Interferes with AKT/p-AKT and PI3K/p-PI3K Pathway to Inhibit HIF and Destroy Cell Energy Metabolism

The purpose of this study was to explore the mechanism of Solanine disrupting energy metabolism in human renal cancer ACHN cells and to clarify its target. The specific method was to culture human renal cancer ACHN cell lines, and to intervene with Solanine of high, medium and low concentrations. The content of ATP in cells was measured by ELISA method. The expression of HIF-1α protein and the expression of PI3K, AKT, p-PI3K, p-AKT in PI3K/AKT pathway were detected by Western blotting. The results showed that compared with the control group, the relative expression of p-PI3K and p-AKT showed a downward trend with the increase of Solanine concentration (P < 0.05), while the relative expression of PI3K and AKT showed no significant change (P > 0.05). In addition, the relative expression of HIF-1α also showed a downward trend (P < 0.05). According to the above results, it is suggested that Solanine can significantly inhibit the energy metabolism of renal cancer cells, the main mechanism of which is the down-regulation of HI-1αf downstream of the PI3K/Akt pathway by inhibiting the phosphorylation process of PI3K/p-PI3K and Akt/p-Akt.

Network pharmacology and experimental validation of Maxing Shigan decoction in the treatment of influenza virus-inducedferroptosis

Influenza is an acute viral respiratory infection that has caused high morbidity and mortality worldwide.Influenza A virus(IAV)has been found to activate multiple programmed cell death pathways,including ferroptosis.Ferroptosis is a novel form of programmed cell death in which the accumulation of intracellular iron promotes lipid peroxidation,leading to cell death.However,little is known about how influenza viruses induce ferroptosis in the host cells.In this study,based on network pharmacology,we predicted the mechanism of action of Maxing Shigan decoction(MXSGD)in IAV-induced ferroptosis,and found that this process was related to biological processes,cellular components,molecular function and multiple signaling pathways,where the hypoxia inducible factor-1(HIF-1)signaling pathway plays a significant role.Subsequently,we constructed the mouse lung epithelial(MLE-12)cell model by IAV-infected in vitro cell experiments,and revealed that IAV infection induced cellular ferroptosis that was characterized by mitochondrial damage,increased reactive oxygen species(ROS)release,increased total iron and iron ion contents,decreased expression of ferroptosis marker gene recombinant glutathione peroxidase 4(GPX4),increased expression of acyl-CoA synthetase long chain family member 4(ACSL4),and enhanced activation of hypoxia inducible factor-1α(HIF-1α),induced nitric oxide synthase(iNOS)and vascular endothelial growth factor(VEGF)in the HIF-1 signaling pathway.Treatment with MXSGD effectively reduced intracellular viral load,while reducing ROS,total iron and ferrous ion contents,repairing mitochondrial results and inhibiting the expression of cellular ferroptosis and the HIF-1 signaling pathway.Finally,based on animal experiments,it was found that MXSGD effectively alleviated pulmonary congestion,edema and inflammation in IAV-infected mice,and inhibited the expression of ferroptosis-related protein and the HIF-1 signaling pathway in lung tissues.

Extracts of Celastrus Orbiculatus Inhibit Cancer Metastasis by Down-regulating Epithelial-Mesenchymal Transition in Hypoxia-Induced Human Hepatocellular Carcinoma Cells

Objective: To evaluate the effects of Celastrus Orbiculatus extracts(COE) on metastasis in hypoxiainduced hepatocellular carcinoma cells(Hep G2) and to explore the underlying molecular mechanisms. Methods:The effect of COE(160, 200 and 240 μg/mL) on cell viability, scratch-wound, invasion and migration were studied by 3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide(MTT), scratch-wound and transwell assays, respectively. Co Cl2 was used to establish a hypoxia model in vitro. Effects of COE on the expressions of E-cadherin, vimentin and N-cadherin were investigated with Western blot and immuno?uorescence analysis,respectively. Results: COE inhibited proliferation and metastasis of hypoxia-induced hepatocellular carcinoma cells in a dose-dependent manner(P<0.01). Furthermore, the expression of epithelial-mesenchymal transition(EMT) related markers were also remarkably suppressed in a dose-dependent manner(P<0.01). In addition, the upstream signaling pathways, including the hypoxia-inducible factor 1α(Hif-1α) and Twist1 were suppressed by COE. Additionally, the Hif-1α inhibitor 3-5'-hydroxymethyl-2'-furyl)-1-benzylindazole(YC-1), potently suppressed cell invasion and migration as well as expression of EMT in hypoxia-induced Hep G2 cells. Similarly, the combined treatment with COE and YC-1 showed a synergistic effect(P<0.01) compared with the treatment with COE or YC-1 alone in hypoxia-induced Hep G2 cells. Conclusions: COE signi?cantly inhibited the tumor metastasis and EMT by suppressing Hif-1α/Twist1 signaling pathway in hypoxia-induced Hep G2 cell. Thus, COE might have potential effect to inhibit the progression of Hep G2 in the context of tumor hypoxia.