The binding of PKCε and MEG2 to STAT3 regulates IL-6-mediated microglial hyperalgesia during inflammatory pain.

Studies have suggested that microglial IL-6 modulates inflammatory pain; however, the exact mechanism of action remains unclear. We therefore hypothesized that PKCε and MEG2 competitively bind to STAT3 and contribute to IL-6-mediated microglial hyperalgesia during inflammatory pain. Freund's complete adjuvant (FCA) and lipopolysaccharide (LPS) were used to induce hyperalgesia model mice and microglial inflammation. Mechanical allodynia was evaluated using von Frey tests in vivo. The interaction among PKCε, MEG2, and STAT3 was determined using ELISA and immunoprecipitation assay in vitro. The PKCε, MEG2, t-STAT3, pSTAT3Tyr705, pSTAT3Ser727, IL-6, GLUT3, and TREM2 were assessed by Western blot. IL-6 promoter activity and IL-6 concentration were examined using dual luciferase assays and ELISA. Overexpression of PKCε and MEG2 promoted and attenuated inflammatory pain, accompanied by an increase and decrease in IL-6 expression, respectively. PKCε displayed a stronger binding ability to STAT3 when competing with MEG2. STAT3Ser727 phosphorylation increased STAT3 interaction with both PKCε and MEG2. Moreover, LPS increased PKCε, MEG2, pSTAT3Tyr705, pSTAT3Ser727, IL-6, and GLUT3 levels and decreased TREM2 during microglia inflammation. IL-6 promoter activity was enhanced or inhibited by PKCε or MEG2 in the presence of STAT3 and LPS stimulation, respectively. In microglia, overexpression of PKCε and/or MEG2 resulted in the elevation of tSTAT3, pSTAT3Tyr705, pSTAT3Ser727, IL-6, and TREM2, and the reduction of GLUT3. PKCε is more potent than MEG2 when competitively binding to STAT3, displaying dual modulatory effects of IL-6 production, thus regulating the GLUT3 and TREM2 in microglia during inflammatory pain sensation.

Dexamethasone inhibits IL-8 via glycolysis and mitochondria-related pathway to regulate inflammatory pain.

BACKGROUND: Dexamethasone (Dexa) has been recently found to exert an analgesic effect, whose action is closely related to IL-8. However, whether dexamethasone induces antinociception via glycolysis and mitochondria-related pathways is still unclear. METHODS: Right hind paw inflammatory pain in mice was induced by intraplantar injection of Freund's Complete Adjuvant (FCA). Von Frey test was then used to measure the paw withdrawal threshold. The detection of glycolysis and mitochondrial pathway-related proteins and IL-8 were determined by Western blot and ELISA. The potential interaction between Dexa and fructose-1,6-bisphosphate (FBP, a PKM2 activator) was examined by simulation predictions using molecular docking. RESULTS: Intrathecal administration of Dexa (20 µg/20 µL) had an obvious analgesic effect in FCA-treated mice, which was counteracted by the glycolysis inhibitor 2-deoxyglucose (2-DG, 5 mg/20 µL) or the mitochondria-related pathway inhibitor oligomycin complex (Oligo, 5 µg/20 µL). In the glycolysis pathway, Dexa decreased GLUT3 and had no impact on HIF-1α expression during FCA-induced inflammation. Additionally, Dexa further increased the PKM2 level, accompanied by the formation of hydrogen bonds between Dexa and the PKM2 activator fructose-1,6-bisphosphate (FBP). In the mitochondrial pathway, Dexa downregulated the expression of Mfn2 protein but not the PGC-1α and SIRT-1 levels in the spinal cord. Moreover, both 2-DG and Oligo decreased Mfn2 expression. Finally, IL-8 level was reduced by the single or combined administration of Dexa, 2-DG, and Oligo. CONCLUSION: Dexa attenuated IL-8 expression via glycolysis and mitochondrial pathway-related proteins, thus mediating the analgesic effect during inflammatory pain.

An Fgr kinase inhibitor attenuates sepsis-associated encephalopathy by ameliorating mitochondrial dysfunction, oxidative stress, and neuroinflammation via the SIRT1/PGC-1α signaling pathway.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is characterized by diffuse brain dysfunction, long-term cognitive impairment, and increased morbidity and mortality. The current treatment for SAE is mainly symptomatic; the lack of specific treatment options and a poor understanding of the underlying mechanism of disease are responsible for poor patient outcomes. Fgr is a member of the Src family of tyrosine kinases and is involved in the innate immune response, hematologic cancer, diet-induced obesity, and hemorrhage-induced thalamic pain. This study investigated the protection provided by an Fgr kinase inhibitor in SAE and the underlying mechanism(s) of action. METHODS: A cecal ligation and puncture (CLP)-induced mouse sepsis model was established. Mice were treated with or without an Fgr inhibitor and a PGC-1α inhibitor/activator. An open field test, a novel object recognition test, and an elevated plus maze were used to assess neurobehavioral changes in the mice. Western blotting and immunofluorescence were used to measure protein expression, and mRNA levels were measured using quantitative PCR (qPCR). An enzyme-linked immunosorbent assay was performed to quantify inflammatory cytokines. Mitochondrial membrane potential and morphology were measured by JC-1, electron microscopy, and the MitoTracker Deep Red probe. Oxidative stress and mitochondrial dysfunction were analyzed. In addition, the regulatory effect of Fgr on sirtuin 1 (SIRT1) was assessed. RESULTS: CLP-induced sepsis increased the expression of Fgr in the hippocampal neurons. Pharmacological inhibition of Fgr attenuated CLP-induced neuroinflammation, the survival rate, cognitive and emotional dysfunction, oxidative stress, and mitochondrial dysfunction. Moreover, Fgr interacted with SIRT1 and reduced its activity and expression. In addition, activation of SIRT1/PGC-1α promoted the protective effects of the Fgr inhibitor on CLP-induced brain dysfunction, while inactivation of SIRT1/PGC-1α counteracted the benefits of the Fgr inhibitor. CONCLUSIONS: To our knowledge, this is the first report of Fgr kinase inhibition markedly ameliorating SAE through activation of the SIRT1/PGC-1α pathway, and this may be a promising therapeutic target for SAE.

Dexamethasone and potassium canrenoate alleviate hyperalgesia by competitively regulating IL-6/JAK2/STAT3 signaling pathway during inflammatory pain in vivo and in vitro.

BACKGROUND: Dexamethasone (Dexa) and potassium canrenoate (Cane) modulate nociceptive behavior via glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) by two mechanisms (genomic and nongenomic pathways). This study was designed to investigate the Dexa- or Cane-mediated nongenomic and genomic effects on mechanical nociception and inflammation-induced changes in interleukin-6 (IL-6) mediated signaling pathway in rats. METHODS: Freund's complete adjuvant (FCA) was used to trigger an inflammation of the right hind paw in male Sprague-Dawley rats. First, the mechanical nociceptive behavioral changes were examined following intraplantar administration of GR agonist Dexa and/or MR antagonist Cane in vivo. Subsequently, the protein levels of IL-6, IL-6Rα, JAK2, pJAK2, STAT3, pSTAT3Ser727 , migration inhibitory factor, and cyclooxygenase-2 were assessed by Western blot following intraplantar injection of Dexa or Cane or the combination. Moreover, the molecular docking studies determined the interaction between Dexa, Cane, and IL-6. The competition binding assay was carried out using enzyme-linked immunosorbent assays (ELISA). RESULTS: Administration of Dexa and Cane dose-dependently attenuated FCA-induced inflammatory pain. The sub-additive effect of Dexa/Cane combination was elucidated by isobologram analysis, accompanied by decrease in the spinal levels of IL-6, pJAK2, and pSTAT3Ser727 . The molecular docking study demonstrated that both Dexa and Cane displayed a firm interaction with THR138 binding site of IL-6 via a strong hydrogen bond. ELISA revealed that Dexa has a higher affinity to IL-6 than Cane. CONCLUSIONS: There was no additive or negative effect of Dexa and Cane, and they modulate the IL-6/JAK2/STAT3 signaling pathway through competitive binding with IL-6 and relieves hypersensitivity during inflammatory pain.

Phosphorylation at Ser 727 Increases STAT3 Interaction with PKCε Regulating Neuron-Glia Crosstalk via IL-6-Mediated Hyperalgesia In Vivo and In Vitro.

METHODS: A rat hyperalgesia model was induced using an intraplantar injection of Freund's complete adjuvant (FCA) or an intrathecal injection of IL-6. Mechanical allodynia was evaluated using von Frey filament tests after intrathecal injections of T-5224 (c-Fos/AP-1 inhibitor), minocycline (Mino, a specific microglia inhibitor), L-2-aminoadipic acid (LAA, an astroglial toxin), PKCε inhibitor peptide, APTSTAT3-9R (STAT3 inhibitor), or anti-IL-6 antibody. The c-Fos, GFAP, Iba-1, PKCε, STAT3, pSTAT3Tyr705 and pSTAT3Ser727, and IL-6 expression at the spinal cord level was assessed by Western blot analysis. The interactive effects of PKCε and STAT3 were determined using immunofluorescence staining and immunoprecipitation in vivo and in vitro. Interleukin-6 promoter activity was examined using luciferase assays. RESULTS: T-5224, Mino, and LAA attenuated FCA- or IL-6-mediated inflammatory pain, with a decrease in c-Fos, GFAP, Iba-1, PKCε, and IL-6 expression. PKCε inhibitor peptide and APTSTAT3-9R reversed FCA-induced nociceptive behavior, while decreasing pSTAT3Ser727, IL-6, c-Fos, GFAP, and Iba-1 expression and PKCε and STAT3 coexpression. Interleukin-6 promoter activity increased in the presence of PKCε and STAT3. The interaction with PKCε increased on phosphorylating STAT3 at Ser727 but not at Tyr705. CONCLUSION: STAT3 phosphorylation at Ser 727 and the interaction with PKCε contribute to hyperalgesia via the IL-6-mediated signaling pathway, thus regulating neuron-glia crosstalk during inflammatory pain.

Sigma-1 Receptor and Binding Immunoglobulin Protein Interact with Ulinastatin Contributing to a Protective Effect in Rat Cerebral Ischemia/Reperfusion.

OBJECTIVE: To investigate impact of ulinastatin (UTI) on sigma-1 receptor (σ1R) and binding immunoglobulin protein (BiP) after cerebral ischemia/reperfusion injury. METHODS: The middle cerebral artery occlusion (MCAO) model was used to induce cerebral ischemia/reperfusion injury. Eighty male Sprague Dawley rats were randomly divided into 6 groups: control, MCAO, MCAO+50,000 U/kg UTI, MCAO+100,000 U/kg UTI, MCAO+200,000 U/kg UTI, MCAO+300,000 U/kg UTI. At 24 and 48 hours after MCAO, infarct volume, neurological dysfunction, and grip strength test were measured, and level of σ1R and BiP proteins was further detected using Western blot. Molecular docking assays were carried out to verify interaction between σ1R, BiP, and UTI. The serum concentration of BiP and the binding assay between σ1R, BiP, and UTI were determined using enzyme-linked immunosorbent assay. RESULTS: UTI increased the modified neurological severity score and upregulated σ1R and BiP expression in the cerebral cortex after MCAO. The grip strength of forelimbs increased significantly in the MCAO+200,000 U/kg UTI and MCAO+300,000 U/kg UTI groups compared with the MCAO group, while BiP serum levels remained unchanged. The molecular docking assay indicated putative binding between σ1R, BiP, and UTI. The binding assay also revealed that both σ1R and BiP could be combined with UTI. CONCLUSIONS: UTI displays a neuroprotective effect via upregulation of σ1R and BiP during ischemia/reperfusion injury, suggesting that UTI modulates σ1R and BiP and their interaction may provide a novel insight into potential therapeutic mechanisms for stroke.

Roles of Long Non-coding RNAs in the Development of Chronic Pain.

Chronic pain, a severe public health issue, affects the quality of life of patients and results in a major socioeconomic burden. Only limited drug treatments for chronic pain are available, and they have insufficient efficacy. Recent studies have found that the expression of long non-coding RNAs (lncRNAs) is dysregulated in various chronic pain models, including chronic neuropathic pain, chronic inflammatory pain, and chronic cancer-related pain. Studies have also explored the effect of these dysregulated lncRNAs on the activation of microRNAs, inflammatory cytokines, and so on. These mechanisms have been widely demonstrated to play a critical role in the development of chronic pain. The findings of these studies indicate the significant roles of dysregulated lncRNAs in chronic pain in the dorsal root ganglion and spinal cord, following peripheral or central nerve lesions. This review summarizes the mechanism underlying the abnormal expression of lncRNAs in the development of chronic pain induced by peripheral nerve injury, diabetic neuropathy, inflammatory response, trigeminal neuralgia, spinal cord injury, cancer metastasis, and other conditions. Understanding the effect of lncRNAs may provide a novel insight that targeting lncRNAs could be a potential candidate for therapeutic intervention in chronic pain.

Antinociceptive effects of IL-6R vs. glucocorticoid receptors during rat hind paw inflammatory pain.

BACKGROUND: The glucocorticoid receptor (GR) plays a role in inflammatory pain modulation. However, the specific role played by interleukin 6 receptor (IL-6R) in these processes remains elusive. The present study aimed to investigate the extent of inflammation induced by IL-6R and GR. METHODS: Male Wistar rats were treated with Freund's complete adjuvant to induce right hind paw inflammation. The levels of IL-6Rα and GR were evaluated in the spinal cord and dorsal root ganglion using Western blot and immunofluorescence assays. Subsequently, we examined the nociceptive behavioral changes following the binding of IL-6R with a GR agonist and/or antagonist, as well as the concentration levels of IL-6 and soluble IL-6R (sIL-6R) in the serum and cerebrospinal fluid. Moreover, the spinal levels of IL-6, IL-6Rα, gp130, JAK2, pJAK2, STAT3, pSTAT3, c-fos, GFAP, and Iba-1 were assessed following anti-IL-6R antibody, sgp130, and dexamethasone intrathecal administration. RESULTS: Right hind paw inflammation resulted in significant upregulation of IL-6Rα expression in spinal nociceptive neurons, astrocytes, and microglia cells, as well as increased of IL-6Rα and GR colocalization. Notably, anti-IL-6R or dexamethasone attenuated the nociceptive behavior in a dose-dependent manner. Isobologram analysis indicated the sub-additive effects with a concomitant decrease in the spinal levels of IL-6, pJAK2, pSTAT3, c-fos, GFAP, and Iba-1 and increase in the sIL-6R level. CONCLUSION: The enhanced mechanical sensitivity accompanying the increase of IL-6Rα and GR was attenuated by anti-IL-6R and dexamethasone application, and the sub-additive effects were regulated by the decreased activation of neurons and glial cells and modulated by IL-6/JAK2/STAT3 signaling pathway, which might be attributed to IL-6 induced trans-signaling.

Neuronal aldosterone elicits a distinct genomic response in pain signaling molecules contributing to inflammatory pain.

BACKGROUND: Recently, mineralocorticoid receptors (MR) were identified in peripheral nociceptive neurons, and their acute antagonism was responsible for immediate and short-lasting (non-genomic) antinociceptive effects. The same neurons were shown to produce the endogenous ligand aldosterone by the enzyme aldosterone synthase. METHODS: Here, we investigate whether endogenous aldosterone contributes to inflammation-induced hyperalgesia via the distinct genomic regulation of specific pain signaling molecules in an animal model of Freund's complete adjuvant (FCA)-induced hindpaw inflammation. RESULTS: Chronic intrathecal application of MR antagonist canrenoate-K (over 4 days) attenuated nociceptive behavior in rats with FCA hindpaw inflammation suggesting a tonic activation of neuronal MR by endogenous aldosterone. Consistently, double immunofluorescence confocal microscopy showed abundant co-localization of MR with several pain signaling molecules such as TRPV1, CGRP, Nav1.8, and trkA whose enhanced expression of mRNA and proteins during inflammation was downregulated following i.t. canrenoate-K. More importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by continuous intrathecal delivery of a specific aldosterone synthase inhibitor prevented the inflammation-induced enhanced transcriptional expression of TRPV1, CGRP, Nav1.8, and trkA and subsequently attenuated nociceptive behavior. Evidence for such a genomic effect of endogenous aldosterone was supported by the demonstration of an enhanced nuclear translocation of MR in peripheral sensory dorsal root ganglia (DRG) neurons. CONCLUSION: Taken together, chronic inhibition of local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons may contribute to long-lasting downregulation of specific pain signaling molecules and may, thus, persistently reduce inflammation-induced hyperalgesia.

Aldosterone Synthase in Peripheral Sensory Neurons Contributes to Mechanical Hypersensitivity during Local Inflammation in Rats.

BACKGROUND: Recent emerging evidence suggests that extra-adrenal synthesis of aldosterone occurs (e.g., within the failing heart and in certain brain areas). In this study, the authors investigated evidence for a local endogenous aldosterone production through its key processing enzyme aldosterone synthase within peripheral nociceptive neurons. METHODS: In male Wistar rats (n = 5 to 8 per group) with Freund's complete adjuvant hind paw inflammation, the authors examined aldosterone, aldosterone synthase, and mineralocorticoid receptor expression in peripheral sensory neurons using quantitative reverse transcriptase-polymerase chain reaction, Western blot, immunohistochemistry, and immunoprecipitation. Moreover, the authors explored the nociceptive behavioral changes after selective mineralocorticoid receptor antagonist, canrenoate-K, or specific aldosterone synthase inhibitor application. RESULTS: In rats with Freund's complete adjuvant-induced hind paw inflammation subcutaneous and intrathecal application of mineralocorticoid receptor antagonist, canrenoate-K, rapidly and dose-dependently attenuated nociceptive behavior (94 and 48% reduction in mean paw pressure thresholds, respectively), suggesting a tonic activation of neuronal mineralocorticoid receptors by an endogenous ligand. Indeed, aldosterone immunoreactivity was abundant in peptidergic nociceptive neurons of dorsal root ganglia and colocalized predominantly with its processing enzyme aldosterone synthase and mineralocorticoid receptors. Moreover, aldosterone and its synthesizing enzyme were significantly upregulated in peripheral sensory neurons under inflammatory conditions. The membrane mineralocorticoid receptor consistently coimmunoprecipitated with endogenous aldosterone, confirming a functional link between mineralocorticoid receptors and its endogenous ligand. Importantly, inhibition of endogenous aldosterone production in peripheral sensory neurons by a specific aldosterone synthase inhibitor attenuated nociceptive behavior after hind paw inflammation (a 32% reduction in paw pressure thresholds; inflammation, 47 ± 2 [mean ± SD] vs. inflammation + aldosterone synthase inhibitor, 62 ± 2). CONCLUSIONS: Local production of aldosterone by its processing enzyme aldosterone synthase within peripheral sensory neurons contributes to ongoing mechanical hypersensitivity during local inflammation via intrinsic activation of neuronal mineralocorticoid receptors.

Pro- versus Antinociceptive Nongenomic Effects of Neuronal Mineralocorticoid versus Glucocorticoid Receptors during Rat Hind Paw Inflammation.

BACKGROUND: In naive rats, corticosteroids activate neuronal membrane-bound glucocorticoid and mineralocorticoid receptors in spinal cord and periphery to modulate nociceptive behavior by nongenomic mechanisms. Here we investigated inflammation-induced changes in neuronal versus glial glucocorticoid and mineralocorticoid receptors and their ligand-mediated nongenomic impact on mechanical nociception in rats. METHODS: In Wistar rats (n = 5 to 7/group) with Freund's complete adjuvant hind paw inflammation, we examined glucocorticoid and mineralocorticoid receptor expression in spinal cord and peripheral sensory neurons versus glial using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot, immunohistochemistry, and radioligand binding. Moreover, we explored the expression of mineralocorticoid receptors protecting enzyme 11-betahydroxysteroid dehydrogenase type 2 as well as the nociceptive behavioral changes after glucocorticoid and mineralocorticoid receptors agonist or antagonist application. RESULTS: Hind paw inflammation resulted in significant upregulation of glucocorticoid receptors in nociceptive neurons of spinal cord (60%) and dorsal root ganglia (15%) as well as mineralocorticoid receptors, while corticosteroid plasma concentrations remained unchanged. Mineralocorticoid (83 ± 16 fmol/mg) but not glucocorticoid (104 ± 20 fmol/mg) membrane binding sites increased twofold in dorsal root ganglia concomitant with upregulated 11-betahydroxysteroid dehydrogenase type 2 (43%). Glucocorticoid and mineralocorticoid receptor expression in spinal microglia and astrocytes was small. Importantly, glucocorticoid receptor agonist dexamethasone or mineralocorticoid receptor antagonist canrenoate-K rapidly and dose-dependently attenuated nociceptive behavior. Isobolographic analysis of the combination of both drugs showed subadditive but not synergistic or additive effects. CONCLUSIONS: The enhanced mechanical sensitivity of inflamed hind paws accompanied with corticosteroid receptor upregulation in spinal and peripheral sensory neurons was attenuated immediately after glucocorticoid receptor agonist and mineralocorticoid receptor antagonist administration, suggesting acute nongenomic effects consistent with detected membrane-bound corticosteroid receptors.

Membrane-bound glucocorticoid receptors on distinct nociceptive neurons as potential targets for pain control through rapid non-genomic effects.

Glucocorticoids were long believed to primarily function through cytosolic glucocorticoid receptor (GR) activation and subsequent classical genomic pathways. Recently, however, evidence has emerged that suggests the presence of rapid non-genomic GR-dependent signaling pathways within the brain, though their existence in spinal and peripheral nociceptive neurons remains elusive. In this paper, we aim to systemically identify GR within the spinal cord and periphery, to verify their putative membrane location and to characterize possible G protein coupling and pain modulating properties. Double immunofluorescence confocal microscopy revealed that GR predominantly localized in peripheral peptidergic and non-peptidergic nociceptive C- and Aδ-neurons and existed only marginally in myelinated mechanoreceptive and proprioreceptive neurons. Within the spinal cord, GR predominantly localized in incoming presynaptic nociceptive neurons, in pre- and postsynaptic structures of the dorsal horn, as well as in microglia. GR saturation binding revealed that these receptors are linked to the cell membrane of sensory neurons and, upon activation, they trigger membrane targeted [35S]GTPγS binding, indicating G protein coupling to a putative receptor. Importantly, subcutaneous dexamethasone immediately and dose-dependently attenuated acute nociceptive behavior elicited in an animal model of formalin-induced pain hypersensitivity compared to naive rats. Overall, this study provides firm evidence for a novel neuronal mechanism of GR agonists that is rapid, non-genomic, dependent on membrane binding and G protein coupling, and acutely modulates nociceptive behavior, thus unraveling a yet unconsidered mechanism of pain relief.

Acute mechanical sensitization of peripheral nociceptors by aldosterone through non-genomic activation of membrane bound mineralocorticoid receptors in naive rats.

Recently, there is increasing interest in the role of peripheral mineralocorticoid receptors (MR) to modulate pain, but their localization in neurons and glia of the periphery and their distinct involvement in pain control remains elusive. In naive Wistar rats our double immunofluorescence confocal microscopy of the spinal cord, dorsal root ganglia, sciatic nerve and innervated skin revealed that MR predominantly colocalized with calcitonin-gene-related peptide (CGRP)- and trkA-immunoreactive (IR) nociceptive neurons and only marginally with myelinated trkB-IR mechanoreceptive and trkC-IR proprioreceptive neurons underscoring a pivotal role for MR in the modulation of pain. MR could not be detected in Schwann cells, satellite cells, and astrocytes and only scarcely in spinal microglia cells excluding a relevant functional role of glia-derived MR at least in naïve rats. Intrathecal (i.t.) and intraplantar (i.pl.) application of increasing doses of the MR selective agonist aldosterone acutely increased nociceptive behavior which was reversible by a MR selective antagonist and most likely due to non-genomic effects. This was further substantiated by the first identification of membrane bound MR specific binding sites in sensory neurons of dorsal root ganglia and spinal cord. Therefore, a crucial role of MR on nociceptive neurons but not on glia cells and their impact on nociceptive behavior most likely due to immediate non-genomic effects has to be considered under normal but more so under pathological conditions in future studies.

Ulinastatin attenuates cerebral ischemia-reperfusion injury in rats.

UNLABELLED: To investigate the effects of Ulinastatin (UTI) in cerebral ischemia-reperfusion (IR) injury in rats and whether this effect might be related to Aquaporin 4 (AQP4), one hundred and eighty adult male Sprague Dawley (SD) rats, weighing 230-280 g, were randomly divided into 3 groups: sham (S) group, IR group and UTI (U) group. Every group was further divided into 3 sub-groups: 6 h group, 24 h group and 48 h group. The transient focal IR injury was induced by inserting a silicone-coater monofilament nylon suture (0.28 mm) from the right external carotid artery to the origin of the left middle cerebral artery. The suture was removed after 2 h to allow reperfusion. UTI treatment group was injected with UTI 100000 u/kg at the beginning of the reperfusion period, while S group and IR group were injected with the same volume of saline. Samples were taken according to the reperfusion time (6 h, 12 h and 24 h). Infract volume was measured by triphenyl tetrazolium chloride staining, and brain water content was determined by wet-dry weight method and neurological scores were checked with a five-point scale. Expression levels of AQP4 were checked with immunohistochemistry and Western blot. RESULTS: Compared with S group, the infarct volume, water content, neurological scores and AQP4 levels in the rat brain tissues were significantly increased in IR group. Administration of UTI significantly decreased the infarct volume, water content of the brain tissue and neurological scores. Moreover, the expression levels of AQP4 were also down-regulated by UTI treatment. CONCLUSION: UTI improves cerebral IR injury in rats potentially via decreasing the expression levels of AQP4.

Tetrandrine attenuated cerebral ischemia/reperfusion injury and induced differential proteomic changes in a MCAO mice model using 2-D DIGE.

Ischemic stroke is the most common type of stroke and brings about a big disease burden because of high mortality and disability in China. Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the Chinese herb Radix Stephania tetrandra, has been demonstrated to possess anti-inflammatory and free radical scavenging effects and even regulate astrocyte activation, but the possible role of tetrandrine in ameliorating cerebral ischemia/reperfusion injury of ischemic stroke remains unknown. The aim of this study was to determine the effects of tetrandrine on neurological injury and differential proteomic changes induced by transient reversible middle cerebral artery occlusion (MCAO) in mice. Male Balb/c mice were divided into sham (n = 30), MCAO + saline as control (n = 30), and MCAO + Tet as tetrandrine-treated (n = 30) groups. Mice in the control and tetrandrine-treated groups underwent 120 min of MCAO following reperfusion. Immediately and 2 h after MCAO, the mice received either normal saline (sham operated and control groups) or tetrandrine (tetrandrine-treated group) intraperitoneally. Neurological defects, brain water content, and infarct volume at 24 h after stoke were used to evaluate neurological injury extent. Treatment with tetrandrine not only mitigated cerebral neurological deficits (P < 0.05) and infarct size (P < 0.01), but also decreased brian edema in the ischemic brain (P < 0.05). Then, fluorescence two-dimensional difference in gel electrophoresis was used to detect our systematic differential profiling of proteomic changes responding to tetrandrine administration. We validated that the expression of GRP78, DJ-1 and HYOU1 was associated with neuroprotective effect of tetrandrine in MCAO model by Western blotting. These findings indicate a potential neuroprotective role of tetrandrine for ischemic stroke and yield insights into cellular and molecular mechanisms of tetrandrine taking place in ischemic stroke.