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Aims: This study aimed to investigate the potential therapeutic applications of stigmasterol for treating neuropathic pain. Methods: Related mechanisms were investigated by DRG single‐cell sequencing analysis and the use of specific inhibitors in cellular experiments. In animal experiments, 32 male Sprague–Dawley rats were randomly divided into the sham operation group, CCI group, ibuprofen group, and stigmasterol group. We performed behavioral tests, ELISA, H&E staining and immunohistochemistry, and western blotting. Results: Cell communication analysis by single‐cell sequencing reveals that after peripheral nerve injury, Schwann cells secrete IL‐34 to act on CSF1R in macrophages. After peripheral nerve injury, the mRNA expression levels of CSF1R pathway and NLRP3 inflammasome in macrophages were increased in DRG. In vitro studies demonstrated that stigmasterol can reduce the secretion of IL‐34 in LPS‐induced RSC96 Schwann cells; stigmasterol treatment of LPS‐induced Schwann cell‐conditioned medium (L‐S‐CM) does not induce the proliferation and migration of RAW264.7 macrophages; L‐S‐CM reduces CSF1R signaling pathway (CSF1R, P38MAPK, and NFκB) activation, NLRP3 inflammasome activation, and ROS production. In vivo experiments have verified that stigmasterol can reduce thermal and cold hyperalgesia in rat chronic compressive nerve injury (CCI) model; stigmasterol can reduce IL‐1β, IL‐6, TNF‐α, CCL2, SP, and PGE2 in serum of CCI rats; immunohistochemistry and western blot confirmed that stigmasterol can reduce the levels of IL‐34/CSF1R signaling pathway and NLRP3 inflammasome in DRG of CCI rats. Conclusion: Stigmasterol alleviates neuropathic pain by reducing Schwann cell‐macrophage cascade in DRG by modulating IL‐34/CSF1R axis. [ABSTRACT FROM AUTHOR]

(Switching from the microglial M1 phenotype to the M2 phenotype is a promising therapeutic strategy for neuropathic pain (NP). This study aimed to investigate the potential use of stigmasterol for treating NP. In animal experiments, 32 male Sprague–Dawley rats were randomly divided into the sham operation group, chronic constriction injury (CCI) group, CCI + ibuprofen group, and CCI + stigmasterol group. We performed behavioral tests, enzyme‐linked immunosorbent assay, hematoxylin‐esoin staining (H&E) staining and immunohistochemistry, immunofluorescence, and Western blotting. In cell experiments, we performed flow cytometry, immunofluorescence, Western blotting, and qRT‐PCR. Stigmasterol reduced thermal and mechanical hyperalgesia and serum IL‐1β and IL‐8 levels and increased serum IL‐4 and TGF‐β levels in CCI rats. Stigmasterol reduced IL‐1β, COX‐2, and TLR4 expression in the right sciatic nerve and IL‐1β expression in the spinal cord. Stigmasterol reduced the expression of Iba‐1, TLR4, MyD88, pNF‐κB, pP38 MAPK, pJNK, pERK, COX‐2, IL‐1β, and CD32 in the spinal cord of CCI rats while increasing the expression of IL‐10 and CD206. Stigmasterol decreased M1 polarization markers and increased M2 polarization markers in lipopolysaccharide (LPS)‐induced microglia and decreased the expression of Iba‐1, TLR4, MyD88, pNF‐κB, pP38 MAPK, pJNK, pERK, iNOS, COX‐2, and IL‐1β in LPS‐treated microglia while increasing the expression of Arg‐1 and IL‐10. Stigmasterol regulates microglial M1/M2 polarization via the TLR4/NF‐κB pathway to alleviate NP. [ABSTRACT FROM AUTHOR]

Switching microglial polarization from the M1 to M2 phenotype is a promising therapeutic strategy for neuropathic pain (NP). Toll‐like receptor 4 (TLR4) is activated by lipopolysaccharide (LPS). Uncontrolled activation of TLR4 has been proven to trigger chronic inflammation. Kaempferol, a dietary flavonoid, is known to have anti‐inflammatory properties. This study is aimed to investigate the analgesic and anti‐inflammatory effects and the underlying mechanisms of kaempferol, which were explored with an NP model in vivo and LPS‐induced injury in microglial BV2 cells in vitro. The levels of proinflammatory cytokines were evaluated. H&E staining and immunohistochemistry were used to assess the sciatic nerve condition after chronic constriction injury surgery. Western blotting and immunofluorescence were used to determine whether TLR4/NF‐ĸB signaling pathway plays a major role in kaempferol‐mediated alleviation of neuroinflammation. Quantitative real‐time polymerase chain reaction and flow cytometry were used to examine the modulator effect of kaempferol on microglial M1/M2 polarization. We found that kaempferol treatment can significantly reduce NP and proinflammatory cytokine production. Kaempferol attenuated the activation of TLR4/NF‐κB pathways in LPS‐activated BV2 cells. The analgesic effects of kaempferol on NP may be due to inhibition of microglia activation and switching the M1 to M2 phenotype. [ABSTRACT FROM AUTHOR]

Pyroptosis is defined as inflammation-induced programmed cell death. However, gene expression levels related to pyroptosis and their role in neuropathic pain (NP) remain unclear. The present study aimed to develop and validate an NP-predictive signature based on the genes associated with pyroptosis. Gene expression level profiles were downloaded from the Gene Expression Omnibus database. Weighted gene co-expression network analysis was used to identify the pyroptotic genes most highly associated with NP. NP-related pyroptosis gene signature was constructed using multivariate logistic regression. A rat model of neuropathic pain was established through chronic constriction injury to analyse the inflammatory infiltration and myelin damage around the sciatic nerve, and examine the expression levels of macrophage markers S100 calcium-binding protein β (S100β) and ionized calcium-binding adapter molecule 1 (Iba-1). Finally, flow cytometry analysis was used to examine the lipopolysaccharide (LPS)-induced cell death ratio of RSC96 cells (Schwann cells), while the expression levels of LPS-induced pyroptosis-related genes in RSC96 cells were measured via reverse transcription-quantitative PCR. The results demonstrated that pyroptosis-related genes (gasdermin D, NLR family pyrin domain containing 3, neuronal apoptosis inhibitory protein and NLR family CARD domain containing 4) were identified to increase the risk of NP. NP-related pyroptosis signatures were constructed based on these four genes. Moreover, the high-risk group had a higher level of macrophage infiltration compared with the low-risk group, as determined by the CIBERSORT algorithm. H&E staining results showed that the myelin structure of the sciatic nerve tissue of chronic constriction injury (CCI) rats was destroyed and inflammatory cells infiltrated around neurons. The results of immunohistochemistry showed that compared with in the sham group, the expression levels of Iba-1 and sS100β in the sciatic nerve of the CCI group were increased. Furthermore, the expression levels of cell death and pyroptosis-related genes in Schwann cells induced by LPS were increased compared with in the control group. In conclusion, an NP-related pyroptosis gene signature was constructed based on four pyroptosis-related genes and it was found that the expression of pyroptosis-related genes was upregulated in the early steps of the neuroinflammatory process in RSC96 cells. [ABSTRACT FROM AUTHOR]

The aim of the present study was to assess the protective effects of 18β-GA against hydrogen peroxide (H2O2)-induced injury. First, the SMILES annotation for 18β-GA was used to search PubChem and for reverse molecular docking in Swiss Target Prediction, the Similarity Ensemble Approach Search Server and the TargetNet database to obtain potential targets. Injury-related molecules were obtained from the GeneCards database and the predicted targets of 18β-GA for injury treatment were selected by Wayne diagram analysis. Subsequently, Kyoto Encyclopedia of Genes and Genomes analysis was performed by WebGestalt. The experimental cells were assorted into control, model, 10 µM SB203580-treated, 5 µM 18β-GA-treated and 10 µM 18β-GA-treated groups. Hoechst 33258 staining was performed and intracellular reactive oxygen species (ROS) levels, cell apoptosis, Bcl-xl, Bcl-2, Bad, Bax, cleaved-caspase 3, cleaved-caspase 7, transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) levels, as well as p38 MAPK phosphorylation were measured. The 'Inflammatory mediator regulation of TRP channels' pathway was selected for experimental verification. The results indicated that 10 µM 18β-GA significantly increased cell viability as compared with the H2O2-treated model group. As suggested by the difference in intracellular ROS fluorescence intensity, 18β-GA inhibited H2O2-induced ROS production in Schwann cells. Hoechst 33258 staining indicated that 18β-GA reversed chromatin condensation and the increase in apoptotic nuclei following H2O2 treatment. Furthermore, flow cytometry suggested that 18β-GA substantially inhibited H2O2-induced apoptosis. Pre-treatment with 18β-GA obviously reduced Bad, Bax, cleaved-caspase3, cleaved-caspase 7, TRPA1 and TRPV1 levels and p38 MAPK phosphorylation after H2O2 treatment and increased Bcl-2 and Bcl-xl levels. In conclusion, 18β-GA inhibited Schwann cell injury and apoptosis induced by H2O2 and may be a potential drug to prevent peripheral nerve injury. [ABSTRACT FROM AUTHOR]

This study explored the therapeutic effect of α-asarone on chronic sciatica. Thirty-two Sprague-Dawley (SD) rats were divided into four groups: the sham group, chronic constriction injury (CCI) group, pregabalin group, and α-asarone group. Hot hyperalgesia was induced after the CCI operation, and α-asarone was found to relieve chronic neuralgia. Furthermore, α-asarone reduced IL1β, IL6, TNF-α, CRP, and LPS levels and increased IL10 levels in serum. α-Asarone decreased the protein levels of TRPA1, TRPM8, and TRPV1-4 and the mRNA levels of TRPA1, TRPM8, TRPV1-4, IL1β, and TNF-α in dorsal root ganglion neurons. In the sciatic nerve, α-asarone treatment reduced the number of inflammatory cells and promoted the proliferation of Schwann cells, favouring recovery of the nerve structure. In cellular experiments, LPS induced Schwann cell apoptosis via TLR4/p38MAPK signalling; α-asarone attenuated LPS-induced Schwann cell apoptosis by decreasing TLR4, p-p38MAPK, cleaved-caspase3, and cleaved-caspase7 levels and increasing Bcl-2 and Bcl-xl expression. Overall, these findings suggest that α-asarone relieves chronic sciatica by decreasing the levels of inflammatory factors, inhibiting peripheral sensitization, and favouring the repair of damaged nerves., (© 2022 John Wiley & Sons Ltd.)

The aim of the present study was to assess the protective effects of 18β-GA against hydrogen peroxide (H 2 O 2 )-induced injury. First, the SMILES annotation for 18β-GA was used to search PubChem and for reverse molecular docking in Swiss Target Prediction, the Similarity Ensemble Approach Search Server and the TargetNet database to obtain potential targets. Injury-related molecules were obtained from the GeneCards database and the predicted targets of 18β-GA for injury treatment were selected by Wayne diagram analysis. Subsequently, Kyoto Encyclopedia of Genes and Genomes analysis was performed by WebGestalt. The experimental cells were assorted into control, model, 10 µM SB203580-treated, 5 µM 18β-GA-treated and 10 µM 18β-GA-treated groups. Hoechst 33258 staining was performed and intracellular reactive oxygen species (ROS) levels, cell apoptosis, Bcl-xl, Bcl-2, Bad, Bax, cleaved-caspase 3, cleaved-caspase 7, transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) levels, as well as p38 MAPK phosphorylation were measured. The 'Inflammatory mediator regulation of TRP channels' pathway was selected for experimental verification. The results indicated that 10 µM 18β-GA significantly increased cell viability as compared with the H 2 O 2 -treated model group. As suggested by the difference in intracellular ROS fluorescence intensity, 18β-GA inhibited H 2 O 2 -induced ROS production in Schwann cells. Hoechst 33258 staining indicated that 18β-GA reversed chromatin condensation and the increase in apoptotic nuclei following H 2 O 2 treatment. Furthermore, flow cytometry suggested that 18β-GA substantially inhibited H 2 O 2 -induced apoptosis. Pre-treatment with 18β-GA obviously reduced Bad, Bax, cleaved-caspase3, cleaved-caspase 7, TRPA1 and TRPV1 levels and p38 MAPK phosphorylation after H 2 O 2 treatment and increased Bcl-2 and Bcl-xl levels. In conclusion, 18β-GA inhibited Schwann cell injury and apoptosis induced by H 2 O 2 and may be a potential drug to prevent peripheral nerve injury., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Zhang et al.)