Elucidation of the mechanism of Yiqi Tongluo Granule against cerebral ischemia/reperfusion injury based on a combined strategy of network pharmacology, multi-omics and molecular biology.

BACKGROUND: Ischemic stroke is caused by local lesions of the central nervous system and is a severe cerebrovascular disease. A traditional Chinese medicine, Yiqi Tongluo Granule (YQTL), shows valuable therapeutic effects. However, the substances and mechanisms remain unclear. PURPOSE: We combined network pharmacology, multi-omics, and molecular biology to elucidate the mechanisms by which YQTL protects against CIRI. STUDY DESIGN: We innovatively created a combined strategy of network pharmacology, transcriptomics, proteomics and molecular biology to study the active ingredients and mechanisms of YQTL. We performed a network pharmacology study of active ingredients absorbed by the brain to explore the targets, biological processes and pathways of YQTL against CIRI. We also conducted further mechanistic analyses at the gene and protein levels using transcriptomics, proteomics, and molecular biology techniques. RESULTS: YQTL significantly decreased the infarction volume percentage and improved the neurological function of mice with CIRI, inhibited hippocampal neuronal death, and suppressed apoptosis. Fifteen active ingredients of YQTL were detected in the brains of rats. Network pharmacology combined with multi-omics revealed that the 15 ingredients regulated 19 pathways via 82 targets. Further analysis suggested that YQTL protected against CIRI via the PI3K-Akt signaling pathway, MAPK signaling pathway, and cAMP signaling pathway. CONCLUSION: We confirmed that YQTL protected against CIRI by inhibiting nerve cell apoptosis enhanced by the PI3K-Akt signaling pathway.

A network-based method for mechanistic investigation and neuroprotective effect on treatment of tanshinone â against ischemic stroke in mouse.

ETHNOPHARMACOLOGICAL RELEVANCE: Tanshinone-Ⅰ (TSNⅠ), a member of the mainly active components of Salvia miltiorrhiza Bunge (Dan Shen), which is widely used for the treatment for modern clinical diseases including cardiovascular and cerebrovascular diseases, has been reported to show the properties of anti-oxidation, anti-inflammation, neuroprotection and other pharmacological actions. However, whether TSNⅠ can improve neuron survival and neurological function against transient focal cerebral ischemia (tMCAO) in mice is still a blank field. AIM OF THE STUDY: This study aims to investigate the neuroprotective effects of TSNⅠ on ischemic stroke (IS) induced by tMCAO in mice and explore the potential mechanism of TSNⅠ against IS by combining network pharmacology approach and experimental verification. MATERIALS AND METHODS: In this study, the pivotal candidate targets of TSNⅠ against IS were screened by network pharmacology firstly. Enrichment analysis and molecular docking of those targets were performed to identify the possible mechanism of TSNⅠ against IS. Afterwards, experiments were carried out to further verify the mechanism of TSNⅠ against IS. The infarct volume and neurological deficit were evaluated by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Longa respectively. Immunohistochemistry was used to observe neuronal death in the hippocampus and cortical regions by detecting the change of NeuN. The predicting pathways of signaling-related proteins were assessed by Western blot in vitro and in vivo experiments. RESULTS: In vivo, TSNⅠ was found to dose-dependently decrease mice's cerebral infarct volume induced by tMCAO. In vitro, pretreatment with TSNⅠ could increase cell viability of HT-22 cell following oxygen-glucose deprivation (OGD/R). Moreover, the results showed that 125 candidate targets were identified, Protein kinase B (AKT) signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and mitogen-activated protein kinases 1 (MAPK1) and AKT1 could be bound to TSNⅠ more firmly by molecular docking analysis, which implies that TSNⅠ may play a role in neuroprotection through activating AKT and MAPK signaling pathways. Meanwhile, TSNⅠ was confirmed to significantly protect neurons from injury induced by IS through activating AKT and MAPK signaling pathways. CONCLUSION: In conclusion, our study clarifies that the mechanism of TSNⅠ against IS might be related to AKT and MAPK signaling pathways, which may provide the basic evidence for further development and utilization of TSNⅠ.

Hyperoside alleviates epilepsy-induced neuronal damage by enhancing antioxidant levels and reducing autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum perforatum L. (genus Hypericum, family Hypericaceae), a plant commonly used in traditional Chinese medicine, is believed to confer a wide range of benefits, including fever reduction, detoxification, calming, and pain relief via decoctions of its stems and leaves. Hyperoside (HYP), a natural compound extracted from Hypericum perforatum L., has been shown to demonstrate a wide array of bioactivities including antioxidative, anti-inflammatory, and anti-apoptotic effects. In this study, we investigated the effects of HYP on epilepsy-induced neuronal damage in mice and the associated regulatory factors. AIM OF THE STUDY: This study examined the potential therapeutic use of HYP for the treatment of neuronal damage in a mouse model of epilepsy and explored the relationships of the potential neuroprotective effects of HYP pretreatment with antioxidant levels and autophagy. MATERIALS AND METHODS: ICR mice were randomly divided into six groups: sham group, sham-HYP group, KA group, KA-HYP group, KA-HYP-DDC group and KA-CQ group. Immunohistochemical staining was used to assess changes in NeuN, IBA-1, and GFAP expression in the CA3 region of the hippocampus. Immunofluorescence staining was used to assess the effects of HYP on the number of autophagosomes that accumulated in neurons in the hippocampal CA3 region. The levels of SOD1, SOD2, LC3I/II, Beclin1, and PI3K/AKT and MAPK signaling-related proteins were detected by Western blot. RESULTS: Pretreatment with 50 mg/kg HYP protected against epilepsy-induced neuronal damage in the hippocampal CA3 region. Additionally, HYP enhanced antioxidant levels and reduced the levels of autophagy-related proteins via the PI3K/AKT and MAPK pathways. CONCLUSION: HYP protected the hippocampal CA3 region against epilepsy-induced neuronal damage via enhancing antioxidant levels and reducing autophagy. The mechanism of action may be related to the maintenance of antioxidant levels and the suppression of autophagy via the PI3K/Akt and MAPK pathways.

Pretreatment of Populus tomentiglandulosa protects hippocampal CA1 pyramidal neurons from ischemia-reperfusion injury in gerbils via increasing SODs expressions and maintaining BDNF and IGF-I expressions.

To examine the effects of Populus tomentiglandulosa (PT) extract on the expressions of antioxidant enzymes and neurotrophic factors in the cornu ammonis 1 (CA1) region of the hippocampus at 5 min after inducing transient global cerebral ischemia (TGCI) in gerbils, TGCI was induced by occlusion of common carotid arteries for 5 min. Before ischemic surgery, 200 mg·kg-1 PT extract was orally administrated once daily for 7 d. We performed neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B staining. Furthermore, we determined in situ production of superoxide anion radical, expression levels of SOD1 and SOD2 as antioxidant enzymes and brain-derived neurotrophic factor (BDNF) and insulin-like growth factor I (IGF-I) as neurotrophic factors. Pretreatment with 200 mg·kg-1 PT extract prevented neuronal death (loss). Furthermore, pretreatment with 200 mg·kg-1 PT extract significantly inhibited the production of superoxide anion radical, increased expressions of SODs and maintained expressions of BDNF and IGF-I. Such increased expressions of SODs were maintained in the neurons after IRI. In summary, pretreated PT extract can significantly increase levels of SODs and protect the neurons against TGCI, suggesting that PT can be a useful natural agent to protect against TGCI.

A Network-Based Method for Mechanistic Investigation and Neuroprotective Effect on Post-treatment of Senkyunolid-H Against Cerebral Ischemic Stroke in Mouse.

Senkyunolide-H (SEH), a major bioactive compound extracted from Ligusticum chuanxiong, has been reported to be effective in preventing cerebral ischemic stroke (CIS). In this study, we employed network pharmacology to reveal potential mechanism of SEH against CIS on a system level and confirmed the therapeutic effects of SEH on CIS by models of cerebral ischemia-reperfusion in vivo and in vitro. Through protein-protein interaction networks construction of SEH- and CIS-related targets, a total of 62 key targets were obtained by screening topological indices and analyzed for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment. Gene Ontology analysis indicated that SEH might have a role in treating CIS via regulating some biological processes including regulation of transcription from RNA polymerase II promoter, epidermal growth factor receptor signaling pathway, phosphatidylinositol-mediated signaling, and some molecular function, such as transcription factor and protein phosphatase binding and nitric oxide synthase regulator activity. Meanwhile, the Kyoto Encyclopedia of Genes and Genomes analysis showed that phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was significantly enriched. In addition, our result showed that SEH posttreatment significantly decreased the neurological scores, infarct volume, and neuronal death in the middle cerebral artery occlusion mice. Moreover, the PI3K/Akt/nuclear factor kappa B signaling pathway was activated by intragastric administration of 40 mg/kg SEH, as verified by Western blot. In vitro, treatment of PC12 cells with 100 µM SEH markedly reduced cell death induced by oxygen-glucose deprivation through the activation of PI3K/Akt/nuclear factor kappa B pathway, and the therapeutic effect of SEH was obviously inhibited by 10 µM LY294002. In summary, these results suggested that SEH carries a therapeutic potential in CIS involving multiple targets and pathways, and the most crucial mechanism might be through the activation of PI3K/Akt/nuclear factor kappa B (NF-κB) signaling pathway to inhibit inflammatory factor releases and increase the antiapoptosis capacity. Our study furnishes the future traditional Chinese medicine research with a network pharmacology framework.

Icariin Improves Cognitive Impairment after Traumatic Brain Injury by Enhancing Hippocampal Acetylation.

OBJECTIVE: To examine the effect of icariin (ICA) on the cognitive impairment induced by traumatic brain injury (TBI) in mice and the underlying mechanisms related to changes in hippocampal acetylation level. METHODS: The modifified free-fall method was used to establish the TBI mouse model. Mice with post-TBI cognitive impairment were randomly divided into 3 groups using the randomised block method (n=7): TBI (vehicle-treated), low-dose (75 mg/kg) and high-dose (150 mg/kg) of ICA groups. An additional sham-operated group (vehicle-treated) was employed. The vehicle or ICA was administrated by gavage for 28 consecutive days. The Morris water maze (MWM) test was conducted. Acetylcholine (ACh) content, mRNA and protein levels of choline acetyltransferase (ChAT), and protein levels of acetylated H3 (Ac-H3) and Ac-H4 were detected in the hippocampus. RESULTS: Compared with the sham-operated group, the MWM performance, hippocampal ACh content, mRNA and protein levels of ChAT, and protein levels of Ac-H3 and Ac-H4 were signifificantly decreased in the TBI group (P<0.05). High-dose of ICA signifificantly ameliorated the TBI-induced weak MWM performance, increased hippocampal ACh content, and mRNA and protein levels of ChAT, as well as Ac-H3 protein level compared with the TBI group (P<0.05). CONCLUSION: ICA improved post-TBI cognitive impairment in mice by enhancing hippocampal acetylation, which improved hippocampal cholinergic function and ultimately improved cognition.

Pretreated Glehnia littoralis Extract Prevents Neuronal Death Following Transient Global Cerebral Ischemia through Increases of Superoxide Dismutase 1 and Brain-derived Neurotrophic Factor Expressions in the Gerbil Hippocampal Cornu Ammonis 1 Area.

BACKGROUND: Glehnia littoralis, as a traditional herbal medicine to heal various health ailments in East Asia, displays various therapeutic properties including antioxidant effects. However, neuroprotective effects of G. littoralis against cerebral ischemic insults have not yet been addressed. Therefore, in this study, we first examined its neuroprotective effects in the hippocampus using a gerbil model of transient global cerebral ischemia (TGCI). METHODS: Gerbils were subjected to TGCI for 5 min. G. littoralis extract (GLE; 100 and 200 mg/kg) was administrated orally once daily for 7 days before ischemic surgery. Neuroprotection was examined by neuronal nuclear antigen immunohistochemistry and Fluoro-Jade B histofluorescence staining. Gliosis was observed by immunohistochemistry for glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1. For neuroprotective mechanisms, immunohistochemistry for superoxide dismutase (SOD) 1 and brain-derived neurotrophic factor (BDNF) was done. RESULTS: Pretreatment with 200 mg/kg of GLE protected pyramidal neurons in the cornu ammonis 1 (CA1) area from ischemic insult area (F = 29.770, P < 0.05) and significantly inhibited activations of astrocytes (F = 22.959, P < 0.05) and microglia (F = 44.135, P < 0.05) in the ischemic CA1 area. In addition, pretreatment with GLE significantly increased expressions of SOD1 (F = 28.561, P < 0.05) and BDNF (F = 55.298, P < 0.05) in CA1 pyramidal neurons of the sham- and ischemia-operated groups. CONCLUSIONS: Our findings indicate that pretreatment with GLE can protect neurons from ischemic insults, and we suggest that its neuroprotective mechanism may be closely associated with increases of SOD1 and BDNF expressions as well as attenuation of glial activation.

Anti-inflammatory effect of tanshinone I in neuroprotection against cerebral ischemia-reperfusion injury in the gerbil hippocampus.

Tanshinone I (TsI) is an important lipophilic diterpene extracted from Danshen (Radix Salvia miltiorrhizae) and has been used in Asia for the treatment of cerebrovascular diseases such as ischemic stroke. In this study, we examined the neuroprotective effect of TsI against ischemic damage and its neuroprotective mechanism in the gerbil hippocampal CA1 region (CA1) induced by 5 min of transient global cerebral ischemia. Pre-treatment with TsI protected pyramidal neurons from ischemic damage in the stratum pyramidale (SP) of the CA1 after ischemia-reperfusion. The pre-treatment with TsI increased the immunoreactivities and protein levels of anti-inflammatory cytokines [interleukin (IL)-4 and IL-13] in the TsI-treated-sham-operated-groups compared with those in the vehicle-treated-sham-operated-groups; however, the treatment did not increase the immunoreactivities and protein levels of pro-inflammatory cytokines (IL-2 and tumor necrosis factor-α). On the other hand, in the TsI-treated-ischemia-operated-groups, the immunoreactivities and protein levels of all the cytokines were maintained in the SP of the CA1 after transient cerebral ischemia. In addition, we examined that IL-4 injection into the lateral ventricle did not protect pyramidal neurons from ischemic damage. In conclusion, these findings indicate that the pre-treatment with TsI can protect against ischemia-induced neuronal death in the CA1 via the increase or maintenance of endogenous inflammatory cytokines, and exogenous IL-4 does not protect against ischemic damage.

Neuroprotection of a novel synthetic caffeic acid-syringic acid hybrid compound against experimentally induced transient cerebral ischemic damage.

We investigated effects of caffeic acid, syringic acid, and their synthesis on transient cerebral ischemic damage in the gerbil hippocampal CA1 region. In the 10 mg/kg caffeic acid-, syringic acid-, and 20 mg/kg syringic-treated ischemia groups, we did not find any significant neuroprotection in the ischemic hippocampal CA region. In the 20 mg/kg caffeic acid- and 10 mg/kg caffeic acid-syringic acid-treated ischemia groups, moderate neuroprotection was found in the hippocampal CA1 region. In the 20 mg/kg caffeic acid-syringic acid-treated ischemia group, a strong neuroprotective effect was found in the ischemic hippocampal CA1 region: about 89 % of hippocampal CA1 region pyramidal neurons survived. We also observed changes in glial cells (astrocytes and microglia) in the ischemic hippocampal CA1 region in all the groups. Among them, the distribution pattern of the glial cells was only in the 20 mg/kg caffeic acid-syringic acid-treated ischemia group similar to that in the sham group (control). In brief, 20 mg/kg caffeic acid-syringic acid showed a strong neuroprotective effect with an inhibition of glia activation in the hippocampal CA1 region induced by transient cerebral ischemia.

Comparison of neuroprotective effects of five major lipophilic diterpenoids from Danshen extract against experimentally induced transient cerebral ischemic damage.

We observed neuroprotective effects of five major lipophilic diterpenes derived from Danshen (Radix Salvia miltiorrhiza) extract, such as cryptotanshinone (CTs), dihydrotanshinone I (DTsI), tanshinone I (TsI), tanshinone IIA (TsIIA) and tanshinone IIB (TsIIB), in the hippocampal CA1 region (CA1) against transient ischemic damage in gerbils. These diterpenes were administered 30min before ischemia-reperfusion and the animals were sacrificed 4days after ischemia-reperfusion. In the vehicle-treated-group, cresyl violet positive (CV(+)) cells and neuronal nuclei (NeuN)(+) neurons were significantly decreased in the CA1. However, in the TsI- and CTs-treated-ischemia-groups, CV(+) and NeuN(+) neurons were abundant in the CA1. In the other groups, the number of CV(+) and NeuN(+) neurons was less than the TsI- and CTs-treated-ischemia-groups. In addition, gliosis induced by ischemic damage was apparently blocked in the TsI- and CTs-treated-ischemia-groups. These results suggest that TsI and CTs among five major lipophilic diterpenes have strong potentials for neuroprotection against ischemic damage.