Rea regulates microglial polarization and attenuates neuronal apoptosis via inhibition of the NF-κB and MAPK signalings for spinal cord injury repair.

Inflammation and neuronal apoptosis aggravate the secondary damage after spinal cord injury (SCI). Rehmannioside A (Rea) is a bioactive herbal extract isolated from Rehmanniae radix with low toxicity and neuroprotection effects. Rea treatment inhibited the release of pro-inflammatory mediators from microglial cells, and promoted M2 polarization in vitro, which in turn protected the co-cultured neurons from apoptosis via suppression of the NF-κB and MAPK signalling pathways. Furthermore, daily intraperitoneal injections of 80 mg/kg Rea into a rat model of SCI significantly improved the behavioural and histological indices, promoted M2 microglial polarization, alleviated neuronal apoptosis, and increased motor function recovery. Therefore, Rea is a promising therapeutic option for SCI and should be clinically explored.

Duraplasty of PHBV/PLA/Col membranes promotes axonal regeneration by inhibiting NLRP3 complex and M1 macrophage polarization in rats with spinal cord injury.

Duraplasty after decompression decreases the lesion size and scar formation, promoting better functional recovery, but the underlying mechanism has not been clarified. Here, we fabricated a series of poly(hydroxybutyrate-co-hydroxyvalerate)/polylactic acid/collagen (PHBV/PLA/Col) membranes and cultured them with VSC4.1 motor neurons. The material characteristics and in vitro biological characteristics were evaluated. In the subcutaneous implantation test, PHBV/PLA/COl scaffolds supported the cellular infiltration, microvasculature formation, and decreased CD86-positive macrophage aggregation. Following contusion spinal cord injury at T10 in Sprague-Dawley rats, durotomy was performed with allograft dura mater or PHBV/PLA or PHBV/PLA/Col membranes. At 3 days post-injury, Western blot assay showed decreased the expression of the NLRP3, ASC, cleaved-caspase-1, IL-1ß, TNF-α, and CD86 expression but increased the expression of CD206. Immunofluorescence demonstrated that duraplasty with PHBV/PLA/Col membranes reduced the infiltration of CD86-positive macrophages in the lesion site, decreased the glial fibrillary acidic protein expression, and increased the expression of NF-200. Moreover, duraplasty with PHBV/PLA/Col membranes improved locomotor functional recovery at 8 weeks post-injury. Thus, duraplasty with PHBV/PLA/Col membranes decreased the glial scar formation and promoted axon growth by inhibiting inflammasome activation and modulating macrophage polarization in acute spinal cord injury.

Injectable exosome-functionalized extracellular matrix hydrogel for metabolism balance and pyroptosis regulation in intervertebral disc degeneration.

Exosome therapy is a promising therapeutic approach for intervertebral disc degeneration (IVDD) and achieves its therapeutic effects by regulating metabolic disorders, the microenvironment and cell homeostasis with the sustained release of microRNAs, proteins, and transcription factors. However, the rapid clearance and disruption of exosomes are the two major challenges for the application of exosome therapy in IVDD. Herein, a thermosensitive acellular extracellular matrix (ECM) hydrogel coupled with adipose-derived mesenchymal stem cell (ADSC) exosomes (dECM@exo) that inherits the superior properties of nucleus pulposus tissue and ADSCs was fabricated to ameliorate IVDD. This thermosensitive dECM@exo hydrogel system can provide not only in situ gelation to replenish ECM leakage in nucleus pulposus cells (NPCs) but also an environment for the growth of NPCs. In addition, sustained release of ADSC-derived exosomes from this system regulates matrix synthesis and degradation by regulating matrix metalloproteinases (MMPs) and inhibits pyroptosis by mitigating the inflammatory response in vitro. Animal results demonstrated that the dECM@exo hydrogel system maintained early IVD microenvironment homeostasis and ameliorated IVDD. This functional system can serve as a powerful platform for IVD drug delivery and biotherapy and an alternative therapy for IVDD.

Scar Tissue-Targeting Polymer Micelle for Spinal Cord Injury Treatment.

Spinal cord injury (SCI) is a devastating disorder, leading to permanent motor and sensory deficit. Despite recent advances in neurosciences, the treatment efficacy on SCI patients remains unsatisfactory, mainly due to the poor accumulation, short retention, and lack of controlled release of therapeutics in lesion tissue. Herein, an injured spinal cord targeting prodrug polymer micelle is built. An esterase-responsive bond is used to link apocynin (APO) monomer, because of the enhanced esterase activity found in microglia cells after activation, which ensures a controlled degradation of APO prodrug (Allyloxypolyethyleneglycol-b-poly [2-(((4-acetyl-2-methoxyphenoxy)carbonyl)oxy)ethyl methacrylate], APEG-PAPO or PAPO) by activated microglia cells. A scar tissue-homing peptide (cysteine-alanine-glutamine-lysine, CAQK) is introduced to the PAPO to endow the polymer micelle the lesion tissue-targeting ability. As a result, this CAQK-modified prodrug micelle (cPAM) exhibits an improved accumulation and prolonged retention in lesion tissue compared to the control micelle. The cPAM also leads to superior tissue protection and sustained motor function recovery than the control groups in a mouse model of SCI. In conclusion, the cPAM induces an effective treatment of SCI by the lesion tissue specific delivery of the prodrug polymer via its robust scar binding effect, making the scar tissue a drug releasing platform for sustained treatment of SCI.

An injectable recombinant human milk fat globule-epidermal growth factor 8-loaded copolymer system for spinal cord injury reduces inflammation through NF-κB and neuronal cell death.

Spinal cord injury (SCI) is a common disease and a major cause of paralysis, carrying much burden around the world. Despite the progress made with growth factors therapy, the response rate of acute SCI treatment still remains unsatisfactory, due largely to complex and severe inflammatory reactions. Herein, we prepare a MFG-E8-loaded copolymer system-based anti-inflammation therapy for SCI treatment. It is shown that the MFG-E8-loaded copolymer system can decrease pro-inflammatory cytokine expression and neuron death. In a rat model of crush-caused SCI, the copolymer system shows significant therapeutic efficacy by ameliorating inflammation, decreasing fibrotic scar, promoting myelin regeneration and suppressing overall SCI severity.

Comparative transcriptomics reveals eyestalk ablation induced responses of the neuroendocrine-immune system in the Pacific white shrimp Litopenaeus vannamei.

In decapod crustaceans, eyestalk ablation is widely used to expedite ovarian maturation and spawning because of the removal of a gonad inhibiting hormone produced by the X-organ sinus gland. However, eyestalk ablation also results in negative impacts on the immunocompetence of the eyestalk-ablated females. In the current study, we investigated the impact of eyestalk ablation on the transcriptomic responses of three major nervous organs of shrimp, including the eyestalk ganglion, brain and thoracic ganglion, using the Illumina Hiseq™ 4000 platform. A total of 48,249 unigenes with an average length of 1253 bp and a N50 value of 2482 bp were obtained. Following eyestalk ablation treatment, a total of 2,983, 6325 and 6575 unigenes were detected as differentially expressed (log2Ratio >1 and FDR <0.05) from the eyestalk, brain and thoracic ganglia, respectively. Functional GO and KEGG analysis of these differential expression genes (DEGs) showed that these DEGs were associated with a wide variety of biological processes and pathways. The distribution of DEGs among three comparison groups was similar, and many DEGs were mapped to the phagosome pathway, indicating that eyestalk ablation triggers activation of the neuroendocrine-immune (NEI) system. Interestingly, several important pathways were uniquely enriched in the brain tissue, suggesting that the brain may play a crucial role in the NEI system in response to eyestalk ablation. This is the first report on the transcriptomic regulation of the nervous system in response to eyestalk ablation in L. vannamei. The genes and pathways identified in this study will help to elucidate the molecular mechanisms of neuroendocrine-immune responses to eyestalk ablation in penaeid shrimp.

Research into the hemocyte immune response of Fenneropenaeus merguiensis under decapod iridescent virus 1 (DIV1) challenge using transcriptome analysis.

The banana shrimp (Fenneropenaeus merguiensis) is a common cultural species worldwide. With the development of the shrimp farming industry, increasing number of diseases have emerged and cause huge impacts. Decapod iridescent virus 1 (DIV1) is a new virus of the family Iridoviridae isolated in China that causes very high mortality in shrimp. In this study, DIV1 and PBS were injected into two groups of shrimp, and hemocytes were collected for comparative transcriptomic analysis. We confirmed that F. merguiensis was the new host of DIV1 by nested PCR. A total of 100,759 unigenes were assembled from the control group and the DIV1 infected group, with an average length of 733.06 bp and N50 of 1136 bp. Significant hits were found in 21,465 unigenes compared to known sequences in major databases including COG (33.30%), GO (42.17%), KEGG (46.76%), KOG (61.37%), Pfam (66.90%), Swissprot (54.21%) and Nr (93.86%). A total of 1003 differentially expressed genes (DEGs) were identified, including 929 up-regulated genes and 74 down-regulated genes. Several known immune-related genes, including caspase, C-type lectin, Wnt5 and integrin, were among the differentially expressed transcripts. A total of 14,459 simple sequence repeats, including 8128 monomers, 3276 dimers, 1693 trimers, 150 quadmers, 4 pentamers and 16 hexamers, were found in the transcriptomic dataset. Our study is the first comprehensive investigation of the transcriptomic response to DIV1 infection in F. merguiensis. Collectively, these results not only provide valuable information for characterizing the immune mechanisms of the shrimp responses to DIV1 infection, they open new ways for the study of the molecular mechanisms of DIV1 infection in F. merguiensis.

Melatonin protects vertebral endplate chondrocytes against apoptosis and calcification via the Sirt1-autophagy pathway.

Melatonin is reportedly associated with intervertebral disc degeneration (IDD). Endplate cartilage is vitally important to intervertebral discs in physiological and pathological conditions. However, the effects and mechanism of melatonin on endplate chondrocytes (EPCs) are still unclear. Herein, we studied the effects of melatonin on EPC apoptosis and calcification and elucidated the underlying mechanism. Our study revealed that melatonin treatment decreases the incidence of apoptosis and inhibits EPC calcification in a dose-dependent manner. We also found that melatonin upregulates Sirt1 expression and activity and promotes autophagy in EPCs. Autophagy inhibition by 3-methyladenine reversed the protective effect of melatonin on apoptosis and calcification, while the Sirt1 inhibitor EX-527 suppressed melatonin-induced autophagy and the protective effects of melatonin against apoptosis and calcification, indicating that the beneficial effects of melatonin in EPCs are mediated through the Sirt1-autophagy pathway. Furthermore, melatonin may ameliorate IDD in vivo in rats. Collectively, this study revealed that melatonin reduces EPC apoptosis and calcification and that the underlying mechanism may be related to Sirt1-autophagy pathway regulation, which may help us better understand the association between melatonin and IDD.

Functional characterization of a protein inhibitor of activated STAT (PIAS) gene in Litopenaeus vannamei.

Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway as a negative regulator in mammals and Drosophila, but the function of PIAS in crustaceans is still unclear. In this study, a PIAS termed LvPIAS was cloned and characterized from Litopenaeus vannamei. The full length of LvPIAS was 3065 bp, including a 2361 bp open reading frame (ORF) coding for a protein of 786 aa. LvPIAS expression was most abundant in muscle and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly I: C and white spot syndrome virus (WSSV). LvPIAS could be induced by the transcription factor LvSTAT, but LvPIAS could inhibit the transcriptional activity of LvSTAT to the LvPIAS promoter conversely, which indicated that there was a negative feedback loop between LvSTAT and LvPIAS. Furthermore, RNAi-mediated knockdown of LvPIAS shrimps showed higher survival rate to WSSV infection than those in the control group (dsGFP injection), suggesting that LvPIAS may play a negatively role against WSSV infection.

Potential role for microRNA in facilitating physiological adaptation to hypoxia in the Pacific whiteleg shrimp Litopenaeus vannamei.

Hypoxia is one of the most common physiological stressors in shrimp farming. Post-transcriptional regulation by microRNAs has been recognized as a ubiquitous strategy to enable transient phenotypic plasticity and adaptation to stressful environment, but involvement of microRNAs in hypoxia stress response of penaeid shrimp remains elusive. In this study, small RNA sequencing and comparative transcriptomic analysis was conducted to construct a comprehensive microRNA dataset for the whiteleg shrimp Litopenaeus vannamei exposed to hypoxia challenge. A total of 3324 known miRNAs and 8 putative novel miRNAs were identified, providing a valuable resource for future investigation on the functional mechanism of miRNAs in shrimp. Upon hypoxia, 1213 miRNAs showed significant differential expression, and many well-known miRNAs involved in hypoxia tolerance such as miR-210, let-7, miR-143 and miR-101 were identified. Remarkably, the vast majority of these miRNAs were up-regulated, suggesting that up-regulation of miRNAs may represent an effective strategy to inhibit protein translation under stressful hypoxic condition. The differentially expressed miRNAs were potentially targeting a wide variety of genes, including those with essential roles in hypoxia tolerance such as HIF1a and p53. GO and KEGG enrichment analysis further revealed that a broad range of biological processes and metabolic pathways were over-represented. Several GO terms associated with gene transcription and translation and KEGG pathways related to cytoskeleton remodeling, immune defense and signaling transduction were enriched, highlighting the crucial roles of these cellular events in the adaptation to hypoxia. Taken together, our study revealed that the differentially expressed miRNAs may regulate host response to hypoxia by modulating the expression of stress response genes such as HIF1a and p53 and affecting key cellular events involved in hypoxia adaptation. The findings would expand our knowledge of the biochemical and molecular underpinnings of hypoxia response strategies used by penaeid shrimp, and contribute to a better understanding of the molecular mechanisms of hypoxia tolerance in decapod crustaceans.

Monotropein promotes angiogenesis and inhibits oxidative stress-induced autophagy in endothelial progenitor cells to accelerate wound healing.

Attenuating oxidative stress-induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress-induced mitochondrial dysfunction and stimulate bone marrow-derived EPC (BM-EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM-EPCs and prevented tert-butyl hydroperoxide (TBHP)-induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM-EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury-related wounds.

Salidroside attenuates neuroinflammation and improves functional recovery after spinal cord injury through microglia polarization regulation.

Spinal cord injury (SCI) is a severe neurological disease; however, few drugs have been proved to treat SCI effectively. Neuroinflammation is the major pathogenesis of SCI secondary injury and considered to be the therapeutic target of SCI. Salidroside (Sal) has been reported to exert anti-inflammatory effects in airway, adipose and myocardial tissue; however, the role of Sal in SCI therapeutics has not been clarified. In this study, we showed that Sal could improve the functional recovery of spinal cord in rats as revealed by increased BBB locomotor rating scale, angle of incline, and decreased cavity of spinal cord injury and apoptosis of neurons in vivo. Immunofluorescence double staining of microglia marker and M1/M2 marker demonstrated that Sal could suppress M1 microglia polarization and activate M2 microglia polarization in vivo. To verify how Sal exerts its effects on microglia polarization and neuron protection, we performed the mechanism study in vitro in microglia cell line BV-2 and neuron cell line PC12. The results showed that Sal prevents apoptosis of PC12 cells in coculture with LPS-induced M1 BV-2 microglia, also the inflammatory secretion phenotype of M1 BV-2 microglia was suppressed by Sal, and further studies demonstrated that autophagic flux regulation through AMPK/mTOR pathway was involved in Sal regulated microglia polarization after SCI. Overall, our study illustrated that Sal could promote spinal cord injury functional recovery in rats, and the mechanism may relate to its microglia polarization modulation through AMPK-/mTOR-mediated autophagic flux stimulation.

Differential expression of microRNAs in hemocytes from white shrimp Litopenaeus vannamei under copper stress.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate diverse cellular processes, including organismal stress response, through posttranscriptional repression of gene transcripts. They are known to have antiviral functions in aquatic crustacean species, but little is known about the role of miRNAs against environmental stress caused by Cu, a common chemical contaminant in aquatic environment. We performed small RNA sequencing to characterize the differentially expressed microRNAs in Cu exposed shrimp. A total of 4524 known miRNAs and 73 novel miRNAs were significantly (P < .05) differentially expressed after Cu exposure. The peak size of miRNAs was 22 nt. Among them, 218 miRNAs were conserved across 115 species. The validation of 12 miRNAs by stem-loop quantitative RT-PCR were found to be coherent with the expression profile of deep sequencing data as evaluated with Pearson's correlation coefficient (r = 0.707). Target genes of these differentially expressed miRNAs related to immune defense, apoptosis, and xenobiotics metabolism also showed significant changes in expression under Cu stress. The present study provides the first characterization of L. vannamei miRNAs and some target genes expression in response to Cu stress, and the findings support the hypothesis that certain miRNAs along with their target genes might be essential in the intricate adaptive response regulation networks. Our current study will provide valuable information to take an insight into molecular mechanism of L. vannamei against environmental stress.

Asperosaponin VI promotes angiogenesis and accelerates wound healing in rats via up-regulating HIF-1α/VEGF signaling.

Wound therapy remains a clinical challenge due to the complexity of healing pathology and high demand of achieving functional and aesthetically satisfactory scars. Newly formed blood vessels are essential for tissue repair since they can support cells at the wound site with nutrition and oxygen. In this study, we investigated the effects of Asperosaponin VI (ASA VI) isolated from a traditional Chinese medicine, the root of Dipsacus asper Wall, in promoting angiogenesis, as well as its function in wound therapeutics. Treatment of human umbilical vein endothelial cells (HUVECs) with ASA VI (20-80 µg/mL) dose-dependently promoted the proliferation, migration and enhanced their angiogenic ability in vitro, which were associated with the up-regulated HIF-1α/VEGF signaling. Full-thickness cutaneous wound model rats were injected with ASA VI (20 mg·kg-1·d-1, iv) for 21 d. Administration of ASA VI significantly promoted the cutaneous wound healing, and more blood vessels were observed in the regenerated tissue. Due to rapid vascularization, the cellular proliferation status, granulation tissue formation, collagen matrix deposition and remodeling processes were all accelerated, resulting in efficient wound healing. In summary, ASA VI promotes angiogenesis of HUVECs in vitro via up-regulating the HIF-1α/VEGF pathway, and efficiently enhances the vascularization in regenerated tissue and facilitates wound healing in vivo. The results reveal that ASA VI is a potential therapeutic for vessel injury-related wounds.

Metformin ameliorates BSCB disruption by inhibiting neutrophil infiltration and MMP-9 expression but not direct TJ proteins expression regulation.

Blood-spinal cord barrier (BSCB) disruption is a major process for the secondary injury of spinal cord injury (SCI) and is considered to be a therapeutic target for SCI. Previously, we demonstrated that metformin could improve functional recovery after SCI; however, the effect of metformin on BSCB is still unknown. In this study, we found that metformin could prevent the loss of tight junction (TJ) proteins at day 3 after SCI in vivo, but in vitro there was no significant difference of these proteins between control and metformin treatment in endothelial cells. This indicated that metformin-induced BSCB protection might not be mediated by up-regulating TJ proteins directly, but by inhibiting TJ proteins degradation. Thus, we investigated the role of metformin on MMP-9 and neutrophils infiltration. Neutrophils infiltration is the major source of the enhanced MMP-9 in SCI. Our results showed that metformin decreased MMP-9 production and blocked neutrophils infiltration at day 1 after injury, which might be related to ICAM-1 down-regulation. Also, our in vitro study showed that metformin inhibited TNF-α-induced MMP-9 up-regulation in neutrophils, which might be mediated via an AMPK-dependent pathway. Together, it illustrated that metformin prevented the breakdown of BSCB by inhibiting neutrophils infiltration and MMP-9 production, but not by up-regulating TJ proteins expression. Our study may help to better understand the working mechanism of metformin on SCI.

Gill transcriptomes reveal involvement of cytoskeleton remodeling and immune defense in ammonia stress response in the banana shrimp Fenneropenaeus merguiensis.

The banana shrimp, Fenneropenaeus merguiensis, is an important fishery species in the Indo-West Pacific region. As the shrimp is very sensitive to stressors such as ammonia stress in water, understanding the molecular mechanisms of stress tolerance in F. merguiensis is of pivotal importance for improving its farming performance. In the current study, by using the RNA sequencing platform and comparative transcriptomic analysis, we conducted a comprehensive study on the transcriptomic changes of F. merguiensis in response to ammonia stress. A total of 106,996 unique transcripts (or unigenes) with an average length of 672 bp and a N50 value of 1164 bp were recovered, and a large number of potential SSR loci in the transcriptome were identified. Totally, 55,529 transcripts can find significant hits when compared to known sequences in major databases including the nr, nt, SWISSPROT, GO, COG, and KEGG databases. Analysis of differential gene expression between the ammonia-challenged group and the control group revealed that 9190 annotated transcripts were differentially expressed upon ammonia exposure. Among them, 3712 were significantly induced while 5478 of them were repressed. Functional enrichment analysis of these differentially expressed genes further showed that 22 Gene Ontology terms and 62 KEGG pathways were significantly over-represented. Remarkably, many of the genes showing the largest magnitude of expression changes were related to cytoskeleton remodeling and immune response, highlighting the involvement of these biological processes in the ammonia stress response of F. merguiensis. Our study is the first comprehensive investigation on the transcriptomic response to ammonia stress in F. merguiensis. The genes and pathways identified here not only represent valuable genetic resources for development of molecular markers and genetic breeding studies, but open new avenues for studies on the molecular mechanisms of ammonia stress tolerance in penaeid shrimp.

Comparison of traditional surgery and surgery assisted by three dimensional printing technology in the treatment of tibial plateau fractures.

PURPOSE: This study was conducted to compare traditional surgery and surgery assisted by 3D printing technology in the treatment of tibial plateau fractures. In addition, we also investigated the effect of 3D printing technology on the communication between doctors and patients. METHODS: Seventy two patients with tibial plateau fractures were enrolled in the study from April 2014 to October 2015. They were divided into two groups: 34 cases of 3D model group, 38 cases of traditional surgery group. The individual models were used to simulate the surgical procedures and carry out the surgery according to plan. Operation time, blood loss, and number of intra-operative fluoroscopy were recorded. Through the follow-up, the recovery of patients were observed. Besides, we designed questionnaires to verify the satisfaction for both surgeons and patients. RESULTS: The average operation time, average amount of blood loss, and number of intra-operative fluoroscopy for 3D model group was 85.2±0.9 minutes, 186.3± 5.5ml, 5.3± 0.2 times, and for traditional surgery group was 99.2±1.0 minutes, 216.2 ±6.9 ml,7.1 ± 0.2 times respectively. There was statistically significant difference between the traditional surgery group and 3D model group (P < 0.05). Via follow-up, we can see that the 3D printing group has a better clinical efficacy. The average score of the questionnaires to Patient and doctors were 7.3 ± 0.1 points and 8.5± 0.1 points respectively. CONCLUSION: This study suggested the clinical feasibility of 3D printing technology in treatment of tibial plateau fractures.

Ginsenoside-Rh2 Promotes Functional Recovery after Spinal Cord Injury by Enhancing TFEB-Mediated Autophagy.

Background: Following spinal cord injury (SCI), autophagy plays a positive role in neuronal protection, whereas pyroptosis triggers an inflammatory response. Ginsenoside-Rh2 (GRh2), known for its neuroprotective effects, is considered a promising drug. However, the exact molecular mechanisms underlying these protective effects remain unclear. Aim of the Study: Explore the therapeutic value of GRh2 in SCI and its potential mechanisms of action. Materials and Methods: An SCI mouse model was established, followed by random grouping and drug treatments under different conditions. Subsequently, the functional recovery of SCI mice after GRh2 treatment was assessed using hematoxylin and eosin, Masson's trichrome, and Nissl staining, footprint analysis, Basso Mouse Scale scoring, and inclined plane tests. The expression levels of relevant indicators in the mice were detected using Western blotting, immunofluorescence, and a quantitative polymerase chain reaction. Network pharmacology analysis was used to identify the relevant signaling pathways through which GRh2 exerts its therapeutic effects. Results: GRh2 promoted functional recovery after SCI. GRh2 significantly inhibits pyroptosis by enhancing autophagy in SCI mice. Simultaneously, the neuroprotective effect of GRh2, achieved through the inhibition of pyroptosis, is partially reversed by 3-methyladenine, an autophagy inhibitor. Additionally, the increase in autophagy induced by GRh2 is mediated by the promotion of transcription factor EB (TFEB) nuclear translocation and dephosphorylation. Partial attenuation of the protective effects of GRh2 was observed after TFEB knockdown. Additionally, GRh2 can modulate the activity of TFEB in mice post-SCI through the EGFR-MAPK signaling pathway, and NSC228155 (an EGFR activator) can partially reverse the effect of GRh2 on the EGFR-MAPK signaling pathway. Conclusions: GRh2 improves functional recovery after SCI by upregulating TFEB-mediated autophagic flux and inhibiting pyroptosis, indicating its potential clinical applicability.

Anlotinib potentiates anti-PD1 immunotherapy via transferrin receptor-dependent CD8+ T-cell infiltration in hepatocellular carcinoma.

BACKGROUND: The therapeutic potential of immune checkpoint blockade (ICB) extends across various cancers; however, its effectiveness in treating hepatocellular carcinoma (HCC) is frequently curtailed by both inherent and developed resistance. OBJECTIVE: This research explored the effectiveness of integrating anlotinib (a broad-spectrum tyrosine kinase inhibitor) with programmed death-1 (PD-1) blockade and offers mechanistic insights into more effective strategies for treating HCC. METHODS: Using patient-derived organotypic tissue spheroids and orthotopic HCC mouse models, we assessed the effectiveness of anlotinib combined with PD-1 blockade. The impact on the tumour immune microenvironment and underlying mechanisms were assessed using time-of-flight mass cytometry, RNA sequencing, and proteomics across cell lines, mouse models, and HCC patient samples. RESULTS: The combination of anlotinib with an anti-PD-1 antibody enhanced the immune response against HCC in preclinical models. Anlotinib remarkably suppressed the expression of transferrin receptor (TFRC) via the VEGFR2/AKT/HIF-1α signaling axis. CD8+ T-cell infiltration into the tumour microenvironment correlated with low expression of TFRC. Anlotinib additionally increased the levels of the chemokine CXCL14, crucial for attracting CD8+ T cells. CXCL14 emerged as a downstream effector of TFRC, exhibiting elevated expression following the silencing of TFRC. Importantly, low TFRC expression was also associated with a better prognosis, enhanced sensitivity to combination therapy, and a favourable response to anti-PD-1 therapy in patients with HCC. CONCLUSIONS: Our findings highlight anlotinib's potential to augment the efficacy of anti-PD-1 immunotherapy in HCC by targeting TFRC and enhancing CXCL14-mediated CD8+ T-cell infiltration. This study contributes to developing novel therapeutic strategies for HCC, emphasizing the role of precision medicine in oncology. HIGHLIGHTS: Synergistic effects of anlotinib and anti-PD-1 immunotherapy demonstrated in HCC preclinical models. Anlotinib inhibits TFRC expression via the VEGFR2/AKT/HIF-1α pathway. CXCL14 upregulation via TFRC suppression boosts CD8+ T-cell recruitment. TFRC emerges as a potential biomarker for evaluating prognosis and predicting response to anti-PD-1-based therapies in advanced HCC patients.