Investigate mechanism of Jinzhen Oral Liquid for prevention COVID-19 based on network pharmacology and molecular docking technology / 中草药

Objective: To investigate the mechanism of Jinzhen Oral Liquid (JOL) for prevention COVID-19 through network pharmacology and molecular docking technology. Methods: The protein targets related to COVID-19 were searched by literature mining and retrieving in DisGeNET, OMIM, KEGG and UniProt databases. With the aid of Traditional Chinese Medicine Network Pharmacology Intelligent Information Platform (TCMN) searching JOL chemical components and targets, the "herb-compound-target network" was constructed using Cytoscape-3.2.1 software to predict the main active ingredients and action targets of JOL in the treatment of COVID-19. The crystal structure of novel coronavirus (SARS-CoV-2) 3CL hydrolase (3CLpro) and angiotensin converting enzyme II (ACE2) was retrieved from the RCSB PDB database, and the active compounds were docked with the two proteins by using AutoDock Vina software. Results: The herb-compound-target network contained 75 compounds including isoglabrolide, peimisine, and sennoside B, etc., which are from the three medicinal materials of Glycyrrhiza uralensis, Rheum officinale, and Fritillaria ussuriensis, and 28 targets including mammalian target of rapamycin (mTOR), Janus kinase 3 (JAK3) and mitogen-activated protein kinase 1 (MEK1). Furthermore, nine key compounds (isoglabrolide, glabrolide, ebeiedinone, desoxo- glabrolid-acetate, peimisine, verticinone, imperialine, ussuriedinone and euchrenone A5) and 10 potential targets (mTOR, JAK3, ACE2, TNFA, AKT2, PIK3CA, MEK1, BRD2, ACE and ANPEP) of JOL were predicted for treating COVID-19 by network characteristic analysis. The molecular docking results showed that some core compounds of JOL had a certain degree of affinity for 3CLpro and ACE2. Conclusion: JOL may inhibit the occurrence and development of cytokine storm in COVID-19 by regulating the expression of Brd2, CD13, and ACE2 and interfering with the PI3K/Akt, Jak-STAT, TNF and MAPK signaling pathways, and inhibit virus replication by binding with 3CLpro, thus exerting a preventive or therapeutic effect on COVID-19.

Effects of ginkgolide K on platelet aggregation activity and neuroprotection / 中国中药杂志

To investigate the antagonism effects of different concentrations of ginkgolide K(GK) on platelet activating factor (PAF)-induced platelet aggregation and neuroprotective effect on cells and animal models of ischemia-reperfusion injury. GK-containing serum in rabbit was prepared, and the effects of GK-containing serum on PAF-induced platelet aggregation was observed by platelet aggregation assay. The effect of different concentrations of GK on apoptosis of SH-SY5Y cells injured by oxygen-glucose deprivation/reoxygenation (OGD/R) was investigated by Hoechst 33342/PI double staining in OGD/R cell model. The focal cerebral ischemia-reperfusion model (I/R)was established in rats to detect the effects of GK on neurobehavioral scores and cerebral infarction volume. GK could inhibit PAF-induced platelet aggregation, reverse the apoptosis induced by OGD/R injury and improve the neurobehavioral score and cerebral infarction volume after cerebral ischemia-reperfusion injury in rats in a dose-dependent manner. GK can inhibit PAF-induced platelet aggregation and improve nerve injury after cerebral ischemia-reperfusion.

Study on molecular mechanism of Paeoniae Rubra Radix and Phellodendri Cortex intervening Alzheimer's disease using network pharmacology methods / 中草药

Objective: To conduct computing network pharmacological studies on Paeoniae Rubra Radix (Chishao) and Phellodendri Cortex (Huangbai), and to explore their mechanism for intervening Alzheimer's disease (AD). Methods: The interactions among 199 compounds from the two kinds of Chinese Herbs (Chishao and Huangbai) and 23 approved drug targets related to AD were studied with molecular docking and network pharmacological analysis methods. Results: The most of the compounds in Chishao and Huangbai exhibit good drug-like properties. The mechanism of Chishao and Huangbai may be that they modulate the expression of GSK-3β, caspase-7, BchE, and mTOR to resist AD. Conclusion: The method of network pharmacological studies is helpful to explore the possible active molecules in Chishao and Huangbai and elucidate the mechanism of action.

Protective effect of Annexin 5 on human sperm membrane and DNA integrity / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the role of Annexin 5 in protecting human sperm membrane and DNA integrity.</p><p><b>METHODS</b>We collected 53 semen samples based on the criteria of sperm density > 20 x 10(6)/ml and motility > 60%, and divided them into an experimental group (2.5 microl 10(-6) mol/L Annexin 5 added to 47.5 microl semen), a negative control group (2.5 microl 1 mol/L Tris-HCl [pH 8.0, 25 degrees C] added to 47.5 microl semen), and a blank control group (2.5 microl 0.01 mol/L PBS [pH 7.4] added to 47.5 microl semen). After 20 minutes of incubation, we evaluated the sperm membrane integrity using the hypoosmotic swelling test and, after another 60 minutes of treatment with H2O2 at 2.5 microl 10.02 mol/L, measured the sperm nuclear DNA integrity by acridine orange fluorescent staining.</p><p><b>RESULTS</b>After 20 minutes of treatment with Annexin 5, the experimental group showed extremely significant difference in the percentage of hypoosmotic swelling sperm ([66.17 +/- 12.02] %) from the blank control ([58.13 +/- 13.08]%, P < 0.01) and the negative control group ([59.94 +/- 11.91]%, P < 0.01), but there was no significant difference between the latter two. Treatment with H2O2 remarkably increased DFI in the experimental group (6.39 +/- 1.07) as compared with the blank control (11.16 +/- 1.16) and the negative control group (10.86 +/- 1.05, P < 0.01), but no significant difference was observed between the latter two.</p><p><b>CONCLUSION</b>Annexin 5 can increase the percentage of hypoosmotic swelling sperm in vitro and protect sperm membrane integrity, and it can also protect sperm DNA from H2O2 damage.</p>

Effects of GnRH analogues on MAPK pathway in rat Leydig cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the effects of GnRH analogues GnRHa and GnRHant on the MAPK pathway in rat Leydig cells.</p><p><b>METHODS</b>Rat Leydig cells were primarily cultured for 24 hours in vitro and serum-starved for 2 hours, followed by treatment with GnRHa (10(-7) mol/L) or GnRHant (10(-6) mol/L) for 0, 5, 15, 30, 60 and 90 minutes, with the 0 min group as the control. Then the protein levels of phosphorylated ERK (p-ERK) and phosphorylated p38 (p-p38) were detected by Western blot, and that of p-ERK determined by the same means after co-incubation of GnRHa or GnRHant with the PKC inhibitor GF109203X at 1, 5, 10 and 20 micromol/L.</p><p><b>RESULTS</b>After stimulation of the Leydig cells with GnRHa or GnRHant for different times, the protein level of p-p38 showed no significant difference from that of the control group (P > 0.05). Then the Leydig cells were treated with GF109203X at different concentrations for 20 minutes and with addition of GnRHa for another 10 minutes. The level of p-ERK was significantly decreased (P < 0.05) by GF109203X at 10 and 20 micromol/L. Compared with the control, the p-ERK expression was increased by 65% at 15 minutes (P < 0.05) in the GnRHant stimulation group, by 81% (to the peak) at 30 minutes (P < 0.05), began to fall at 60 minutes, and returned to the base level at 90 minutes. The p-ERK level exhibited no significant difference from that of the control (P > 0.05) after treatment of the Leydig cells with different concentrations of GF109203X for 20 minutes and then with GnRHant for 30 minutes.</p><p><b>CONCLUSION</b>The ERK MAPK activation induced by GnRHa depends on the PKC pathway, but not that induced by GnRHant. The p-38 MAPK pathway may not be involved in the effect of GnRH analogues on rat Leydig cells.</p>

Expression of rat annexin 5 and its effect on human sperm motility in vitro / 中华男科学杂志

<p><b>OBJECTIVE</b>Gonadotropin releasing hormones (GnRH) regulate the expression of annexin 5 in Leydig cells, and annexin 5 is supposed to be a signal molecule in regulating testosterone secretion. This study aimed to investigate the function of annexin 5 in male reproduction by observing its effect on human sperm motility in vitro.</p><p><b>METHODS</b>The encoding sequence of rat annexin 5 was chemically synthesized and inserted into the HIS fusion expression vector pET28a. The expression of the fusion protein HIS-annexin 5 was induced by isopropyl-beta-D-thiogalactoside (IPTG) under the control of the T7 promoter, and the products were purified by affinity chromatography. The anticoagulant activity of annexin 5 was determined by the modified activated partial thromboplastin time (APTT) test. Semen samples from 15 donors were assigned to a control and an annexin 5 group, the latter treated with recombinant annexin 5 at the concentration of 10(-8) mol/L. Sperm motility and the percentage of grade a + b sperm were measured by computer-assisted semen analysis (CASA) after 20 and 60 min exposure, and the sperm ascending experiment was done after 20 min treatment.</p><p><b>RESULTS</b>The product of the synthesized target gene was 947 bp in length, and the inserted sequence corresponded to the published encoding sequence of rat annexin 5. The plasmid pET28a-annexin 5 was transformed into E. coli BL21(DE3) and IPTG induced a fusion protein with a relative molecular weight of about 36,000, a purity of 95% and a high anticoagulant activity. Compared with the control group, sperm motility and the percentage of grade a + b sperm were increased by 40% (P < 0.01) and 21% (P < 0.01), respectively, after 20 min treatment with annexin 5, but neither showed any significant improvement after 60 min. The sperm ascending altitude was remarkably elevated after annexin 5 treatment, with extremely significant difference from the control group (37.84 +/- 6.35 vs. 49.5 +/- 12.27, P < 0.01).</p><p><b>CONCLUSION</b>An annexin 5 recombinant expression vector was successfully constructed. The protein annexin 5 can be efficiently expressed in E. coli and effectively improve human sperm motility in vitro.</p>