Advances of phytotherapy in ischemic stroke targeting PI3K/Akt signaling.

The pathogenesis of ischemic stroke is complex, and PI3K/Akt signaling is considered to play a crucial role in it. The PI3K/Akt pathway regulates inflammation, oxidative stress, apoptosis, autophagy, and vascular endothelial homeostasis after cerebral ischemia; therefore, drug research targeting the PI3K/Akt pathway has become the focus of scientists. In this review, we analyzed the research reports of antiischemic stroke drugs targeting the PI3K/Akt pathway in the past two decades. Because of the rich sources of natural products, increasing studies have explored the value of natural compounds, including Flavonoids, Quinones, Alkaloids, Phenylpropanoids, Phenols, Saponins, and Terpenoids, in alleviating neurological impairment and achieved satisfactory results. Herbal extracts and medicinal formulas have been applied in the treatment of ischemic stroke for thousands of years in East Asian countries. These precious clinical experiences provide a new avenue for research of antiischemic stroke drugs. Finally, we summarize and discuss the characteristics and shortcomings of the current research and put forward prospects for further in-depth exploration.

Novel synergistic mechanism of 11-keto-ß-boswellic acid and Z-Guggulsterone on ischemic stroke revealed by single-cell transcriptomics.

Although strides have been made, the challenge of preventing and treating ischemic stroke continues to persist globally. For thousands of years, the natural substances Frankincense and Myrrh have been employed in Chinese and Indian medicine to address cerebrovascular diseases, with the key components of 11-keto-ß-boswellic acid (KBA) and Z-Guggulsterone (Z-GS) being the active agents. In this study, the synergistic effect and underlying mechanism of KBA and Z-GS on ischemic stroke were examined using single-cell transcriptomics. Fourteen cell types were identified in KBA-Z-GS-treated ischemic penumbra, and microglia and astrocytes account for the largest proportion. They were further re-clustered into six and seven subtypes, respectively. GSVA analysis reflected the distinct roles of each subtype. Pseudo-time trajectory indicated that Slc1a2 and Timp1 were core fate transition genes regulated by KBA-Z-GS. In addition, KBA-Z-GS synergistically regulated inflammatory reactions in microglia and cellular metabolism and ferroptosis in astrocytes. Most notably, we established an innovative drug-gene synergistic regulation pattern, and genes regulated by KBA-Z-GS were divided into four categories based on this pattern. Finally, Spp1 was demonstrated as the hub target of KBA-Z-GS. Taken together, this study reveals the synergistic mechanism of KBA and Z-GS on cerebral ischemia, and Spp1 may be the synergistic target for that. Precise drug development targeting Spp1 may offer a potential therapeutic approach for treating ischemic stroke.

Frankincense-Myrrh treatment alleviates neuropathic pain via the inhibition of neuroglia activation mediated by the TLR4/MyD88 pathway and TRPV1 signaling.

BACKGROUND: Neuroglia are important modulators of neuronal functionality, and thus play an integral role in the pathogenesis and treatment of neuropathic pain (NP). According to traditional Chinese medicine, Frankincense-Myrrh is capable of "activating blood and dissipating blood stasis", and as such these two biological compounds are commonly used to treat NP, however, the mechanisms underlying the efficacy of such treatment are unclear. PURPOSE: This study aimed to further elucidate the protective effects associated with the Frankincense-Myrrh treatment of NP. METHODS: A chronic sciatic nerve compression injury (CCI) model of NP was established, after which animals were gavaged with Frankincense, Myrrh, Frankincense-Myrrh, or the positive control drug pregabalin for 14 days. Network pharmacology approaches were used to identify putative pathways and targets associated with the Frankincense-Myrrh-mediated treatment of NP, after which these targets were subjected to in-depth analyses. The impact of TLR4 blockade on NP pathogenesis was assessed by intrathecally administering a TLR4 antagonist (LRU) or the MyD88 homodimerization inhibitory peptide (MIP). RESULTS: Significant alleviation of thermal and mechanical hypersensitivity in response to Frankincense and Myrrh treatment was observed in NP model mice, while network pharmacology analyses suggested that the pathogenesis of NP may be related to TLR4/MyD88-mediated neuroinflammation. Consistently, Frankincense-Myrrh treatment was found to reduce TLR4, MyD88, and p-p65 expression in spinal dorsal horn neuroglia from treated animals, in addition to inhibiting neuronal TRPV1 and inflammatory factor expression. Intrathecal LRU and MIP delivery were sufficient to alleviate thermal and mechanical hyperalgesia in these CCI model mice, with concomitant reductions in neuronal TRPV1 expression and neuroglial activation in the spinal dorsal horn. CONCLUSION: These data suggest that Frankincense-Myrrh treatment was sufficient to alleviate NP in part via inhibiting TLR4/MyD88 pathway and TRPV1 signaling activity. Blocking TLR4 and MyD88 activation may thus hold value as a means of treating NP.

Component-target network and mechanism of Qufeng Zhitong capsule in the treatment of neuropathic pain.

ETHNOPHARMACOLOGICAL RELEVANCE: Qufeng Zhitong capsule (QFZTC) is a traditional Chinese medicine (TCM) clinically used for treating pain. However, the active ingredients of QFZTC and its pharmacological mechanism in the treatment of neuropathic pain (NP) remain unclear. AIM OF THE STUDY: We aimed to identify the active ingredients of QFZTC and reveal its target genes and underlying mechanism of action in NP. MATERIALS AND METHODS: High-performance liquid chromatography (HPLC) was used to identify the active ingredients of QFZTC. Network pharmacology analysis was conducted to determine the core targets and pathway enrichment of QFZTC. An NP mice model was established through chronic compression injury (CCI) surgery of the sciatic nerve, while von Frey instrumentation and a thermal stimulator were employed to measure the sensitivity of mice to mechanical and thermal stimuli. Immunofluorescence was used to observe the expression of TLR4 and p-P65 in microglia. Western blotting was used to detect the levels of protein expression of Iba-1, TLR4, MyD88, P65, p-P65, and c-Fos, while ELISA kits were used to detect the release of TNF-α, IL-6, and IL-1ß. RESULTS: Seven active ingredients were identified in QFZTC: gallic acid, loganylic acid, syringin, corilagin, loganin, ellagic acid, and osthole. Network analysis identified TLR4, TNF, IL6, IL1ß, and c-Fos as core targets, and Toll-like receptors and NF-κB as core signaling pathways. Treatment with QFZTC significantly relieved mechanical allodynia and thermal hyperalgesia in CCI mice models. CCI induced an increase in the expression of TLR4 and p-P65 in microglia, whereas QFZTC dose-dependently reduced the expression of Iba-1, TLR4, MyD88, and p-P65 in the spinal cord. QFZTC inhibited the expression of the c-Fos pain marker and reduced the expression of the TNF-α, IL-6, and IL-1ß inflammatory factors. CONCLUSION: We combined the active ingredients of QFZTC with network pharmacology research to clarify its biological mechanism in the treatment of NP. We demonstrated that QFZTC reduced NP in mice probably through regulating the spinal microglia via the TLR4/MyD88/NF-κB signaling pathway. Hence, QFZTC could be regarded as a potential drug for relieving NP.

A Network Pharmacology Approach to Predict the Proangiogenesis Mechanism of Huangqi-Honghua Herb Pair after Cerebral Ischemia.

OBJECTIVE: Huangqi-Honghua herb pair is known for its medicinal value to treat Qi deficiency and blood stasis syndrome with a long history in clinical practice. To understand its possible mechanism in a systematic study, a network pharmacological method was addressed. METHODS: Detailed information on the HH compounds was obtained from two public databases, and oral bioavailability (OB) and drug-like (DL) of the compounds were evaluated. A correlation between HH compounds, its potential targets, and known targets was extrapolated, and the herb-compound-target-disease (H-C-T-D) network was established. Next, the pathway enrichment and essential genes were analyzed. Then, three key genes (VEGFA, VEGFR2, and eNOS), highly associated with angiogenesis, were screened and verified through western blot assay. RESULTS: Out of 276 compounds, 21 HH compounds and 78 target genes regulating the major pathways associated with CI in the network are analyzed. The bioactive compounds in HH were active in various signal transduction pathways such as the toll-like receptor signaling pathway, VEGF signaling pathway, TNF signaling pathway, and HIF-1 signaling pathway are important pathways that may regulate anti-inflammatory, antiapoptotic, immune correlation, and antioxidative effects. The core genes are PTGS2, TNF, NOS2, IL6, BCL2, IL1B, SOD2, NOS3, SOD1, MMP9, and VEGFA. The in vitro results suggested that HH treatment could significantly elevate the expression of proangiogenic genes such as VEGFA, VEGFR2, and eNOS compared with OGD groups. CONCLUSIONS: Our results predict that HH may regulate the expression of VEGFA, VEGFR2, and eNOS via the VEGF and HIF-1 signaling pathway to promote angiogenesis and alleviate cerebral ischemia injury.

Combination of Systems Pharmacology and Experimental Evaluation to Explore the Mechanism of Synergistic Action of Frankincense-Myrrh in the Treatment of Cerebrovascular Diseases.

Frankincense-Myrrh is a classic drug pair that promotes blood circulation, and eliminates blood stasis. The combination of the two drugs has a definite clinical effect on the treatment of cerebrovascular diseases (CBVDs), but its mechanism of action and compatibility have not been elucidated. In this study, the bioactive components, core targets, and possible synergistic mechanisms of Frankincense-Myrrh in the treatment of CBVDs are explored through systems pharmacology combined with in vivo and in vitro experiments. Comparing target genes of components in Frankincense and Myrrh with CBVD-related genes, common genes were identified; 15 core target genes of Frankincense-Myrrh for the treatment of CBVDs were then identified using protein-protein interaction (PPI) analysis. It was also predicted through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis that the molecular mechanism of Frankincense-Myrrh action on CBVDs was mainly related to the regulation of neurotrophic factors and inflammatory responses. Frankincense-Myrrh significantly improved neurological function, decreased infarct volume, alleviated histopathological damage, inhibited microglial expression, and promoted the expression of neurons in middle cerebral artery occlusion (MCAO)-induced rats. The results of this study not only provide important theoretical support and experimental basis for the synergistic effect of Frankincense-Myrrh, but also provide new ideas for the prevention and treatment of cerebral ischemic injuries.

A Network Pharmacology Technique to Investigate the Synergistic Mechanisms of Salvia miltiorrhiza and Radix puerariae in Treatment of Cardio-Cerebral Vascular Diseases.

OBJECTIVE: This study is aimed to analyze the active ingredients, drug targets, and related pathways in the combination of Salvia miltiorrhiza (SM) and Radix puerariae (RP) in the treatment of cardio-cerebral vascular diseases (CCVDs). METHOD: The ingredients and targets of SM and RP were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), and the disease targets were obtained from Therapeutic Target Database (TTD), National Center for Biotechnology Information (NCBI), and Online Mendelian Inheritance in Man (OMIM) Database. The synergistic mechanisms of the SM and RP were evaluated by gene ontology (GO) enrichment analyses and Kyoto encyclopedia of genes and genomes (KEGG) path enrichment analyses. RESULT: A total of 61 active ingredients and 58 common targets were identified in this study. KEGG pathway enrichment analysis results showed that SM- and RP-regulated pathways were mainly inflammatory processes, immunosuppression, and cardiovascular systems. The component-target-pathway network indicated that SM and RP exert a synergistic mechanism for CCVDs through PTGS2 target in PI3k-Akt, TNF, and Jak-STAT signaling pathways. CONCLUSION: In summary, this study clarified the synergistic mechanisms of SM and RP, which can provide a better understanding of effect in the treatment of CCVDs.

Ellagic acid protects mice against sleep deprivation-induced memory impairment and anxiety by inhibiting TLR4 and activating Nrf2.

Sleep disorder has become a prevalent issue in current society and is connected with the deterioration of neurobehaviors such as mood, cognition and memory. Ellagic acid (EA) is a phenolic phytoconstituent extracted from grains and fruits that has potent neuroprotective properties. This research aimed to study the alleviative effect and mechanism of EA on memory impairment and anxiety caused by sleep deprivation (SD). EA ameliorated behavioral abnormalities in SD mice, associated with increased dendritic spine density, and reduced shrinkage and loss of hippocampal neurons. EA reduced the inflammatory response and oxidative stress injury caused by SD, which may be related to activation of the Nrf2/HO-1 pathway and mitigation of the TLR4-induced inflammatory response. In addition, EA significantly reduced the mortality and ROS levels in glutamate (Glu)-induced hippocampal neuron injury, and these effects of EA were enhanced in TLR4 siRNA-transfected neurons. However, knockdown of Nrf2 dramatically restrained the protective impact of EA on Glu-induced toxicity. Taken together, EA alleviated memory impairment and anxiety in sleep-deprived mice potentially by inhibiting TLR4 and activating Nrf2. Our findings suggested that EA may be a promising nutraceutical ingredient to prevent cognitive impairment and anxiety caused by sleep loss.

Corilagin ameliorates sleep deprivation-induced memory impairments by inhibiting NOX2 and activating Nrf2.

Sleep deprivation (SD) can induce cognitive and memory impairments. This impairment is in part due to oxidative stress damage in the hippocampus region of the brain. Corilagin (CL), a polyphenol belonging to the tannin family and extracted from Terminalia chebula and Phyllanthus emblica, shows strong antioxidant and neuroprotective effects. NF-E2-related factor (Nrf2)/heme oxygenase-1 (HO-1) and NADPH oxidase (NOX) are critical targets involved in cellular defense mechanisms against oxidative injury. Thus, we hypothesized that CL could be a preventive treatment for SD-induced memory impairments by inhibiting NOX2 and activating Nrf2. The results from behavioral tests showed that administration of CL resulted in significantly better performance compared to the SD mice. CL significantly normalized the elevated MDA level and the reduced activity of GPx and SOD (P <0.05, p<0.01) caused by SD. In hippocampal tissues, CL effectively activated Nrf2/HO-1 signaling and downregulated NOX2 protein expression compared with SD (P <0.05, P <0.01). Meanwhile, in vitro findings showed that knockdown of Nrf2 blocked the protective effect of CL versus Glu-induced toxicity, while the effect of CL was enhanced in NOX2 siRNA-transfected neurons. Overall, these findings provided evidence that CL ameliorates SD-induced memory impairments in mice by inhibiting NOX2 and activating Nrf2.

A network pharmacology approach to reveal the protective mechanism of Salvia miltiorrhiza-Dalbergia odorifera coupled-herbs on coronary heart disease.

Salvia miltiorrhiza-Dalbergia odorifera coupled-herbs (SMDOCH) has been used to treat coronary heart disease (CHD) for thousands of years, but its unclear bioactive components and mechanisms greatly limit its clinical application. In this study, for the first time, we used network pharmacology to elucidate the mechanisms of action of SMDOCH on CHD. We collected 270 SMDOCH-related targets from 74 bioactive components and 375 CHD-related targets, with 58 overlapping common targets. Next, we performed enrichment analysis for common-target network and protein-protein interaction (PPI) network. The results showed that SMDOCH affected CHD mainly through 10 significant signaling pathways in three biological processes: 'vascular endothelial function regulation', 'inflammatory response', and 'lipid metabolism'. Six pathways belonged to the 'vascular endothelial function regulation' model, which primarily regulated hormone (renin, angiotensin, oestrogen) activity, and included three key upstream pathways that influence vascular endothelial function, namely KEGG:04933, KEGG:05418, and KEGG:04066. Three pathways, namely KEGG:04668, KEGG:04064, and KEGG:04620, belonged to the 'inflammatory response' model. One pathway (KEGG:04920) belonged to the 'lipid metabolism' model. To some extent, this study revealed the potential bioactive components and pharmacological mechanisms of SMDOCH on CHD, and provided a new direction for the development of new drugs for the treatment of CHD.

Neuroprotective effects of protocatechuic aldehyde through PLK2/p-GSK3ß/Nrf2 signaling pathway in both in vivo and in vitro models of Parkinson's disease.

Mitochondrial dysfunction and oxidative damage are closely related to the pathogenesis of Parkinson's disease (PD). The pharmacological mechanism of protocatechuic aldehyde (PCA) for PD treatment have retained unclear. The purposes of the present study were to clarify the neuroprotective effects of post-treatment of PCA for PD treatment by mitigating mitochondrial dysfunction and oxidative damage, and to further determine whether its effects were mediated by the polo-like kinase 2/phosphorylated glycogen synthase kinase 3 ß/nuclear factor erythroid-2-related factor 2 (PLK2/p-GSK3ß/Nrf2) pathways. We found that PCA improved 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced behavioral deficits and dopaminergic cell loss. Moreover, PCA increased the expressions of PLK2, p-GSK3ß and Nrf2, following the decrease of α-synuclein (α-Syn) in MPTP-intoxicated mice. Cell viability was increased and the apoptosis rate was reduced by PCA in 1-methyl-4-phenylpyridinium iodide (MPP+)-incubated cells. Mitochondrial membrane potential (MMP), mitochondrial complex I activity and reactive oxygen species (ROS) levels in MPP+-incubated cells were also ameliorated by treatment with PCA. The neuroprotective effects of PCA were abolished by inhibition or knockdown of PLK2, whereas overexpression of PLK2 strengthened the protection of PCA. Furthermore, GSK3ß and Nrf2 were involved in PCA-induced protection. These results indicated that PCA has therapeutic effects on PD by the PLK2/p-GSK3ß/Nrf2 pathway.

Study on the multi-targets mechanism of triphala on cardio-cerebral vascular diseases based on network pharmacology.

BACKGROUND & AIMS: Numerous references made clear that Triphala is revered as a multiuse therapeutic and perhaps even panacea historically. Nevertheless, the protective mechanism of Triphala on cardio-cerebral vascular diseases (CCVDs) remains not comprehensive understanding. Hence, a network pharmacology-based method was suggested in this study to address this problem. METHODS: This study was based on network pharmacology and bioinformatics analysis. Information on compounds in herbal medicines of Triphala formula was acquired from public databases. Oral bioavailability as well as drug-likeness were screened by using absorption, distribution, metabolism, and excretion (ADME) criteria. Then, components of Triphala, candidate targets of each component and known therapeutic targets of CCVDs were collected. Compound-target gene and compounds-CCVDs target networks were created through network pharmacology data sources. In addition, key targets and pathway enrichment were analyzed by STRING database and DAVID database. Moreover, we verified three of the key targets (PTGS2, MMP9 and IL6) predicted by using western blot analysis. RESULTS: Network analysis determined 132 compounds in three herbal medicines that were subjected to ADME screening, and 23 compounds as well as 65 genes formed the principal pathways linked to CCVDs. And 10 compounds, which actually linked to more than three genes, are determined as crucial chemicals. Core genes in this network were IL6, TNF, VEGFA, PTGS2, CXCL8, TP53, CCL2, IL10, MMP9 and SERPINE1. And pathways in cancer, TNF signaling pathway, neuroactive ligand-receptor interaction, etc. related to CCVDs were identified. In vitro experiments, the results indicated that compared with the control group (no treatment), PTGS2, MMP9 and IL6 were up-regulated by treatment of 10 ng/mL TNF-α, while pretreatment with 20-80 µg/mL Triphala could significantly inhibit the expression of PTGS2, MMP9 and IL6. With increasing Triphala concentration, the expression of PTGS2, MMP9 and IL6 decreased. CONCLUSIONS: This study revealed the complex components and pharmacological mechanism of Triphala, and obtained some potential therapeutic targets of CCVDs, which could provide theoretical basis for the research and development of new drugs for treating CCVDs.

Development of a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for simultaneous determination of epigallocatechin-3-gallate, silibinin, and curcumin in plasma and different tissues after oral dosing of Protandim in rats and its application in pharmacokinetic and tissue distribution studies.

Protandim is an over-the-counter herbal dietary supplement. The key components of Protandim, i.e., epigallocatechin-3-gallate (EGCG), silibinin (SIL), and curcumin (CUR) were simultaneously analyzed through a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method in plasma and different tissues after administration of Protandim in rats. The developed and validated method was employed to assess the pharmacokinetic profiles and the accumulation of EGCG, SIL, CUR in rat plasma and tissue homogenates. The plasma and tissue homogenates were subjected to liquid-liquid extraction and separated on a Hypurity C18 column (50 × 4.6 mm) with a gradient elution of water and acetonitrile. Mass spectrometric detection was performed in the multiple reaction monitoring mode (MRM) following the transitions: m/z 457.3/169.3, m/z 481.3/125.0, m/z 367.3/149.3 and m/z 609.4 /300.2 for EGCG, SIL, CUR, and RU (rutin), respectively. The concentrations of all the analytes in the range from 2 to 1000 ng/mL showed linear relationships with respective peak areas in different matrices. For all matrices, the values of inter-day and intra-day precisions and accuracies were less than 10.3% of the nominal concentration. The matrix effect, extraction recovery, dilution integrity, and stability values were all within acceptable levels. This method was successfully applied for determining the pharmacokinetics and tissue distribution of the components in rats after the intragastrical administration of a single-dose (364.5 mg/kg) or multiple-doses (1458 mg/kg) of Protandim. The data showed that EGCG, SIL, and CUR did not accumulate in rats after multiple doses of Protandim, and the three main components were distributed mainly in the small intestine.

Neuroprotection of hydroxysafflor yellow A in experimental cerebral ischemia/reperfusion injury via metabolic inhibition of phenylalanine and mitochondrial biogenesis.

Stroke is the second most frequent cause of mortality, resulting in a huge societal burden worldwide. Timely reperfusion is the most effective therapy; however, it is difficult to prevent ischemia/reperfusion (I/R) injury. In traditional Chinese medicine, hydroxysafflor yellow A (HSYA) has been widely used for the treatment of cerebrovascular disease and as a protective therapy against I/R injury. Evidence has demonstrated that HSYA could reduce the levels of reactive oxygen species and suppress cellular apoptosis; however, whether HSYA alters the metabolic profile as its underlying mechanism for neuroprotection remains unknown. In the present study, using a metabolomic screening, phenylalanine was identified to significantly increase in an experimental model of mouse cerebral I/R injury. Notably, western blotting and qPCR analysis were conducted to test the expression level of apoptosis­associated factors, and HSYA was identified to be able to protect neuronal cells by reducing phenylalanine level associated with I/R injury. Additionally, these findings were confirmed in primary mouse neurons and PC12 cells exposed to oxygen and glucose deprivation/reoxygenation (OGD/R) stress. Of note, HSYA was observed to regulate the mRNA expression of key metabolic enzymes, phenylalanine hydroxylase, tyrosine aminotransferase and aspartate aminotransferase, which are responsible for phenylalanine metabolism. Furthermore, by performing mitochondrial labeling and JC­1 fluorescence assay, HSYA was identified to promote mitochondrial function and biogenesis suppressed by OGD/R. The findings of the present study demonstrated that I/R injury could increase the levels of phenylalanine, and HSYA may inhibit phenylalanine synthesis to enhance mitochondrial function and biogenesis for neuroprotection. The present study proposed a novel metabolite biomarker for cerebral I/R injury and the evaluated the efficacy of HSYA as a potential therapeutic treatment I/R injury.

Proteomic Analysis of Hydroxysafflor Yellow A Against Cerebral Ischemia/Reperfusion Injury in Rats.

Hydroxysafflor yellow A (HSYA), an active component from Chinese medicinal herb, has been applied to the prevention and treatment of cerebral ischemia/reperfusion injury (CIRI). To clarify the comprehensive mechanisms HSYA for stroke, we used label-free quantitative proteomic analysis to investigate the modulated proteins of rats subjected to CIRI and their alteration by HSYA. Neurological examination, infarct assessment, and biochemical assay were performed to validate the effects of HSYA, and the results indicated that HSYA played a significant role in brain protection. A total of 13 proteins were identified as overlapped proteins by label-free quantitative proteomic analysis. Gene Ontology and pathway analysis showed that these differentially expressed proteins were mainly enriched in the hypoxia-inducible factor 1 (HIF-1) signaling pathway. Furthermore, networks were constructed with respect to protein function interactions. The results suggested that seven proteins were identified as hub proteins between model and sham groups, while 25 proteins were identified as hub proteins between HSYA and model groups. In addition, the expressions of three overlapping proteins were validated by Western blot, and their levels were consistent with the results of label-free analysis. In conclusion, Eftud2, mTOR, Rab11, Ppp2r5e, and HIF-1 signaling pathways have been detected as key hub proteins and pathways in HSYA against CIRI through proteomic analysis. Our research has provided convincing explanations for the mechanism of HSYA against CIRI and the identified key proteins and pathways might provide novel therapeutics for CIRI.

Z-Guggulsterone attenuates astrocytes-mediated neuroinflammation after ischemia by inhibiting toll-like receptor 4 pathway.

Inflammatory damage plays a pivotal role in ischemic stroke pathogenesis and may represent one of the therapeutic targets. Z-Guggulsterone (Z-GS), an active component derived from myrrh, has been used to treat various diseases. The traditional uses suggest that myrrh is a good candidate for anti-inflammatory damage. This study was to investigate the anti-inflammatory and neuroprotective effects of Z-GS following cerebral ischemic injury, as well as the exact mechanisms behind them. Rat middle cerebral artery occlusion (MCAO) model and in vitro astrocytes oxygen-glucose deprivation (OGD) model were adopted to simulate ischemic stroke. Z-GS (30 or 60 mg/kg) was administered intraperitoneally immediately after reperfusion, while astrocytes were maintained in 30 or 60 µM Z-GS before OGD treatment. The results indicated that Z-GS significantly alleviated neurological deficits, infarct volume and histopathological damage in vivo, and increased the astrocytes viability in vitro. Moreover, the treatment of Z-GS inhibited the astrocytes activation and down-regulated the mRNA levels of pro-inflammatory cytokines. Furthermore, the activated TLR4-NF-κB signaling pathways induced by MCAO or OGD were significantly suppressed by Z-GS treatment, which was achieved via inhibiting the phosphorylation of JNK. Our results demonstrated that Z-GS exerted neuroprotective and anti-inflammatory properties through preventing activation of TLR4-mediated pathway in the activated astrocytes after ischemia injury. Therefore, Z-GS could be considered as a promising candidate for the treatment of ischemic stroke.

Cardioprotective effects and underlying mechanism of Radix Salvia miltiorrhiza and Lignum Dalbergia odorifera in a pig chronic myocardial ischemia model.

Traditional Chinese medicines, including Radix Salvia miltiorrhiza (SM) and Lignum Dalbergia odorifera (DO) extracts, have historically been used to treat myocardial ischemia and other cardiovascular diseases. The volatile oil of DO (DOO) is one of the main components of DO. The aim of the present study was to assess the cardioprotective effects and possible underlying mechanisms of SM­DOO in pigs with ameroid constriction­induced chronic myocardial ischemia. An ameroid constrictor was placed around the left anterior descending coronary artery of pigs to induce chronic myocardial ischemia. At weeks 2, 6 and 8, myocardial injury markers and blood gas levels were detected. At week 8, coronary angiography, echocardiography and hemodynamics analysis were performed to evaluate myocardial function. Following sacrifice, myocardial tissue was collected and subjected to morphological, histopathological and apoptosis assays. Western blotting was used to detect the protein expression of Bcl­2 associated X (Bax), Bcl­2, Akt, phosphorylated (p)­Akt, glycogen synthase kinase (GSK)­3ß and p­GSK­3ß. It was revealed that SM­DOO treatment following chronic myocardial ischemia significantly downregulated the expression of myocardial injury markers, ameliorated myocardial oxygen consumption, increased collateralization, reduced regional cardiac dysfunction and limited the extent of myocardial damage. Furthermore, the results of an apoptosis assay revealed that the apoptosis rate was decreased, the expression of Bax decreased and Bcl­2 increased, and the ratio of Bcl­2/Bax was increased. Further experiments indicated that treatment with SM­DOO increased the phosphorylation of Akt and GSK­3ß. These findings suggest that SM­DOO treatment ameliorates myocardial injury in a chronic myocardial ischemia model, and that the underlying mechanisms responsible may be associated with the activation of the Akt/GSK­3ß signal pathway. Thus, experimental evidence that SM­DOO may be an effective drug for the prevention and treatment of chronic myocardial ischemia in clinical applications has been provided.

Ultra Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry-Based Metabonomics Reveal Protective Effect of Terminalia chebula Extract on Ischemic Stroke Rats.

Terminalia chebula (TC), a kind of Combretaceae, is a widely used herb in India and East Asia to treat cerebrovascular diseases. However, the potential mechanism of the neuroprotective effects of TC at the metabonomics level is still not clear. The present study focused on the effects of TC on metabonomics in a stroke model. Rats were divided randomly into sham, model, and TC groups. Rats in the TC group were intragastrically administered with TC for 7 days after a middle cerebral artery occlusion (MCAO) operation. The sham and the model groups received vehicle for the same length of time. Subsequently, the neuroprotective effects of TC were examined by evaluation of neurological defects, assessment of infarct volume, and identification of biochemical indicators for antioxidant and anti-inflammatory activities. Further, metabonomics technology was employed to evaluate the endogenous metabolites profiling systematically. Consist with the results of biochemical and histopathological assays, pattern recognition analysis showed a clear separation of the model group and the sham group, indicating the recovery impact of TC on the MCAO rats. Moreover, 12 potential biomarkers were identified in the MCAO model group, involving energy (lactic acid, succinic acid, and fumarate), amino acids (leucine, alanine, and phenylalanine), and glycerophospholipid (PC [16:0/20:4], PC [20:4/20:4], LysoPC [18:0], and LysoPC [16:0]) metabolism, as well as other types of metabolism (arachidonic acid and palmitoylcarnitine). Notably, it was found that metabolite levels of TC group were partially reversed to normal. In conclusion, TC could ameliorate MCAO in rats by affecting energy metabolism (glycolysis and the TCA cycle), amino acid metabolism, glycerophospholipid metabolism, and other types of metabolism.

[Ruyi Jinhuang Plaster suppresses BPH by reducing the expressions of P38, JNK2, NF-кBP65 and STAT3 in the prostate tissue of rats].

OBJECTIVE: To investigate the effect of Ruyi Jinhuang Plaster (RJP) on testosterone propionate-induced BPH in the rat model and its action mechanisms. METHODS: Forty-eight SD male rats were randomly divided into six groups of equal number: normal control, BPH model control, finasteride, and high-, medium- and low-dose RJP. The BPH model was made in the latter five groups by hypodermic injection of testosterone propionate. From the first day of modeling, the rats of the normal control and BPH model control groups were treated with blank plasters and those of the high-, medium- and low-dose RJP groups with RJPs at 42.0, 21.0 and 10.5 cm2/kg applied to the dehaired area of the back, and those of the finasteride group by gavage of finasteride at 4.5 mg/kg, all once a day for 30 successive days. Then the prostates of the animals were harvested for observation of histopathological changes by HE staining, measurement of the areas of interstitial and epithelial cells and prostatic glandular cavity, and determination of the expressions of P38, JNK2, NF-кBP65 and STAT3 proteins in the prostate tissue by Western blot. RESULTS: Compared with the BPH model controls, the high-dose RJP group showed significantly decreased proliferation and area proportion of prostatic epithelial cells (P < 0.05), increased area proportion of the prostatic glandular cavity (P < 0.05), and reduced expressions of P38, p-P38, NF-кBP65, P-NF-кBP65, STAT3, P-STAT3 and JNK2 in the prostate tissue (P < 0.05); the medium-dose RJP group exhibited markedly down-regulated expressions of JNK2 and NF-кBP65 (P < 0.05) but an up-regulated level of p-JNK (P < 0.05); while the low-dose RJP group displayed a remarkably reduced expression of JNK2 (P < 0.05) but an elevated level of p-JNK (P < 0.05). CONCLUSIONS: RJP suppresses BPH in the model rat by down-regulating the expressions of P38, p-P38, NF-кBP65, P-NF-кBP65, STAT3, P-STAT3 and JNK2 or up-regulating that of p-JNK in the prostate tissue.