Harnessing developmental dynamics of spinal cord extracellular matrix improves regenerative potential of spinal cord organoids.

Neonatal spinal cord tissues exhibit remarkable regenerative capabilities as compared to adult spinal cord tissues after injury, but the role of extracellular matrix (ECM) in this process has remained elusive. Here, we found that early developmental spinal cord had higher levels of ECM proteins associated with neural development and axon growth, but fewer inhibitory proteoglycans, compared to those of adult spinal cord. Decellularized spinal cord ECM from neonatal (DNSCM) and adult (DASCM) rabbits preserved these differences. DNSCM promoted proliferation, migration, and neuronal differentiation of neural progenitor cells (NPCs) and facilitated axonal outgrowth and regeneration of spinal cord organoids more effectively than DASCM. Pleiotrophin (PTN) and Tenascin (TNC) in DNSCM were identified as contributors to these abilities. Furthermore, DNSCM demonstrated superior performance as a delivery vehicle for NPCs and organoids in spinal cord injury (SCI) models. This suggests that ECM cues from early development stages might significantly contribute to the prominent regeneration ability in spinal cord.

Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R.

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-1ß, 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.

Pathogenesis or a response to lithium? A novel perspective for mitochondrial mass fluctuation of naïve T cells in patients with bipolar disorder.

BACKGROUND: Immune imbalances are associated with the pathogenesis and pharmacological efficacy of bipolar disorder (BD). The underlying mechanisms remain largely obscure but may involve immunometabolic dysfunctions of T-lymphocytes. METHODS: We investigated if inflammatory cytokines and the immunometabolic function of T-lymphocytes, including frequencies of subsets, mitochondrial mass (MM), and low mitochondrial membrane potential (MMPLow) differed between BD patients (n = 47) and healthy controls (HC, n = 43). During lithium treatment of hospitalized patients (n = 33), the association between weekly T-lymphocyte immune metabolism and clinical symptoms was analyzed, and preliminary explorations on possible mechanisms were conducted. RESULTS: In comparison to HC, BD patients predominantly showed a trend toward CD4+ naïve T (Tn) activation and exhibited mitochondrial metabolic disturbances such as decreased MM and increased MMPLow. Lower CD4+ Tn-MM correlated with elevated IL-6, IL-8, and decreased IL-17 A in BD patients. With lithium treatment effective, MM of CD4+ T/Tn was negatively correlated with depression score HAMD. When lithium intolerance was present, MM of CD4+ T/Tn was positively correlated with depression score HAMD and mania score BRMS. Lithium does not mediate through the inositol depletion hypothesis, but the mRNA level of IMPA2 in peripheral blood is associated with mitochondrial function in CD8+ T cells. LIMITATIONS: The cross-sectional design and short-term follow-up meant that we could not directly examine the causality of BD and immune dysregulation. CONCLUSION: The altered metabolism of CD4+ Tn was strongly associated with remodeling of the inflammatory landscape in BD patients and can also be used to reflect the short-term therapeutic effects of lithium.

Stigmasterol regulates microglial M1/M2 polarization via the TLR4/NF-κB pathway to alleviate neuropathic pain.

(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-1ß and IL-8 levels and increased serum IL-4 and TGF-ß levels in CCI rats. Stigmasterol reduced IL-1ß, COX-2, and TLR4 expression in the right sciatic nerve and IL-1ß expression in the spinal cord. Stigmasterol reduced the expression of Iba-1, TLR4, MyD88, pNF-κB, pP38 MAPK, pJNK, pERK, COX-2, IL-1ß, 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-1ß 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.

Mimicked Spinal Cord Fibers Trigger Axonal Regeneration and Remyelination after Injury.

Spinal cord injury (SCI) causes tissue structure damage and composition changes of the neural parenchyma, resulting in severe consequences for spinal cord function. Mimicking the components and microstructure of spinal cord tissues holds promise for restoring the regenerative microenvironment after SCI. Here, we have utilized electrospinning technology to develop aligned decellularized spinal cord fibers (A-DSCF) without requiring synthetic polymers or organic solvents. A-DSCF preserves multiple types of spinal cord extracellular matrix proteins and forms a parallel-oriented structure. Compared to aligned collagen fibers (A-CF), A-DSCF exhibits stronger mechanical properties, improved enzymatic stability, and superior functionality in the adhesion, proliferation, axonal extension, and myelination of differentiated neural progenitor cells (NPCs). Notably, axon extension or myelination has been primarily linked to Agrin (AGRN), Laminin (LN), or Collagen type IV (COL IV) proteins in A-DSCF. When transplanted into rats with complete SCI, A-DSCF loaded with NPCs improves the survival, maturation, axon regeneration, and motor function of the SCI rats. These findings highlight the potential of structurally and compositionally biomimetic scaffolds to promote axonal extension and remyelination after SCI.

(+)-Catechin Alleviates CCI-Induced Neuropathic Pain in Rats by Modulating the IL34/CSFIR Axis and Attenuating the Schwann Cell-Macrophage Cascade Response in the DRG.

The aim of this study was to investigate the potential therapeutic applications of (+)-catechin in the treatment of neuropathic pain. In vivo study, 32 SD rats were randomly divided into four groups: sham group, chronic constriction injury (CCI) group, CCI + ibuprofen group and CCI+ (+)-catechin group. They were subjected to behavioural tests, ELISA, immunohistochemistry and Western blotting. The mechanisms involved were investigated using specific inhibitors in cell experiments. Results of in vivo experiments showed that (+)-catechin could reduce the cold sensitivity pain in a rat model of CCI; ELISA and immunohistochemistry results showed that (+)-catechin could decrease the levels of IL-8, IL-6, TNF-α, CCL2 and CCL5 in serum and the expression levels of nNOS, COX2, IL6, TNF-α, IBA-1 and CSF1R in DRG of CCI rats. Finally, western blot confirmed that (+)-catechin could diminish the levels of IL-34/CSF1R/JAK2/STAT3 signalling pathway in DRG of CCI rats. In vitro studies showed that (+)-catechin reduced IL-34 secretion in LPS-induced RSC96 cells. Meanwhile, (+)-catechin administration in LPS-induced Schwann cell-conditioned medium (L-CM) significantly inhibited the proliferation and migration of RAW264.7 cells; in addition, L-CM+(+)-catechin reduced the activation of the CSF1R/JAK2/STAT3 signalling pathway. (+)-Catechin attenuated the Schwann cell-macrophage cascade response in the DRG by modulating the IL34/CSFIR axis and inhibiting activation of the JAK2/STAT3 pathway, thereby attenuating CCI-induced neuropathic pain in rats.

The role of the symbiotic microecosystem in cancer: gut microbiota, metabolome, and host immunome.

The gut microbiota is not just a simple nutritional symbiosis that parasitizes the host; it is a complex and dynamic ecosystem that coevolves actively with the host and is involved in a variety of biological activities such as circadian rhythm regulation, energy metabolism, and immune response. The development of the immune system and immunological functions are significantly influenced by the interaction between the host and the microbiota. The interactions between gut microbiota and cancer are of a complex nature. The critical role that the gut microbiota plays in tumor occurrence, progression, and treatment is not clear despite the already done research. The development of precision medicine and cancer immunotherapy further emphasizes the importance and significance of the question of how the microbiota takes part in cancer development, progression, and treatment. This review summarizes recent literature on the relationship between the gut microbiome and cancer immunology. The findings suggest the existence of a "symbiotic microecosystem" formed by gut microbiota, metabolome, and host immunome that is fundamental for the pathogenesis analysis and the development of therapeutic strategies for cancer.

Single-cell analysis reveals region-heterogeneous responses in rhesus monkey spinal cord with complete injury.

Spinal cord injury (SCI) leads to severe sensory and motor dysfunction below the lesion. However, the cellular dynamic responses and heterogeneity across different regions below the lesion remain to be elusive. Here, we used single-cell transcriptomics to investigate the region-related cellular responses in female rhesus monkeys with complete thoracic SCI from acute to chronic phases. We found that distal lumbar tissue cells were severely impacted, leading to degenerative microenvironments characterized by disease-associated microglia and oligodendrocytes activation alongside increased inhibitory interneurons proportion following SCI. By implanting scaffold into the injury sites, we could improve the injury microenvironment through glial cells and fibroblast regulation while remodeling spared lumbar tissues via reduced inhibitory neurons proportion and improved phagocytosis and myelination. Our findings offer crucial pathological insights into the spared distal tissues and proximal tissues after SCI, emphasizing the importance of scaffold-based treatment approaches targeting heterogeneous microenvironments.

Mitochondrial mass of circulating NK cells as a novel biomarker in severe SARS-CoV-2 infection.

BACKGROUND: Severe SARS-CoV-2 infection results in lymphopenia and impaired function of T, B, and NK (TBNK-dominant) lymphocytes. Mitochondria are essential targets of SARS-CoV-2 and the efficacy of lymphocyte mitochondrial function for immunosurveillance in COVID-19 patients has not been evaluated. METHODS: Multi-parametric flow cytometry was used to characterize mitochondrial function, including mitochondrial mass (MM) and low mitochondrial membrane potential (MMPlow), in TBNK-dominant lymphocytes from severe (n = 93) and moderate (n = 77) hospitalized COVID-19 patients. We compared the role of novel lymphocyte mitochondrial indicators and routine infection biomarkers as early predictors of severity and death in COVID-19 patients. We then developed a mortality decision tree prediction model based on immunosurveillance indicators through machine learning. RESULTS: At admission, the MM of circulating NK cells (NK-MM) was the best discriminator of severe/moderate disease (AUC = 0.8067) compared with the routine infection biomarkers. The NK cell count and NK-MM displayed superior diagnostic effects to distinguish patients with non-fatal or fatal outcomes. Interestingly, NK-MM was significantly polarized in non-survivors, with some patients showing a decrease and others showing an abnormal increase. Kaplan-Meier analysis showed that NK-MM had the optimal predictive efficacy (hazard ratio = 11.66). The decision tree model has the highest proportion of importance for NK-MM, which is superior to the single diagnostic effect of the above indicators (AUC = 0.8900). CONCLUSION: NK-MM was not only associated with disease severity, its abnormal increases or decreases also predicted mortality risk. The resulting decision tree prediction model is the first to focus on immune monitoring indicators to provide decision-making clues for COVID-19 clinical management.

Clinical efficacy of prostate PI-RADS V2.1 score combined with serum PSA-related indicators in the detection of gray zone prostate cancer.

PURPOSE: The purpose of this study is to improve the diagnostic accuracy of gray zone prostate cancer evaluation by combining the prostate imaging report and data system version 2.1 (PI-RADS V2.1) score with serum prostate-specific antigen (PSA). METHODS: We analyzed data from 212 men suspected of having prostate cancer and compared PSA-related indicators and PI-RADS V2.1 scores between 96 patients with prostate cancer and 116 without prostate cancer. By contrasting PI-RADS V2.1 scores with serum PSA-related markers, the diagnostic precision in the detection of grey zone prostate cancer was assessed. RESULTS: The median PI-RADS V2.1 scores and serum tPSA levels of patients with prostate cancer were significantly higher (P < 0.05). The PI-RADS V2.1 score correlated positively with serum tPSA, PSA density (PSAD), and prostate health index (PHI) levels (P < 0.05) and negatively correlated with fPSA/tPSA concentrations (P < 0.05). Logistic regression identified risk factors including family history, PI-RADS V2.1 score, tPSA, PSAD, and PHI, with prostate volume and fPSA/tPSA as protective factors (P < 0.05). Combining serum PSA-related indicators with the PI-RADS V2.1 score improved diagnostic accuracy for gray zone prostate cancer (AUC 0.986, specificity 99.14%, sensitivity 92.71%). CONCLUSION: The presence of a family history, a high PI-RADS V2.1 score, and elevated serum PSA-related markers contribute to high prostate cancer risk and development. The combined use of these indicators offers superior predictive value in detecting prostate cancer compared to a single indicator.

Assessing Transboundary Impacts of Energy-Driven Water Footprint on Scarce Water Resources in China: Catchments under Stress and Mitigation Options.

The energy supply chains operating beyond a region's jurisdiction can exert pressure on the availability of water resources in the local area. In China, however, there is a lack of transboundary assessments that investigate the effects of energy consumption on water stress within and across river basins. In this study, we therefore investigate transboundary impacts on scarce water resources that are induced by energy demands (i.e., electricity, petroleum, coal mining, oil and gas extraction, and gas production). We develop a bottom-up high spatial resolution water inventory and link it to a 2017 multiregional input-output (MRIO) table of China to analyze supply chain scarce water use at provincial and river basin levels. We find that the energy-driven water footprint accounts for 21.6% of national water usage, of which 35.7% is scarce water. Nonelectric power energy sectors contribute to around half of the nation's scarce water transfer. We identify three sets of catchments whose water resources are stressed by energy demand, i.e., (a) from the northern Hai River Basin to the eastern part of the Yellow River Basin and the Huai River Basin, (b) the northern area of the Northwest Rivers, and (c) the developed coastal city clusters in the Yangtze River Basin and the Pearl River Basin. We then evaluate the impacts of eight mitigation options, which may potentially shift around half of the moderate- or high-stress areas in the Hai River Basin and the Northwest Rivers to low to moderate (or even low) stress. We highlight the need for transboundary collaboration to sustain water-constrained energy demand and to develop targeted measures to mitigate stress on water resources within a river basin.

T cells: an emerging cast of roles in bipolar disorder.

Bipolar disorder (BD) is a distinctly heterogeneous and multifactorial disorder with a high individual and social burden. Immune pathway dysregulation is an important pathophysiological feature of BD. Recent studies have suggested a potential role for T lymphocytes in the pathogenesis of BD. Therefore, greater insight into T lymphocytes' functioning in patients with BD is essential. In this narrative review, we describe the presence of an imbalance in the ratio and altered function of T lymphocyte subsets in BD patients, mainly in T helper (Th) 1, Th2, Th17 cells and regulatory T cells, and alterations in hormones, intracellular signaling, and microbiomes may be potential causes. Abnormal T cell presence explains the elevated rates of comorbid inflammatory illnesses in the BD population. We also update the findings on T cell-targeting drugs as potentially immunomodulatory therapeutic agents for BD disease in addition to classical mood stabilizers (lithium, valproic acid). In conclusion, an imbalance in T lymphocyte subpopulation ratios and altered function may be involved in the development of BD, and maintaining T cell immune homeostasis may provide an overall therapeutic benefit.

Novel tumor therapy strategies targeting endoplasmic reticulum-mitochondria signal pathways.

Organelles form tight connections through membrane contact sites, thereby cooperating to regulate homeostasis and cell function. Among them, the contact between endoplasmic reticulum (ER), the main intracellular calcium storage organelles, and mitochondria has been recognized for decades, and its main roles in the ion and lipid transport, ROS signaling, membrane dynamic changes and cellular metabolism are basically determined. At present, many tumor chemotherapeutic drugs rely on ER-mitochondrial calcium signal to function, but the mechanism of targeting resident molecules at the mitochondria-associated endoplasmic reticulum membranes (MAM) to sensitize traditional chemotherapy and the new tumor therapeutic targets identified based on the signal pathways on the MAM have not been thoroughly discussed. In this review, we highlight the key roles of various signaling pathways at the ER-mitochondria contact site in tumorigenesis and focus on novel anticancer therapy strategies targeting potential targets at this contact site.

Paeonol alleviates neuropathic pain by modulating microglial M1 and M2 polarization via the RhoA/p38MAPK signaling pathway.

BACKGROUND: This study aimed to investigate the potential mechanism of paeonol in the treatment of neuropathic pain. METHODS: Relevant mechanisms were explored through microglial pseudotime analysis and the use of specific inhibitors in cell experiments. In animal experiments, 32 SD rats were randomly divided into the sham operation group, the chronic constrictive injury (CCI) group, the ibuprofen group, and the paeonol group. We performed behavioral testing, ELISA, PCR, Western blotting, immunohistochemistry, and immunofluorescence analysis. RESULTS: The pseudotime analysis of microglia found that RhoA, Rock1, and p38MAPK were highly expressed in activated microglia, and the expression patterns of these genes were consistent with the expression trends of the M1 markers CD32 and CD86. Paeonol decreased the levels of M1 markers (IL1ß, iNOS, CD32, IL6) and increased the levels of M2 markers (IL10, CD206, ARG-1) in LPS-induced microglia. The expression of iNOS, IL1ß, RhoA, and Rock1 was significantly increased in LPS-treated microglia, while paeonol decreased the expression of these proteins. Thermal hyperalgesia occurred after CCI surgery, and paeonol provided relief. In addition, paeonol decreased the levels of IL1ß and IL8 and increased the levels of IL4 and TGF-ß in the serum of CCI rats. Paeonol decreased expression levels of M1 markers and increased expression levels of M2 markers in the spinal cord. Paeonol decreased IBA-1, IL1ß, RhoA, RhoA-GTP, COX2, Rock1, and p-p38MAPK levels in the spinal dorsal horn. CONCLUSION: Paeonol relieves neuropathic pain by modulating microglial M1 and M2 phenotypes through the RhoA/p38 MAPK pathway.

Engineered human spinal cord-like tissues with dorsal and ventral neuronal progenitors for spinal cord injury repair in rats and monkeys.

Transplanting human neural progenitor cells is a promising method of replenishing the lost neurons after spinal cord injury (SCI), but differentiating neural progenitor cells into the diverse types of mature functional spinal cord neurons in vivo is challenging. In this study, engineered human embryonic spinal cord-like tissues with dorsal and ventral neuronal characters (DV-SC) were generated by inducing human neural progenitor cells (hscNPCs) to differentiate into various types of dorsal and ventral neuronal cells on collagen scaffold in vitro. Transplantation of DV-SC into complete SCI models in rats and monkeys showed better therapeutic effects than undifferentiated hscNPCs, including pronounced cell survival and maturation. DV-SC formed a targeted connection with the host's ascending and descending axons, partially restored interrupted neural circuits, and improved motor evoked potentials and the hindlimb function of animals with SCI. This suggests that the transplantation of pre-differentiated hscNPCs with spinal cord dorsal and ventral neuronal characteristics could be a promising strategy for SCI repair.

Spinal cord tissue engineering using human primary neural progenitor cells and astrocytes.

Neural progenitor cell (NPC) transplantation is a promising approach for repairing spinal cord injury (SCI). However, cell survival, maturation and integration after transplantation are still major challenges. Here, we produced a novel centimeter-scale human spinal cord neural tissue (hscNT) construct with human spinal cord neural progenitor cells (hscNPCs) and human spinal cord astrocytes (hscAS) on a linearly ordered collagen scaffold (LOCS). The hscAS promoted hscNPC adhesion, survival and neurite outgrowth on the LOCS, to form a linearly ordered spinal cord-like structure consisting of mature neurons and glia cells. When transplanted into rats with SCI, the hscNT created a favorable microenvironment by inhibiting inflammation and glial scar formation, and promoted neural and vascular regeneration. Notably, the hscNT promoted neural circuit reconstruction and motor functional recovery. Engineered human spinal cord implants containing astrocytes and neurons assembled on axon guidance scaffolds may therefore have potential in the treatment of SCI.

Air pollution control or economic development? Empirical evidence from enterprises with production restrictions.

Production restriction is an environmental regulation adopted in China to curb the air pollution of industrial enterprises. Frequent production restrictions may cause economic losses for enterprises and further hinder their green transformation. Polluting enterprises are faced with the dilemma of choosing environmental protection or economic development. Using panel data on industrial enterprises in China from 2016 to 2019, this paper evaluates the impact of production restrictions on both enterprises' environmental and economic performance with regression models. The results show that production restrictions significantly drop the concentrations of SO2 and NOx emitted from polluting enterprises. Meanwhile, production restrictions have significant negative effects on operating income, financial expenses, net profit, and environmental protection investment. The mechanism analysis reveals that production restrictions mitigate air pollutant concentrations by increasing the number of green patents and improving total factor productivity, which also verifies the Porter hypothesis. However, there is a masking mediating effect of environmental investment, which indicates that the reduction of environmental investment hinders the enterprise's efforts to control air pollution. In addition, heterogeneous analysis shows that the economic shock on microenterprises is larger than that on small enterprises. Implementing production restrictions for microenterprises may be a way to eliminate their backwards production capacity.

Ferulic acid alleviates sciatica by inhibiting neuroinflammation and promoting nerve repair via the TLR4/NF-κB pathway.

INTRODUCTION: Sciatica causes intense pain. No satisfactory therapeutic drugs exist to treat sciatica. This study aimed to probe the potential mechanism of ferulic acid in sciatica treatment. METHODS: Thirty-two SD rats were randomly divided into 4 groups: sham operation, chronic constriction injury (CCI), mecobalamin, and ferulic acid. We conducted RNA sequencing, behavioral tests, ELISA, PCR, western blotting, and immunofluorescence analysis. TAK-242 and JSH23 were administered to RSC96 and GMI-R1 cells to explore whether ferulic acid can inhibit apoptosis and alleviate inflammation. RESULTS: RNA sequencing showed that TLR4/NF-κB pathway is involved in the mechanism of sciatica. CCI induced cold and mechanical hyperalgesia; destroyed the sciatic nerve structure; increased IL-1ß, IL-6, TNF-α, IL-8, and TGF-ß protein levels and IL-1ß, IL-6, TNF-α, TGF-ß, TLR4, and IBA-1 mRNA levels; and decreased IL-10 and INF-γ protein levels and IL-4 mRNA levels. Immunohistochemistry showed that IBA-1, CD32, IL-1ß, iNOS, nNOS, COX2, and TLR4 expression was increased while S100ß and Arg-1 decreased. CCI increased TLR4, IBA-1, IL-1ß, iNOS, Myd88, p-NF-κB, and p-p38MAPK protein levels. Treatment with mecobalamin and ferulic acid reversed these trends. Lipopolysaccharide (LPS) induced RSC96 cell apoptosis by reducing Bcl-2 and Bcl-xl protein and mRNA levels and increasing Bax and Bad mRNA and IL-1ß, TLR4, Myd88, p-NF-κB, and p-p38MAPK protein levels, while ferulic acid inhibited cell apoptosis by decreasing IL-1ß, TLR4, Myd88, p-NF-κB, and p-p38MAPK levels and increasing Bcl-2 and Bcl-xl levels. In GMI-R1 cells, Ferulic acid attenuated LPS-induced M1 polarization by decreasing the M1 polarization markers IL-1ß, IL-6, iNOS, and CD32 and increasing the M2 polarization markers CD206, IL-4, IL-10 and Arg-1. After LPS treatment, IL-1ß, iNOS, TLR4, Myd88, p-p38MAPK, and p-NF-κB levels were obviously increased, and Arg-1 expression was reduced, while ferulic acid reversed these changes. CONCLUSION: Ferulic acid can promote injured sciatic nerve repair by reducing neuronal cell apoptosis and inflammatory infiltration though the TLR4/NF-κB pathway.

Identification of pyroptosis­related genes in neuropathic pain based on bioinformatics analysis.

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 ß (S100ß) 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 sS100ß 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.