Cerebral ischemia causes severe neurological disorders and neuronal dysfunction. Baicalin (BC), geniposide (GP), and their combination (BC/GP) have been shown to inhibit post-ischemic inflammatory injury by inhibiting the 5-LOX/CysLTs pathway. The aims of this study were to observe the inhibitory effects of BC/GP on the activation of microglial cells induced by oxygen glucose deprivation and reoxygenation (OGD/R) and to investigate whether the 5-LOX/LTB4 pathway was involved in these effects. Molecular docking showed that BC and GP exhibited considerable binding activity with LTB4 synthase LTA4H. BV-2 microglia were transfected with a 5-LOX overexpression lentiviral vector, and then OGD/R was performed. The effects of different concentrations of BC, GP, and BC/GP (6.25 μM, 12.5 μM, and 25 μM) on cell viability and apoptosis of microglia were evaluated by MTT and flow cytometry. The expression of TNF-α, IL-1β, NF-κB, and pNF-κB also was measured by ELISA, Western blots and immunofluorescence. Western blots and qRT-PCR analysis were used to determine the levels of CD11b, CD206, and 5-LOX pathway proteins. Results showed that BC, GP, and BC/GP reduced the apoptosis caused by OGD/R in a dose-dependent manner, and cell viability was significantly increased at a concentration of 12.5 μM. OGD/R significantly increased the release of TNF-α, IL-1β, NF-κB, pNF-κB, and CD11b. These effects were suppressed by BC, GP, and BC/GP, and the OGD/R-induced transfer of NF-κB p65 from the ctytoplasm to the nucleus was inhibited in microglia. Interestingly, the LTB4 inhibitor, U75302, exhibited the same effect. Also, BC, GP, and BC/GP significantly reduced the expression of 5-LOX pathway proteins. These results demonstrated that BC/GP inhibited OGD/R-induced polarization in BV2 microglia by regulating the 5-LOX/LTB4 signaling pathways and attenuating the inflammatory response. Our results supported the theoretical basis for additional in-depth study of the function of BC/GP and the value of determining its unique target, which might provide a new therapeutic strategy for ischemic cerebrovascular disease.

Ischaemic encephalopathy is a common cerebrovascular disease caused by insufficient blood supply to the cerebral vessels. The ischaemic encephalopathy is closely associated with the development of many chronic diseases such as obesity, hypertension and diabetes. Neurotrophic therapy has become the main therapeutic strategy for ischaemic encephalopathy. However, neurotrophic drugs only slightly recover the neurological function of patients, and their long-term efficacy is uncertain. Previous reports revealed that the active ingredients of natural medicines play important roles in the treatment of cerebral ischemia. In this study, we reviewed clearing herbs with anti-ischaemic encephalopathy functions using the data from quantitative statistical and network pharmacological exploration methods. We also discussed the different bioactive components and pharmacological effects of these herbs. First, we collected Chinese herbal prescriptions against ischaemic encephalopathy in four databases. Then, we statistically analysed the frequency of application of heat-clearing herbs to obtain the commonly used heat-clearing herbs against ischaemic encephalopathy, and classified them according to their efficacy according to the statistical results, to summarize the mechanism of anti-ischaemic effects of different bioactive components; Second, the network database was used to obtain the above components of heat-clearing Chinese medicines and their corresponding targets of action, disease targets of ischaemic stroke; Venny 2.1.0 was used to obtain component-disease target intersections; Cytoscape was used to construct the 'Drug-Active Ingredient-Target Network Graph '; DAVID was used for GO and KEGG enrichment analysis. Literature and database screening involved 149 prescriptions, with a total of 269 flavours of Chinese medicines and 20 flavours of single-flavour heat-clearing Chinese medicines; The top nine in terms of frequency of use were Radix Paeoniae Rubra、Rehmanniae Radix Praeparata、Figwort Root、Cortex Moutan、Scutellariae Radix、Coptidis Rhizoma、Gardeniae Fructus、Cassiae Semen、Lonicerae Japonicae Flos. The common components obtained from network pharmacology were beta-sitosterol, quercetin, and stigmasterol, which mainly act on key targets such as RELA, AKT1, JUN, PRKACA, PTGS2, RAF1 and CHUK; and their active ingredients are mainly involved in signalling pathways such as Calcium, PI3K-Ak, MAPK, cAMP, IL-17, HIF-1, TNF, T-cell receptor, NF-kappa B and JAK-STAT. Heat-clearing herbs are useful and promising for the protection against and prevention of ischemic encephalopathy. The results of the network pharmacological studies are similar to the mechanisms of anti-ischemic encephalopathy of the active ingredients of the purgative herbs we have listed; Thin either directly protects cerebrovascular tissues by improving vascular permeability and reducing the area of infarcted tissues, or produces protective effects through molecular signaling pathways. It can be seen that the components of heat-clearing Chinese medicines can exert cerebroprotective effects through multiple pathways, which provides us with a reference for further development and study of heat-clearing Chinese medicines in the treatment of ischemic cerebrovascular diseases.

Diabetic neuropathic pain (DNP) is a common complication of diabetes mellitus (DM) and is characterized by spontaneous pain and neuroinflammation. The Sigma-1 receptor (Sig-1R) has been proposed as a target for analgesic development. It is an important receptor with anti-inflammatory properties and has been found to regulate DNP. However, it is not known whether Sig-1R can ameliorate pathological neuroinflammation in DNP. The present study used a rat model of DNP and a highly selective agonist of Sig-1R to assess the effects of the protein on neuropathic pain in rats with type 2 diabetes mellitus. The rats were divided into Control, Model, Sig-1R agonist PRE-084 (0.3, 0.6, 1mg/kg), and metformin (Met, 20mg/kg) groups, with seven rats per group, and their body weight, fasting blood glucose, mechanical withdrawal threshold and thermal withdrawal latency were tested weekly for two weeks. After treatment with PRE-084, the pain thresholds in the DNP rats were significantly improved, together with pathological changes in the dorsal root ganglion, reductions in the serum levels of TNF-α, IL-1β, IL-6, MOD, and prostaglandin E2 (PGE2), and the activity of superoxide dismutase was increased. The mRNA levels of TNF-α, IL-1β, and cyclooxygenase 2 (COX-2) were reduced. Pharmacological inhibition of Sig-1R with BD1047 (10μM) abolished Sig-1R-mediated activation of lipopolysaccharide-treated BV-2 microglial cells. It was also found that PRE-084 increased phosphorylation of serine/threonine protein kinase B (AKT) and glycogen synthase kinase 3β (GSK-3β) at Ser9, inhibiting nuclear factor kappa B (NF-κB)-mediated neuroinflammation in the dorsal root ganglion, thus reducing DNP. The findings suggest that the effect of Sig-1R agonist PRE-084 on DNP may reduce the level of inflammation through the up-regulation of AKT/GSK-3β and down-regulation of the NF-κB signaling, thereby contributing to the treatment of the disease.