Scorpion venom heat-resistant synthesized peptide ameliorates epileptic seizures and imparts neuroprotection in rats mediated by NMDA receptors.

Finding new and effective natural products for designing antiepileptic drugs is highly important in the scientific community. The scorpion venom heat-resistant peptide (SVHRP) was purified from Buthus martensii Karsch scorpion venom, and subsequent analysis of the amino acid sequence facilitated the synthesis of a peptide known as scorpion venom heat-resistant synthesis peptide (SVHRSP) using a technique for peptide synthesis. Previous studies have demonstrated that the SVHRSP can inhibit neuroinflammation and provide neuroprotection. This study aimed to investigate the antiepileptic effect of SVHRSP on both acute and chronic kindling seizure models by inducing seizures in male rats through intraperitoneal administration of pentylenetetrazole (PTZ). Additionally, an N-methyl-D-aspartate (NMDA)-induced neuronal injury model was used to observe the anti-excitotoxic effect of SVHRSP in vitro. Our findings showed that treatment with SVHRSP effectively alleviated seizure severity, prolonged latency, and attenuated neuronal loss and glial cell activation. It also demonstrated the prevention of alterations in the expression levels of NMDA receptor subunits and phosphorylated p38 MAPK protein, as well as an improvement in spatial reference memory impairment during Morris water maze (MWM) testing in PTZ-kindled rats. In vitro experiments further revealed that SVHRSP was capable of attenuating neuronal action potential firing, inhibiting NMDA receptor currents and intracellular calcium overload, and reducing neuronal injury. These results suggest that the antiepileptic and neuroprotective effects of SVHRSP may be mediated through the regulation of NMDA receptor function and expression. This study provides new insight into therapeutic strategies for epilepsy.

Anti-epileptic and Neuroprotective Effects of Ultra-low Dose NADPH Oxidase Inhibitor Dextromethorphan on Kainic Acid-induced Chronic Temporal Lobe Epilepsy in Rats.

Neuroinflammation mediated by microglia and oxidative stress play pivotal roles in the development of chronic temporal lobe epilepsy (TLE). We postulated that kainic acid (KA)-Induced status epilepticus triggers microglia-dependent inflammation, leading to neuronal damage, a lowered seizure threshold, and the emergence of spontaneous recurrent seizures (SRS). Extensive evidence from our laboratory suggests that dextromethorphan (DM), even in ultra-low doses, has anti-inflammatory and neuroprotective effects in many animal models of neurodegenerative disease. Our results showed that administration of DM (10 ng/kg per day; subcutaneously via osmotic minipump for 4 weeks) significantly mitigated the residual effects of KA, including the frequency of SRS and seizure susceptibility. In addition, DM-treated rats showed improved cognitive function and reduced hippocampal neuronal loss. We found suppressed microglial activation-mediated neuroinflammation and decreased expression of hippocampal gp91phox and p47phox proteins in KA-induced chronic TLE rats. Notably, even after discontinuation of DM treatment, ultra-low doses of DM continued to confer long-term anti-seizure and neuroprotective effects, which were attributed to the inhibition of microglial NADPH oxidase 2 as revealed by mechanistic studies.

Corrigendum: Scorpion Venom Heat-Resistant Peptide Attenuates Microglia Activation and Neuroinflammation.

[This corrects the article DOI: 10.3389/fphar.2021.704715.].

Scorpion Venom Heat-Resistant Peptide Attenuates Microglia Activation and Neuroinflammation.

Background: Intervention of neuroinflammation in central nervous system (CNS) represents a potential therapeutic strategy for a host of brain disorders. The scorpion Buthus martensii Karsch (BmK) and its venom have long been used in the Orient to treat inflammation-related diseases such as rhumatoid arthritis and chronic pain. Scorpion venom heat-resistant peptide (SVHRP), a component from BmK venom, has been shown to reduce seizure susceptibility in a rat epileptic model and protect against cerebral ischemia-reperfusion injury. As neuroinflammation has been implicated in chronic neuronal hyperexcitability, epileptogenesis and cerebral ischemia-reperfusion injury, the present study aimed to investigate whether SVHRP has anti-inflammatory property in brain. Methods: An animal model of neuroinflammation induced by lipopolysacchride (LPS) injection was employed to investigate the effect of SVHRP (125 µg/kg, intraperitoneal injection) on inflammagen-induced expression of pro-inflammatory factors and microglia activation. The effect of SVHRP (2-20 µg/ml) on neuroinflammation was further investigated in primary brain cell cultures containing microglia as well as the immortalized BV2 microglia culture stimulated with LPS. Real-time quantitative PCR were used to measure mRNA levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6 in hippocampus of animals. Protein levels of TNF-α, iNOS, P65 subunit of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) were examined by ELISA or western blot. Microglia morphology in animal hippocampus or cell cultures and cellular distribution of p65 were shown by immunostaining. Results: Morphological study demonstrated that activation of microglia, the main component that mediates the neuroinflammatory process, was inhibited by SVHRP in both LPS mouse and cellular model. Our results also showed dramatic increases in the expression of iNOS and TNF-α in hippocampus of LPS-injected mice, which was significantly attenuated by SVHRP treatment. In vitro results showed that SVHRP attenuated LPS-elicited expression of iNOS and TNF-α in different cultures without cell toxicity, which might be attributed to suppression of NF-κB and MAPK pathways by SVHRP. Conclusion: Our study demonstrates that SVHRP is able to inhibit neuroinflammation and microglia activation, which may underlie the therapeutic effects of BmK-derived materials, suggesting that BmK venom could be a potential source for CNS drug development.

Scorpion venom heat-resistant synthesized peptide ameliorates 6-OHDA-induced neurotoxicity and neuroinflammation: likely role of Nav 1.6 inhibition in microglia.

BACKGROUND AND PURPOSE: Microglia-related inflammation is associated with the pathology of Parkinson's disease. Functional voltage-gated sodium channels (VGSCs) are involved in regulating microglial function. Here, we aim to investigate the effects of scorpion venom heat-resistant synthesized peptide (SVHRSP) on 6-hydroxydopamine (6-OHDA)-induced Parkinson's disease-like mouse model and reveal its underlying mechanism. EXPERIMENTAL APPROACH: Unilateral brain injection of 6-OHDA was performed to establish Parkinson's disease mouse model. After behaviour test, brain tissues were collected for morphological analysis and protein/gene expression examination. Primary microglia culture was used to investigate the role of sodium channel Nav 1.6 in the regulation of microglia inflammation by SVHRSP. KEY RESULTS: SVHRSP treatment attenuated motor deficits, dopamine neuron degeneration, activation of glial cells and expression of pro-inflammatory cytokines induced by 6-OHDA lesion. Primary microglia activation and the production of pro-inflammatory cytokines induced by lipopolysaccharide (LPS) were also suppressed by SVHRSP treatment. In addition, SVHRSP could inhibit mitogen-activated protein kinases (MAPKs) pathway, which plays pivotal roles in the pro-inflammatory response. Notably, SVHRSP treatment suppressed the overexpression of microglial Nav 1.6 induced by 6-OHDA and LPS. Finally, it was shown that the anti-inflammatory effect of SVHRSP in microglia was Nav 1.6 dependent and was related to suppression of sodium current and probably the consequent Na+ /Ca2+ exchange. CONCLUSIONS AND IMPLICATIONS: SVHRSP might inhibit neuroinflammation and protect dopamine neurons via down-regulating microglial Nav 1.6 and subsequently suppressing intracellular Ca2+ accumulation to attenuate the activation of MAPKs signalling pathway in microglia.

GPR50 Distribution in the Mouse Cortex and Hippocampus.

G protein-coupled receptor 50 (GPR50) belongs to the G protein-coupled receptor which is highly homologous with the sequence of melatonin receptor MT1 and MT2. GPR50 expression has previously been reported in many brain regions, like cortex, midbrain, pons, amygdala. But, the distribution of GPR50 in the hippocampus and cortex and the cell types expressing GPR50 is not yet clear. In this study, we examined the distribution of GPR50 in adult male mice by immunofluorescence. Our results showed that GPR50 was localized in the CA1-3 pyramidal cells and the granule cells of the dentate gyrus. GPR50 was also expressed in excitatory and inhibitory neurons. As inhibitory neurons also contain many types, we found that GPR50 was localized in some interneurons in which it was co-expressed with the calcium-binding proteins calbindin, calretinin, and parvalbumin. Besides, similar results were seen in the cortex. The widespread expression of GPR50 in the hippocampus and cortex suggests that GPR50 may be associated with synaptic plasticity and cognitive function.

NF-κB "decoy" inhibits COX-2 expression in epileptic rat brain.

There is a need to investigate the role of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression in the epileptic rat brain and cultured hippocampal neurons. Immunofluorescence and polymerase chain reaction was used to detect the expression of nuclear factor kappa B and cyclooxygenase-2. In cultured hippocampal neurons and rat brain: the control group compared with the normal group, nuclear factor kappa B expression in the hippocampal dentate gyrus, cerebral cortex, the piriform cortex brain regions were significantly increased (P < 0.01). This is accompanied by a significant increase in cyclooxygenase-2 protein and mRNA expressions in the hippocampus (P < 0.01). In the experimental group compared to the control group, the nuclear factor-kappa B expression in the hippocampal dentate gyrus, cerebral cortex, piriform cortex, and other brain regions was significantly lower (P < 0.01), with the accompanying decrease in cyclooxygenase-2 protein and mRNA expression (P < 0.01) in the hippocampus. In conclusion, κB-decoy can inhibit nuclear factor kappa B activation in epileptic rat brain and cyclooxygenase-2 overexpression.

Scorpion Venom Heat-Resistant Peptide is Neuroprotective against Cerebral Ischemia-Reperfusion Injury in Association with the NMDA-MAPK Pathway.

Scorpion venom heat-resistant peptide (SVHRP) is a component purified from Buthus martensii Karsch scorpion venom. Our previous studies have shown that SVHRP is neuroprotective in models of Alzheimer's disease and Parkinson's disease. The present study aimed to explore the potential neuroprotective effects of SVHRP on cerebral ischemia/reperfusion (I/R) injury, using a mouse model of middle cerebral artery occlusion/reperfusion (MCAO/R) and a cellular model of oxygen-glucose deprivation/reoxygenation (OGD/R). Our results showed that SVHRP treatment decreased the neurological deficit scores, edema formation, infarct volume and neuronal loss in the MCAO/R mice, and protected primary neurons against OGD/R insult. SVHRP pretreatment suppressed the alterations in protein levels of N-methyl-D-aspartate receptors (NMDARs) and phosphorylated p38 MAPK as well as some proinflammatory factors in both the animal and cellular models. These results suggest that SVHRP has neuroprotective effects against cerebral I/R injury, which might be associated with inhibition of the NMDA-MAPK-mediated excitotoxicity.

Impaired Cognitive Function and Altered Hippocampal Synaptic Plasticity in Mice Lacking Dermatan Sulfotransferase Chst14/D4st1.

Chondroitin sulfate (CS) and dermatan sulfate (DS) proteoglycans (PGs) are major extracellular matrix (ECM) components of the central nervous system (CNS). A large body of evidence has shown that CSPGs/DSPGs play critical roles in neuronal growth, axon guidance, and plasticity in the developing and mature CNS. It has been proposed that these PGs exert their function through specific interaction of CS/DS chains with its binding partners in a manner that depends on the sulfation patterns of CS/DS. It has been reported that dermatan 4-O-sulfotransferase-1 (Chst14/D4st1) specific for DS, but not chondroitin 4-O-sulfotransferase-1 (Chst11/C4st1) specific for CS, regulates proliferation and neurogenesis of neural stem cells (NSCs), indicating that CS and DS play distinct roles in the self-renewal and differentiation of NSCs. However, it remains unknown whether specific sulfation profiles of DS has any effect on CNS plasticity. In the present study, Chst14/D4st1-deficient (Chst14 -/-) mice was employed to investigate the involvement of DS in synaptic plasticity. First, behavior study using Morris Water Maze (MWM) showed that the spatial learning and memory of Chst14 -/- mice was impaired when compared to their wild type (WT) littermates. Corroborating the behavior result, long-term potentiation (LTP) at the hippocampal CA3-CA1 connection was reduced in Chst14 -/- mice compared to the WT mice. Finally, the protein levels of N-Methyl-D-aspartate (NMDA) receptor, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, postsynaptic density 95 (PSD95), growth associated protein 43 (GAP-43), synaptophysin (SYN) and N-ethylmaleimide sensitive factor (NSF) which are important in synaptic plasticity were examined and Chst14/D4st1 deficiency was shown to significantly reduce the expression of these proteins in the hippocampus. Further studies revealed that Akt/mammalian target rapamycin (mTOR) pathway proteins, including protein kinase B (p-Akt), p-mTOR and p-S6, were significantly lower in Chst14 -/- mice, which might contribute to the decreased protein expression. Together, this study reveals that specific sulfation of DS is critical in synaptic plasticity of the hippocampus and learning and memory, which might be associated with the changes in the expression of glutamate receptors and other synaptic proteins though Akt/mTOR pathway.

Long-term social isolation inhibits autophagy activation, induces postsynaptic dysfunctions and impairs spatial memory.

Social isolation in adolescence leads to lasting deficits in hippocampal-dependent tasks. The reported effects of isolation on learning and memory in the Morris water maze and synaptic-related proteins have been inconsistent. Moreover, the autophagy level and its effect on cognition in the isolation model are also not clear. In the present study, we did an extended isolation period up to six months to establish a stable and appropriate isolation model to investigate the cognitive changes associated with it. The mTOR inhibitor rapamycin was systemically administered to mice to determine the roles of autophagy activation on cognitive changes. We discovered that long-term post-weaning social isolation (L-PWSI) produced marked deficits in spatial learning and memory and inhibited CA1 long-term potentiation (LTP), but paired-pulse facilitation (PPF) and input/output (I/O) curve were unaffected. The results further showed that the L-PWSI significantly decreased the protein expression levels of PSD-95, GluA1, NR1 and NR2B in the hippocampus, and no significant changes in the extracellular release of glutamate and the protein expression levels of synaptophysin, synapsin I, GAP-43, NR2A and GABAA. Moreover, we found that L-PWSI increased the protein expression of p-AKT/AKT, p-mTOR/mTOR and p62, whereas the protein levels of LC3B and Beclin1 were decreased indicating an inhibition in autophagy activity. Intraperitoneal injection of rapamycin significantly potentiated fEPSP slope and cognition-related proteins expression in the L-PWSI mice. These results therefore suggest that L-PWSI induces postsynaptic dysfunction by disrupting the interaction between AMPAR, NMDAR and PSD-95, and inhibit the autophagy activity which led to impaired spatial memory and cognitive function.

Upregulation of Intestinal Barrier Function in Mice with DSS-Induced Colitis by a Defined Bacterial Consortium Is Associated with Expansion of IL-17A Producing Gamma Delta T Cells.

Bacterial consortium transplantation (BCT) is a promising alternative to fecal microbiota transplantation in treating inflammatory bowel disease (IBD). Here, we showed that a defined bacterial consortium derived from healthy mice was able to enhance the intestinal barrier function of mice with dextran sulfate sodium (DSS)-induced colitis. Interestingly, we found that the bacterial consortium significantly promoted the expansion of IL-17A-producing γδT (γδT17) cells in colonic lamina propria, which was closely associated with changing of intestinal microbial composition. The increased IL-17A secretion upon treatment with microbial products derived from the bacterial consortium was accompanied with upregulation of TLR2 expression by γδT cells, and it might be responsible for the upregulation of mucosal barrier function through IL-17R-ACT1-mediated recovery of the disrupted occludin subcellular location. Changing of some specific microbial groups such as Bifidobacterium and Bacillus spp. was closely correlated with the promotion of TLR2+ γδT cells. Our results support that BCT can restore the alliance between commensal microbiota and intestinal γδT cells, which contributes to the improvement of intestinal barrier function. This study provides new insight into the development of bacteria transplantation therapy for the treatment of IBD.

Scorpion venom heat-resistant peptide (SVHRP) enhances neurogenesis and neurite outgrowth of immature neurons in adult mice by up-regulating brain-derived neurotrophic factor (BDNF).

Scorpion venom heat-resistant peptide (SVHRP) is a component purified from Buthus martensii Karsch scorpion venom. Although scorpions and their venom have been used in Traditional Chinese Medicine (TCM) to treat chronic neurological disorders, the underlying mechanisms of these treatments remain unknown. We applied SVHRP in vitro and in vivo to understand its effects on the neurogenesis and maturation of adult immature neurons and explore associated molecular mechanisms. SVHRP administration increased the number of 5-bromo-2'-dexoxyuridine (BrdU)-positive cells, BrdU-positive/neuron-specific nuclear protein (NeuN)-positive neurons, and polysialylated-neural cell adhesion molecule (PSA-NCAM)-positive immature neurons in the subventricular zone (SVZ) and subgranular zone (SGZ) of hippocampus. Furthermore immature neurons incubated with SVHRP-pretreated astrocyte-conditioned medium exhibited significantly increased neurite length compared with those incubated with normal astrocyte-conditioned medium. This neurotrophic effect was further confirmed in vivo by detecting an increased average single area and whole area of immature neurons in the SGZ, SVZ and olfactory bulb (OB) in the adult mouse brain. In contrast to normal astrocyte-conditioned medium, higher concentrations of brain-derived neurotrophic factor (BDNF) but not nerve growth factor (NGF) or glial cell line-derived neurotrophic factor (GDNF) was detected in the conditioned medium of SVHRP-pretreated astrocytes, and blocking BDNF using anti-BDNF antibodies eliminated these SVHRP-dependent neurotrophic effects. In SVHRP treated mouse brain, more glial fibrillary acidic protein (GFAP)-positive cells were detected. Furthermore, immunohistochemistry revealed increased numbers of GFAP/BDNF double-positive cells, which agrees with the observed changes in the culture system. This paper describes novel effects of scorpion venom-originated peptide on the stem cells and suggests the potential therapeutic values of SVHRP.