鲁斯可皂苷元对脑缺血再灌注损伤小鼠时钟基因的调控作用

目的研究鲁斯可皂苷元(Rus)对小鼠脑缺血再灌注(I/R)损伤的改善作用及其机制。方法C57BL/6J小鼠分为假手术组、假手术+Rus组、I/R组和I/R+Rus组,采用大脑中动脉栓塞线栓法制备小鼠I/R模型,小鼠在术前1 h ig给予Rus 10 mg·kg^(-1)或同体积溶媒(0.5%羧甲纤维素钠)。再灌注24 h后,进行神经功能评分并测定脑梗死体积,采用实时荧光定量PCR和Western印迹法检测时钟基因Per1,Clock和Bmal1 mRNA和蛋白表达水平。结果与假手术组相比,I/R组神经功能评分增高(P<0.01),脑梗死体积增大(P<0.01),Per1,Clock和Bmal1 mRNA和蛋白表达水平下调(P<0.05,P<0.01);与I/R组相比,I/R+Rus组的脑梗死体积减小(P<0.01),神经功能评分降低(P<0.01),Per1,Clock和Bmal1 mRNA和蛋白表达水平上调(P<0.05,P<0.01)。结论 Rus可减轻小鼠脑I/R损伤,其机制可能与调控时钟基因的表达有关。

奥希替尼氘代物对EGFR不同表达水平癌细胞的药理活性研究

目的探讨奥希替尼氘代物(RN9591)对肿瘤细胞体外增殖和对荷瘤裸鼠体内肿瘤生长的影响,并与奥希替尼(AZD9291)比较,确定RN9591的开发前景。方法采用MTT比色法观察RN9591、AZD9291对体外培养的A549、H1975、SW9483种不同EGFR表达型癌细胞株体外增殖的影响,计算IC50;建立EGFR双突变吉非替尼耐药非小细胞肺癌细胞H1975的裸鼠移植瘤模型,观察RN9591200、100、50 mg·kg^-13个剂量组,吉非替尼120 mg·kg^-1组,AZD9291100 mg·kg^-1组的治疗作用。观察给药后荷瘤小鼠的肿瘤体积和瘤重,比较药物对肿瘤生长的影响。结果体外抗肿瘤生长结果表明RN9591对A549、H1975、SW948细胞株的IC50分别为11.43、25.61、59.96μmol·L^-1;AZD9291对A549、H1975、SW948细胞株的IC50分别为9.21、25.45、27.05μmol·L^-1。体内抗肿瘤实验结果表明RN9591高、中、低3个剂量组肿瘤相对增殖率(T/C%)分别为60.9%、72.0%、59.6%;吉非替尼组T/C%为63.9%;AZD9291组T/C%为47.6%。结论与AZD9291相比,RN9591保持了对EGFR突变株及吉非替尼耐药株的杀伤力,并降低了对野生型EGFR的抑制作用,体内实验进一步证明RN9591对非小细胞肺癌有明显抑制活性,提示本品具有良好的开发前景。

防治慢性心衰的中药新研发思路与技术要点分析

慢性心力衰竭(chronic heart failure,CHF)为各种心脏病的严重和终末阶段,是21世纪常见的慢性心血管病症,文献资料显示心衰住院患者5年死亡率高达50%。开发心衰治疗药物成为当今热点,中药在慢性心衰的治疗中体现了中医治疗优势。该文提出慢性心衰防治中药开发的4步考虑要点,首先以临床需求为导向,论述中成药开发、临床中药处方筛选思路和方法;其次,阐述参考中药处方已有的临床用药背景,分析处方中各味药材化学成分和药理作用特点,开展制备工艺和质量控制方法研究;进一步通过中药处方临床用药经验和未来临床定位设计药效学、毒理学等非临床评价方案和开展研究工作,并在方案实施过程中逐步调整完善;最后考虑该处方的临床用药经验、临床前研究药学、主要药效学和安全性实验结果,结合临床定位,依据中医理论设计合理的临床试验方案,形成注册申报资料进行临床试验申请(IND)。在通过IND技术审评后正式开展临床试验。以期取得预期结果,获得疗效确切的心衰治疗中成药。

Ge-Gen Decoction attenuates oxytocin-induced uterine contraction and writhing response: potential application in primary dysmenorrhea therapy

The uterine tetanic contraction and uterine artery blood flow reduction are possible reasons for primary dysmenorrhea(PD). In the present study, we aimed to evaluate the uterine relaxant effect and the influence on uterine artery blood velocity of Ge-Gen Decoction(GGD), a well-known Chinese herbal formula. In female ICR mice, uterine contraction was induced by oxytocin exposure following estradiol benzoate pretreatment, and the uterine artery blood velocity was detected by Doppler ultrasound. Histopathological examination of the uterine tissue samples were performed by H&E staining. Ex vivo studies demonstrated that oxytocin, posterior pituitary, or acetylcholine induced contractions in isolated mouse uterus. GGD inhibited both spontaneous and stimulated contractions. In vivo study demonstrated that GGD significantly reduced oxytocin-induced writhing responses with a maximal inhibition of 87%. Further study demonstrated that GGD normalized oxytocin-induced abnormalities of prostaglandins F_2 alpha(PGF_(2α)) and Ca^(2+) in mice. In addition, injection of oxytocin induced a decrease in uterine artery blood flow velocity. Pretreatment with GGD reversed the oxytocin response on blood flow velocity. Histopathological examination showed pretreatment with GGD alleviated inflammation and edema in the uterus when compared with the model group. Both ex vivo and in vivo results indicated that GGD possessed a significant spasmolytic effect on uterine tetanic contraction as well as improvement on uterine artery blood velocity which may involve PGF_(2α) and Ca^(2+) signaling, suggesting that GGD may have a clinic potential in PD therapy.

Advances in the pharmacological activities and mechanisms of diosgenin

Diosgenin, a well-known steroid sapogenin derived from plants, has been used as a starting material for production of steroidal hormones. The present review will summarize published literature concerning pharmacological potential of diosgenin, and the underlying mechanisms of actions. Diosgenin has shown a vast range of pharmacological activities in preclinical studies. It exhibits anticancer, cardiovascular protective, anti-diabetes, neuroprotective, immunomodulatory, estrogenic, and skin protective effects, mainly by inducing apoptosis, suppressing malignant transformation, decreasing oxidative stress, preventing inflammatory events, promoting cellular differentiation/proliferation, and regulating T-cell immune response, etc. It interferes with cell death pathways and their regulators to induce apoptosis. Diosgenin antagonizes tumor metastasis by modulating epithelial-mesenchymal transition and actin cytoskeleton to change cellular motility, suppressing degradation of matrix barrier, and inhibiting angiogenesis. Additionally, diosgenin improves antioxidant status and inhibits lipid peroxidation. Its anti-inflammatory activity is through inhibiting production of pro-inflammatory cytokines, enzymes and adhesion molecules. Furthermore, diosgenin drives cellular growth/differentiation through the estrogen receptor(ER) cascade and transcriptional factor PPARγ. In summary, these mechanistic studies provide a basis for further development of this compound for pharmacotherapy of various diseases.

Network pharmacology-based prediction and verification of the molecular targets and pathways for schisandrin against cerebrovascular disease

AIM: To illuminate the molecular targets for schisandrin against cerebrovascular disease based on the combined methods of network pharmacology prediction and experimental verification. METHOD: A protein database was established through constructing the drug-protein network from literature mining data. The protein-protein network was built through an in-depth exploration of the relationships between the proteins. The computational platform was implemented to predict and extract the sensitive sub-network with significant P-values from the protein-protein network. Then the key targets and pathways were identified from the sensitive sub-network. The most related targets and pathways were also confirmed in hydrogen peroxide(H2O2)-induced PC12 cells by Western blotting. RESULTS: Twelve differentially expressed proteins(gene names: NFKB1, RELA, TNFSF10, MAPK1, CHUK, CASP8, PIGS2, MAPK14, CREB1, IFNG, APP, and BCL2) were confirmed as the central nodes of the interaction network(45 nodes, 93 edges). The NF-κB signaling pathway was suggested as the most related pathway of schisandrin for cerebrovascular disease. Furthermore, schisandrin was found to suppress the expression and phosphorylation of IKKα, as well as p50 and p65 induced by H2O2 in PC12 cells by Western blotting. CONCLUSION: The computational platform that integrates literature mining data, protein-protein interactions, sensitive sub-network, and pathway results in identification of the NF-κB signaling pathway as the key targets and pathways for schisandrin.

Recent advances of traditional Chinese medicine on the prevention and treatment of COVID-19

Coronavirus disease-2019(COVID-19)is a new highly infectious disease caused by a novel coronavirus.Recently,the number of new cases infected pneumonia in the world continues to increase,which has aroused great concern from the international community.At present,there are no small-molecule specific anti-viral drugs for the treatment.The high mortality rate seriously threatens human health.Traditional Chinese medicine(TCM)is a unique health resource in China.The combination of TCM and Western medicine has played a positive and important role in combating COVID-19 in China.In this review,through literature mining and analysis,it was found that TCM has the potential to prevent and treat the COVID-19.Then,the network pharmacological studies demonstrated that TCM played roles of anti-virus,anti-inflammation and immunoregulation in the management of COVID-19 via multiple components acting on multiple targets and multiple pathways.Finally,clinical researches also confirmed the beneficial effects of TCM on the treatment of patients.This review may provide meaningful and useful information on further drug development of COVID-19 and other viral infectious diseases.

Advances in the antitumor activities and mechanisms of action of steroidal saponins

The steroidal saponins are one of the saponin types that exist in an unbound state and have various pharmacological activities,such as anticancer,anti-inflammatory,antiviral,antibacterial and nerves-calming properties.Cancer is a growing health problem worldwide.Significant progress has been made to understand the antitumor effects of steroidal saponins in recent years.According to reported findings,steroidal saponins exert various antitumor activities,such as inhibiting proliferation,inducing apoptosis and autophagy,and regulating the tumor microenvironment,through multiple related signaling pathways.This article focuses on the advances in domestic and foreign studies on the antitumor activity and mechanism of actions of steroidal saponins in the last five years to provide a scientific basis and research ideas for further development and clinical application of steroidal saponins.

A combination of four effective components derived from Sheng-mai san attenuates hydrogen peroxide-induced injury in PC12 cells through inhibiting Akt and MAPK signaling pathways

The present study was designed to investigate whether a combination of four effective components derived from Sheng-mai san(SMXZF; ginsenoside Rb1: ginsenoside Rg1: DT^(–1)3: Schizandrol A as 6 : 9 : 4 : 5) could attenuate hydrogen peroxide(H_2O_2)-induced injury in PC12 cells, focusing on the Akt and MAPK pathways. The PC12 cells were exposed to H_2O_2(400 mmol·L^(–1)) for 1 h in the presence or absence of SMXZF pre-treatment for 24 h. Cell viability was measured by MTT assay. The efflux of lactate dehydrogenase(LDH), the intracellular content of malondialdehyde(MDA), the activities of superoxide dismutase(SOD), and caspase-3 were also determined. Cell apoptosis was measured by Hoechst 33342 staining and Annexin V-FITC/PI staining method. The expression of Bcl-2, Bax, cleaved caspase-3, Akt, and MAPKs were detected by Western blotting analyses. SMXZF pretreatment significantly increased the cell viability and SOD activity and improved the cell morphological changes, while reduced the levels of LDH and MDA at the concentrations of 0.1, 1 and 10 μg·m L^(–1). SMXZF also inhibited H_2O_2-induced apoptosis in PC12 cells. Moreover, SMXZF reduced the activity of caspase-3, up-regulated the protein ratio of Bcl-2 and Bax and inhibited the expression of cleaved caspase-3, p-Akt, p-p38, p-JNK and p-ERK1/2 in H_2O_2-induced PC12 cells. Co-incubation of Akt inhibitor or p38 inhibitor partly attenuated the protection of SMXZF against H_2O_2-injured PC12 cells. In conclusion, our findings suggested that SMXZF attenuated H_2O_2-induced injury in PC12 cells by inhibiting Akt and MAPKs signaling pathways, which might shed insights on its neuroprotective mechanism.

Novel cytotoxic steroidal glycosides from the roots of Liriope muscari

The present study was designed to investigate the chemical constituents and bioactivities of the roots of Liriope muscari(Decne.) L.H. Bailey. The compounds were isolated through various chromatography techniques, including silica gel, Sephadex LH-20, and semi-preparative HPLC. The structures were elucidated by infrared(IR), mass spectrometric(MS), 1D- and 2D-NMR analyses in comparison with reference data. In addition, the cytotoxicity of these compounds against human breast cancer MDA-MB-435 cells was evaluated by the MTT assay. Two new steroidal glycosides, 25(R, S)-ruscogenin-1-O-[β-D-fucopyranosyl(1→2)]-[ β-D-xylopyranosyl(1→3)]-β-D-glucopyranoside(Liriopem I, 1) and 25(R, S)- ruscogenin-1-O-[β-D-fucopyranosyl(1→2)]-[β-D-xylopyranosyl(1→4)]-β-D-fucopyranoside(Liriopem II, 2) and two known compounds LM-S6(3) and DT-13(4) were isolated and identified. Liriopem I(1), liriopem II(2) and DT-13(4) showed remarkable cytotoxicity with IC50 values being(0.58 ± 0.08),(0.05 ± 0.10), and(0.15 ± 0.09) μg·m L-1, respectively. In summary, compounds 1 and 2 identified in the present study exerted cytotoxicity against breast cancer cells, providing a basis for future development of these compounds as novel anticancer agents.

Sheng-Mai-San attenuates contractile dysfunction and structural damage induced by chronic intermittent hypoxia in mice

Sheng-Mai-San(SMS), a well-known Chinese medicinal plant formula, is widely used for the treatment of cardiac diseases characterized by deficiency of Qi and Yin syndrome. A mouse chronic intermittent hypoxia(CIH) model was established to mimic the primary clinical features of deficiency of Qi and Yin syndrome. Mice experienced CIH for 28 days(nadir 7% to peak 8% oxygen, 20 min per day), resulting in left ventricle(LV) dysfunction and structure abnormalities. After administration of SMS(0.55, 1.1, and 5.5 g·kg-1·d-1) for four weeks, improved cardiac function was observed, as indicated by the increase in the ejection fraction from the LV on echocardiography. SMS also preserved the structural integrity of the LV against eccentric hypotrophy, tissue vacuolization, and mitochondrial injury as measured by histology, electron microscopy, and ultrasound assessments. Mechanistically, the antioxidant effects of SMS were demonstrated; SMS was able to suppress mitochondrial apoptosis as indicated by the reduction of several pro-apoptotic factors(Bax, cytochrome c, and cleaved caspase-3) and up-regulation of the anti-apoptosis factor Bcl-2. In conclusion, these results demonstrate that SMS treatment can protect the structure and function of the LV and that the protective effects of this formula are associated with the regulation of the mitochondrial apoptosis pathway.

Ginsenoside Rb1 improves brain,lung,and intestinal barrier damage in middle cerebral artery occlusion/reperfusion(MCAO/R)micevia the PPARγsignaling pathway

Ischemic stroke causes brain inflammation and multi-organ injury,which is closely associated with the peroxisome proliferator-activated receptor-gamma(PPARγ)signaling pathway.Recent studies have indicated that ginsenoside Rb1(GRb1)can protect the integrity of the blood-brain barrier after stroke.In the current study,a mouse model of middle cerebral artery occlusion/reperfusion(MCAO/R)was established to determine whether GRb1 can ameliorate brain/lung/intestinal barrier damage via the PPARγsignaling pathway.Staining(2,3,5-triphenyltetrazolium chloride,hematoxylin,and eosin)and Doppler ultrasonography were employed to detect pathological changes.Endothelial breakdown was investigated with the leakage of Evans Blue dye and the expression of TJs(tight junctions)and AJs(adherent junctions).Western blot and immunofluorescence were used to determine the levels of cell junction proteins,PPARγand NF-κB.Results showed that GRb1 significantly mitigated multi-organ injury and increased the expression of cerebral microvascular,pulmonary vascular,and intestinal epithelial connexins.In brain,lung,and intestinal tissues,GRb1 activated PPARγ,decreased the levels of phospho-NF-κB p65,and inhibited the production of proinflammatory cytokines,thereby maintaining barrier permeability.However,co-treatment with GRb1 and the PPARγantagonist GW9662 reversed the barrier-protective effect of GRb1.These findings indicated that GRb1 can improve stroke-induced brain/lung/intestinal barrier damagevia the PPARγpathway.

An optimized micro-assay of myosin Ⅱ ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors

Myosin Ⅱ plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin Ⅱ ATPase activity based on molybdenum blue method, using a known myosin Ⅱ ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate(ATP) and calcium chloride, p H, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin Ⅱ ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L^(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin Ⅱ ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin Ⅱ ATPase inhibitors in vitro.