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.

[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.

[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.

Molecular cloning and expression of a cucumber (Cucumis sativus L.) heme oxygenase-1 gene, CsHO1, which is involved in adventitious root formation.

Our previous work showed that in cucumber (Cucumis sativus), auxin rapidly induces heme oxygenase (HO) activity and the product of HO action, carbon monoxide (CO), then triggers the signal transduction events leading to adventitious root formation. In this study, the cucumber HO-1 gene (named as CsHO1) was isolated and sequenced. It contains four exons and three introns and encodes a polypeptide of 291 amino acids. Further results show that CsHO1 shares a high homology with plant HO-1 proteins and codes a 33.3 kDa protein with a 65-amino transit peptide, predicting a mature protein of 26.1 kDa. The mature CsHO1 was expressed in Escherichia coli to produce a fusion protein, which exhibits HO activity. The CsHO1:GFP fusion protein was localized in the chloroplast. Related biochemical analyses of mature CsHO1, including Vmax, Km, Topt and pHopt, were also investigated. CsHO1 mRNA was found in germinating seeds, roots, stem, and especially in leaf tissues. Several well-known adventitious root inducers, including auxin, ABA, hemin, nitric oxide donor sodium nitroprusside (SNP), CaCl(2), and sodium hydrosulfide (NaHS), differentially up-regulate CsHO1 transcripts and corresponding protein levels. These results suggest that CsHO1 may be involved in cucumber adventitious rooting.