Multiple Mechanistic Models Reveal the Neuroprotective Effects of Diterpene Ginkgolides against Astrocyte-Mediated Demyelination via the PAF-PAFR Pathway.

Currently, therapies for ischemic stroke are limited. Ginkgolides, unique Folium Ginkgo components, have potential benefits for ischemic stroke patients, but there is little evidence that ginkgolides improve neurological function in these patients. Clinical studies have confirmed the neurological improvement efficacy of diterpene ginkgolides meglumine injection (DGMI), an extract of Ginkgo biloba containing ginkgolides A (GA), B (GB), and K (GK), in ischemic stroke patients. In the present study, we performed transcriptome analyses using RNA-seq and explored the potential mechanism of ginkgolides in seven in vitro cell models that mimic pathological stroke processes. Transcriptome analyses revealed that the ginkgolides had potential antiplatelet properties and neuroprotective activities in the nervous system. Specifically, human umbilical vein endothelial cells (HUVEC-T1 cells) showed the strongest response to DGMI and U251 human glioma cells ranked next. The results of pathway enrichment analysis via gene set enrichment analysis (GSEA) showed that the neuroprotective activities of DGMI and its monomers in the U251 cell model were related to their regulation of the sphingolipid and neurotrophin signaling pathways. We next verified these in vitro findings in an in vivo cuprizone (CPZ, bis(cyclohexanone)oxaldihydrazone)-induced model. GB and GK protected against demyelination in the corpus callosum (CC) and promoted oligodendrocyte regeneration in CPZ-fed mice. Moreover, GB and GK antagonized platelet-activating factor (PAF) receptor (PAFR) expression in astrocytes, inhibited PAF-induced inflammatory responses, and promoted brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) secretion, supporting remyelination. These findings are critical for developing therapies that promote remyelination and prevent stroke progression.

Two new iridoid glycosides from the fruit of Gardenia jasminoides.

Two new iridoid glycosides, 2'-O-cis-coumaroylgardoside (1), and 6'-O-caffeoylioxide (2), were isolated from the fruit of Gardenia jasminoides. The structures of these compounds were elucidated based on spectroscopic analysis (HR-ESI-MS, NMR) and chemical methods. The anti-inflammatory activities of the isolates were evaluated by measuring their inhibitory effects on PGE2 production in LPS stimulated RAW 264.7 macrophages, compounds 1 and 2 could reduce PGE2 levels in LPS-activated RAW 264.7 macrophages with IC50 values of 121.4 and 83.38 µM, respectively.

Diterpene Ginkgolides Meglumine Injection inhibits apoptosis induced by optic nerve crush injury via modulating MAPKs signaling pathways in retinal ganglion cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Diterpene Ginkgolides Meglumine Injection (DGMI) is made of extracts from Ginkgo biloba L, including Ginkgolides A, B, and K and some other contents, and has been widely used as the treatment of cerebral ischemic stroke in clinic. It can be learned from the "Compendium of Materia Medica" that Ginkgo possesses the effect of "dispersing toxin". The ancient Chinese phrase "dispersing toxin" is now explained as elimination of inflammation and oxidative state in human body. And it led to the original ideas for today's anti-oxidation studies of Ginkgo in apoptosis induced by optic nerve crush injury. AIM OF THE STUDY: To investigate the underlying molecular mechanism of the DGMI in retinal ganglion cells (RGCs) apoptosis. MATERIALS AND METHODS: TUNEL staining was used to observe the anti-apoptotic effects of DGMI on the adult rat optic nerve injury (ONC) model, and flow cytometry and hoechst 33,342 staining were used to observe the anti-apoptotic effects of DGMI on the oxygen glucose deprivation (OGD) induced RGC-5 cells injury model. The regulation of apoptosis and MAPKs pathways were investigated with Immunohistochemistry and Western blotting. RESULTS: This study demonstrated that DGMI is able to decrease the conduction time of F-VEP and ameliorate histological features induced by optic nerve crush injury in rats. Immunohistochemistry and TUNEL staining results indicated that DGMI can also inhibit cell apoptosis via modulating MAPKs signaling pathways. In addition, treatment with DGMI markedly improved the morphological structures and decreased the apoptotic index in RGC-5 cells. Mechanistically, DGMI could significantly inhibit cell apoptosis by inhibiting p38, JNK and Erk1/2 activation. CONCLUSION: The study shows that DGMI and ginkgolides inhibit RGCs apoptosis by impeding the activation of MAPKs signaling pathways in vivo and in vitro. Therefore, the present study provided scientific evidence for the underlying mechanism of DGMI and ginkgolides on optic nerve crush injury.

Dissecting the novel mechanism of reduning injection in treating Coronavirus Disease 2019 (COVID-19) based on network pharmacology and experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Reduning injection (RDNI) is a patented Traditional Chinese medicine that contains three Chinese herbal medicines, respectively are the dry aboveground part of Artemisia annua L., the flower of Lonicera japonica Thunb., and the fruit Gardenia jasminoides J.Ellis. RDNI has been recommended for treating Coronavirus Disease 2019 (COVID-19) in the "New Coronavirus Pneumonia Diagnosis and Treatment Plan". AIM OF THE STUDY: To elucidate and verify the underlying mechanisms of RDNI for the treatment of COVID-19. METHODS: This study firstly performed anti-SARS-CoV-2 experiments in Vero E6 cells. Then, network pharmacology combined with molecular docking was adopted to explore the potential mechanisms of RDNI in the treatment for COVID-19. After that, western blot and a cytokine chip were used to validate the predictive results. RESULTS: We concluded that half toxic concentration of drug CC50 (dilution ratio) = 1:1280, CC50 = 2.031 mg crude drugs/mL (0.047 mg solid content/mL) and half effective concentration of drug (EC50) (diluted multiples) = 1:25140.3, EC50 = 103.420 µg crude drugs/mL (2.405 µg solid content/mL). We found that RDNI can mainly regulate targets like carbonic anhydrases (CAs), matrix metallopeptidases (MMPs) and pathways like PI3K/AKT, MAPK, Forkhead box O s and T cell receptor signaling pathways to reduce lung damage. We verified that RDNI could effectively inhibit the overexpression of MAPKs, PKC and p65 nuclear factor-κB. The injection could also affect cytokine levels, reduce inflammation and display antipyretic activity. CONCLUSION: RDNI can regulate ACE2, Mpro and PLP in COVID-19. The underlying mechanisms of RDNI in the treatment for COVID-19 may be related to the modulation of the cytokine levels and inflammation and its antipyretic activity by regulating the expression of MAPKs, PKC and p65 nuclear factor NF-κB.

[Molecular mechanism of Epimedii Folium in treatment of osteoporosis based on network pharmacology].

Osteoporosis is a systematic bone disease,characterized by deterioration in bone mass or micro-architecture,and increasing risk of fragility and fractures. With the development of aging problems,osteoporosis has been a global health problem. At present,due to the undesirable side effects of synthetic osteoporosis inhibitors,more efforts are made in treatment of osteoporosis by traditional Chinese medicine and its prescriptions. Epimedii Folium,one of the most common herbs for osteoporosis,has attracted great attentions worldwide.In this study,network pharmacology was employed to analyze the active components and potential molecular mechanism of Epimedii Folium on osteoporosis. Component-target network analysis showed that those with higher molecular network degree were flavonoids,with estrogen-like activity,antioxidation and free radical-scavenging activities,playing certain roles in preventing and treating osteoporosis. On the other hand,the targets with high degree were mostly related with sex hormone,osteoclast differentiation,bone matrix degradation,and reactive oxygen species in drug-target network. Multiple components of Epimedii Folium could be interacted with these targets. This study shows that Epimedium could prevent and treat osteoporosis through multiple active ingredients acting on multiple targets.

[Effect of compatibility of ginkgolide A, ginkgolide B and ginkgolide K].

To investigate the best active compatibility of ginkgolide A, B and K (GA，GB，GK). The effects of GA, GB, GK alone, combinations of each two of them, and combinations of these three components on platelet-activating factor (PAF)-induced platelet aggregation activity and rat cerebral ischemia reperfusion model (tMCAO) were compared in this study. Different compatibilities of GA, GB and GK could significantly reduce the maximum aggregation rate of PAF-induced platelet aggregation, and the effect was most obvious in combination of the three. Different compatibilities of GA, GB and GK could alleviate the neural function, cerebral infarction volume and cerebral edema in the tMCAO model of rats to different degrees, and the effect of combinations of the three was stronger than those of combinations of two and single use. The combination of all of GA, GB and GK had the strongest effect on nerve injury caused by anti-platelet aggregation in tMCAO rats.

Discovery of a potent angiotensin converting enzyme inhibitor via virtual screening.

Prompted by the prominent role of angiotensin converting enzyme (ACE) in hypertension, heart failures, myocardial infarction and diabetic nephropathy, we have attempted to discover novel ACE inhibitors through ligand-based virtual screening. Molecular docking method and rigorously validated model was utilized to search a natural compounds database. Finally, 36 compounds were randomly selected and subjected to in vitro ACE kinase inhibitory assay using fluorescence assays method. The results showed that three compounds (Licochalcone A, Echinatin and EGCG) have strong potential to be developed as a new class of ACE inhibitors. Further chemical modification via fragment modifications guided by structure and ligand-based computational methodologies can lead to discover better agents as potential clinical candidates.

[Effects of ginkgolide A, B and K on platelet aggregation].

To investigate the effects of ginkgolide A (GA), ginkgolide B (GB) and ginkgolide K (GK) on platelet aggregation in rabbits, and compare the similarities and differences among these three components. The effects of different doses of ginkgolide A, B and K on platelet aggregation induced by platelet activating factor (PAF) were observed by using in vitro experiment. The results showed that three compounds could inhibit platelet aggregation induced by PAF in vitro, and the intensity was GK> GB> GA. It was further found that all of them can mobilize [Ca2+]i and enhance intracellular c-AMP level in a dose-dependent manner, which was consistent to the ability to antagonize PAF receptor. These findings indicated that GK was highly selective for PAF receptor, and may inhibit platelet aggregation by activating cAMP signaling pathway and inhibiting intracellular [Ca2+]i mobilization; GB and GA also had strong antagonism to PAF receptor, but the effect was weaker than that of GK.

[Effects of ginkgolide K on platelet aggregation activity and neuroprotection].

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.

[Protective effects of Ginkgo Terpene Lactones Meglumine Injection on focal cerebral ischemia in rats].

To investigate the protective effects of ginkgo diterpene lactone meglumine injection (GDLMI) on cerebral focal ischemia reperfusion injury induced by middle cerebral artery occlusion (MCAO) in rats, and explore its possible mechanism. One hundred and forty male SD rats were randomly divided into sham operation group, model group, ginkgo biloba extract injection (Ginaton, 1.0 mL•kg⁻¹) group, nimodipine (0.4 mg•kg⁻¹) group, and GDLMI (5.2, 2.6, 1.3 mg•kg⁻¹) groups; All of rats received corresponding drugs by tail vein injection 4 days before operation (normal saline in model group and sham operation group). Except the sham operation group, the cerebral ischemic stroke model was established by MCAO method in right brain of the other rats. After 3 h of ischemia, all the animals received intravenous administration again. The neurobehavioral scores of rats after ischemia-reperfusion were evaluated and the infarct rate of brain tissue was observed by TTC staining. The super oxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA) and lactic acid (LA) contents in brain tissue homogenate and the concentration of Ca2+, glutamate (Glu) and aspartate (Asp), creatine phosphate kinase (CK-BB) and lactate dehydrogenase (LDH) content changes in cerebrospinal fluid were measured. As compared with the sham operation group, the cerebral infarction rate was increased significantly in the model group; the content of MDA and LA in the homogenate of brain tissue was increased, and the content of GSH and SOD was decreased; in cerebrospinal fluid, Ca2+ concentration was decreased, and the content of Glu and Asp, CK-BB and LDH increased significantly. As compared with the model group, the high and medium dose GDLMI groups can significantly reduce the cerebral infarction rate and improve the symptoms of neurological impairment; increase SOD and GSH activity, reduce MDA and LA content in serum; increase Ca2+ concentration in cerebrospinal fluid and decrease the content of neurotransmitter Glu and Asp as well as CK-BB and LDH. GDLMI could obviously improve neurologic impairment in model rats, and the mechanism may be related to recovering the blood brain barrier, scavenging free radicals, decreasing free Ca2+ inflow into the cells and the content of excitatory amino acid in cerebrospinal fluid to improve its protective effect on cerebral ischemia.

[Cerebral vascular protective effect of ginkgo diterpene lactone meglumine injection].

The left middle cerebral artery occlusion (MCAO) model was made by inserting the nylon thread plug into the internal carotid artery. The behavioral score, cerebral infarction area, brain water content, ethidium bromide (EB) spillover, coagulation four indices, occludin and MMP-9 expression in brain tissues were detected after 14 days of administration, to investigate whether the protective effect of ginkgo diterpene lactone meglumine injection (GDLMI) which had obvious protective effect on cerebral ischemic injury in the previous experiment was related to reducing the permeability of the blood-brain barrier (BBB) and reducing the risk of bleeding, and to explore its possible mechanism of action. The results showed that GDLMI could effectively alleviate the behavioral changes caused by MCAO at 24 h, reduce the behavioral score, improve the edema of brain tissue, reduce the EB overflow rate, reduce the bleeding tendency caused by long-term administration, significantly reduce the occlusion deficiency in ischemic brain tissue of model rats, and down-regulate MMP-9 expression. The above results indicate that GDLMI has obvious effect on cerebral ischemia, and the therapeutic effect of GDLMI may mainly depend on lowering the permeability of blood-brain barrier to improve brain edema.

[Antagonistic effect of ginkgolide homologues on PAF-induced platelet aggregation and neuroprotective effect].

To study the antagonistic effect of ginkgolide homologues on platelet-activating factor (PAF)-induced platelet aggregation and investigate its neuroprotective effect. PAF was used as a coagulant, and ginkgolides were added to the rabbit blood samples respectively. The inhibitory effect of each compound on platelet aggregation was detected by turbidimetry. In L-glutamate induced primary cortical neuron cell injury model, MTT assay was used to detect cell viability. Intracellular free Ca2+ concentration in neurons was measured by using the fluorescent Ca2+ indicator Fura-2 AM. Morphological observation and Hoechst 33258 staining were used to detect the inhibitory effect of ginkgolide on neuronal apoptosis. The results showed that the inhibitory effect on PAF-induced platelet aggregation activity in ginkgolide homologues was ginkgolide K (GK), ginkgolide B (GB), ginkgolide A (GA), ginkgolide C (GC), ginkgolide M (GM), ginkgolide J (GJ) and ginkgolide (GL) from high to low. GB and GK (1-100 µmol•L ⁻¹) could significantly reduce the cell damage caused by L-glutamate, with survival rate increasing, intracellular calcium concentration reducing and cell morphology restoring. This paper has identified the activities and characteristics of various compounds of ginkgolide homologues on PAF-induced platelet aggregation as well as its neuroprotective effect.

Luteoloside Acts as 3C Protease Inhibitor of Enterovirus 71 In Vitro.

Luteoloside is a member of the flavonoids family that exhibits several bioactivities including anti-microbial and anti-cancer activities. However, the antiviral activity of luteoloside against enterovirus 71 (EV71) and the potential mechanism(s) responsible for this effect remain unknown. In this study, the antiviral potency of luteoloside against EV71 and its inhibitory effects on 3C protease activity were evaluated. First, we investigated the cytotoxicity of luteoloside against rhabdomyosarcoma (RD) cells, which was the cell line selected for an in vitro infection model. In a subsequent antiviral assay, the cytopathic effect of EV71 was significantly and dose-dependently relieved by the administration of luteoloside (EC50 = 0.43 mM, selection index = 5.3). Using a plaque reduction assay, we administered luteoloside at various time points and found that the compound reduced EV71 viability in RD cells rather than increasing defensive mobilization or viral absorption. Moreover, biochemical studies focused on VP1 (a key structural protein of EV71) mRNA transcript and protein levels also revealed the inhibitory effects of luteoloside on the EV71 viral yield. Finally, we performed inhibition assays using luteoloside to evaluate its effect on recombinant 3C protease activity. Our results demonstrated that luteoloside blocked 3C protease enzymatic activity in a dose-dependent manner (IC50 = 0.36 mM) that was similar to the effect of rutin, which is a well-known C3 protease inhibitor. Collectively, the results from this study indicate that luteoloside can block 3C protease activity and subsequently inhibit EV71 production in vitro.

Drug discovery of neurodegenerative disease through network pharmacology approach in herbs.

Neurodegenerative diseases, referring to as the progressive loss of structure and function of neurons, constitute one of the major challenges of modern medicine. Traditional Chinese herbs have been used as a major preventive and therapeutic strategy against disease for thousands years. The numerous species of medicinal herbs and Traditional Chinese Medicine (TCM) compound formulas in nervous system disease therapy make it a large chemical resource library for drug discovery. In this work, we collected 7362 kinds of herbs and 58,147 Traditional Chinese medicinal compounds (Tcmcs). The predicted active compounds in herbs have good oral bioavailability and central nervous system (CNS) permeability. The molecular docking and network analysis were employed to analyze the effects of herbs on neurodegenerative diseases. In order to evaluate the predicted efficacy of herbs, automated text mining was utilized to exhaustively search in PubMed by some related keywords. After that, receiver operator characteristic (ROC) curves was used to estimate the accuracy of predictions. Our study suggested that most herbs were distributed in family of Asteraceae, Fabaceae, Lamiaceae and Apocynaceae. The predictive model yielded good sensitivity and specificity with the AUC values above 0.800. At last, 504 kinds of herbs were obtained by using the optimal cutoff values in ROC curves. These 504 herbs would be the most potential herb resources for neurodegenerative diseases treatment. This study would give us an opportunity to use these herbs as a chemical resource library for drug discovery of anti-neurodegenerative disease.

One New Conjugate of a Secoiridoid Glucoside with a Sesquiterpene Glucoside from the Flower Buds of Lonicera japonica.

Secosesquside (1), a new secoiridoid glucoside-sesquiterpene conjugate, together with three known secoiridoid derivatives, were isolated from flower buds of Lonicerajaponica. The isolated compounds were elucidated by extensive spectroscopic analyses, especially 2D NMR experiments. The anti-inflammatory activities of the new compound were also evaluated by enzyme-linked immunosorbent assay.

[Study on contribution of main components in Guizhi Fuling capsule based on molecular imprinting technique and activity screening].

To clarify the active components in Guizhi Fuling capsule in treatment of intrinsic dysmenorrhea, pelvic inflammation and hysteromyoma, main components were gradually knocked out from the capsules, the effects of knockout capsules on uterine contraction, TNF-α secretion, murine splenocytes (SPL) and hysteromyoma cells proliferation were evaluated, respectively. The inhibition of capsules on uterine contraction was weakened by gradient knockout of paeoniflorin, paeonol, and amygdalin. The suppression of capsulte on TNF-α secretion was reduced by gradient knockout of gallic acid, cinnamaldehyde, pentagalloylglucose, and pachyman. The promotion of SPL cells proliferation was reversed by gradient knockout of gallic acid, paeoniflorin, cinnamaldehyde, quercetin, and pachyman. The depression of capsules on hysteromyoma cells proliferation was attenuated by gradient knockout of paeoniflorin, paeonol, pentagalloylglucose, and albiflorin. In conclusion, the compounds mentioned-above could be the key active basis of Guizhi Fuling capsule in treatment of intrinsic dysmenorrhea, pelvic inflammation and hysteromyoma.

[Study of effective components and molecular mechanism for Guizhi Fuling formula treatment of dysmenorrhea, pelvic inflammatory disease and uterine fibroids].

In this study, the active components and potential molecular .mechanism of Guizhi Fuling formula in treatment on dysmenorrhea, pelvic inflammation, and hysteromyoma were investigated using network pharmacological methods. Sterols and pentacyclic triterpenes, with high moleculal network degree, revealed promising effects on anti-inflammatory, analgesic, anti-tumor, and immune-regulation, according to D-T network analysis. On the other hand, the targets with high degree were involved in inflammatory, coagulation, angiopoiesis, smooth muscle contraction, and cell reproduction, which showed the novel function in anti-dysmenorrhea, pelvic inflammation, and hysteromyoma. Furthermore, the formula was indicated to play a key role in smooth muscle proliferation, inhibition of new vessels, circulation improvement, reduction of hormone secretion, alleviation of smooth muscle, block of arachidonic acid metabolism, and inflammation in uterus. Thus, the main mechanism of Guizhi Fuling formula was summarized. In conclusion, Guizhi Fuling formula was proven to alleviated dysmenorrhea, pelvic inflammation, and hysteromyoma by acting on multiple targets through several bioactive compounds, regulating 21 biological pathways.

Antiviral effects of Reduning injection against Enterovirus 71 and possible mechanisms of action.

The present study was designed to evaluate the protective effects of Reduning injection against Enterovirus 71 (EV71) in Vero cells and in mice. The Vero cells were infected with 100 and 50 TCID50 (50% tissue culture infective dose) of EV71, respectively. The inhibition of Reduning injection on cytopathic effect (CPE) was detected. Meanwhile, a mouse model produced by intraperitoneal EV71-infection (10(6) TCID50), was used to investigate the protective effects of Reduning injection. The total survival rate, living time, daily survival rate, weight ratio, and score for symptoms were examined. The viral loads in Vero cells and muscle tissues were detected using real-time PCR. Finally, the content of cytokines was analyzed by ELISA. In the Vero cells, 2.5 mg crude drug·mL(-1) of Reduning injection inhibited CPE induced by EV71 infection. In the mice, 1.3 g crude drug·kg(-1) of Reduning injection rescued death triggered by infection, in comparison with model group. Moreover, the survival rate, weight ratio, and clinical scores were also improved. The viral RNA copies in the Vero cells and the mice muscle tissues were reduced. Besides, the steep EV71-induced accumulations of TNF-α and MCP-1 were decreased by Reduning injection. In conclusion, Reduning injection showed promising protective effects against EV71 in Vero cells and in mice.