Transient receptor potential vanilloid four channels modulate inhibitory inputs through differential regulation of GABA and glycine receptors in rat retinal ganglion cells.

The transient receptor potential vanilloid 4 (TRPV4) channel is widely distributed in the retina. Activation of the TRPV4 channel enhances excitatory signaling from bipolar cells to retinal ganglion cells (RGCs), thereby increasing RGC firing rate and membrane excitability. In this study, we investigated the effect of TRPV4 channel activation on the miniature inhibitory postsynaptic current (mIPSC) in rat RGCs. Our results showed that perfusion with HC-067047, a TRPV4-channel antagonist, significantly reduced the amplitude of RGC mIPSCs. Extracellular application of the TRPV4 channel agonist GSK1016790A (GSK101) enhanced the frequency and amplitude of mIPSCs in ON- and OFF-type RGCs; pre-application of HC-067047 blocked the effect of GSK101 on mIPSCs. Furthermore, TRPV4 channels were able to enhance the frequency and amplitude of glycine receptor (GlyR)-mediated mIPSCs and inhibit the frequency of type A γ-aminobutyric acid receptor (GABAA R)-mediated mIPSCs. Upon intracellular administration or intravitreal injection of GSK101, TRPV4 channel activation reduced the release of presynaptic glycine and enhanced the function and expression of postsynaptic GlyRs; however, it inhibited presynaptic release of GABA, but did not affect postsynaptic GABAA Rs. Our study results provide insight regarding the effect of TRPV4 channel activation on RGCs and offer a potential interventional target for retinal diseases involving TRPV4 channels.

Exploring the Targets and Molecular Mechanisms of Thalidomide in the Treatment of Ulcerative Colitis: Network Pharmacology and Experimental Validation.

BACKGROUND: Ulcerative colitis (UC) is a chronic, nonspecific, inflammatory disease of the intestine with an unknown cause. Thalidomide (THA) has been shown to be an effective drug for the treatment of UC. However, the molecular targets and mechanism of action of THA for the treatment of UC are not yet clear. OBJECTIVES: Combining network pharmacology with in vitro experiments, this study aimed to investigate the potential targets and molecular mechanisms of THA for the treatment of UC. METHODS: Firstly, relevant targets of THA against UC were obtained from public databases. Then, the top 10 hub targets and key molecular mechanisms of THA for UC were screened based on the network pharmacology approach and bioinformatics method. Finally, an in vitro cellular inflammation model was constructed using lipopolysaccharide (LPS) induced intestinal epithelial cells (NCM460) to validate the top 10 hub targets and key signaling pathways. RESULTS: A total of 121 relevant targets of THA against UC were obtained, of which the top 10 hub targets were SRC, LCK, MAPK1, HSP90AA1, EGFR, HRAS, JAK2, RAC1, STAT1, and MAP2K1. The PI3K-Akt pathway was significantly associated with THA treatment of UC. In vitro experiments revealed that THA treatment reversed the expression of HSP90AA1, EGFR, STAT1, and JAK2 differential genes. THA was able to up- regulate the mRNA expression of pro-inflammatory factor IL-10 and decrease the mRNA levels of anti-inflammatory factors IL-6, IL-1ß, and TNF-α. Furthermore, THA also exerted anti-inflammatory effects by inhibiting the activation of the PI3K/Akt pathway. CONCLUSION: THA may play a therapeutic role in UC by inhibiting the PI3K-Akt pathway. HSP90AA1, EGFR, STAT1, and JAK2 may be the most relevant potential therapeutic targets for THA in the treatment of UC.

USP20 is a predictor of poor prognosis in colorectal cancer and associated with lymph node metastasis, immune infiltration and chemotherapy resistance.

Background: Colorectal cancer (CRC) is a highly prevalent malignancy with a poor prognosis. USP20 can support progression of variety of tumors. USP20 was shown to promote breast tumor metastasis, and proliferation of oral squamous carcinoma cells. However, the role of USP20 in CRC remains unclear. Methods: We used bioinformatics to analyze the expression and prognosis of USP20 in pan-cancer and explore the relationship between USP20 expression and immune infiltration, immune checkpoints, and chemotherapy resistance in CRC. The differential expression and prognostic role of USP20 in CRC was validated by qRT-PCR and immunohistochemistry. Cox univariate and multivariate analyses were performed to assess risk factors for poor prognosis of CRC, and new prognostic prediction models were constructed and evaluated by decision curve analysis (ROC) and receiver operating characteristic (DCA). USP20 was overexpressed in CRC cell lines to explore the effect of USP20 on the functionalities of CRC cells. Enrichment analyses were used to explore the possible mechanism of USP20 in CRC. Results: The expression of USP20 was lower in CRC tissues than adjacent normal tissues. Compared with low USP20 expression patients, CRC patients with high USP20 expression level had shorter OS. Correlation analysis showed that USP20 expression was associated with lymph node metastasis. Cox regression analysis revealed USP20 as an independent risk factor for poor prognosis in CRC patients. ROC and DCA analyses showed that the performance of the newly constructed prediction model was better than the traditional TNM model. Immune infiltration analysis shown that USP20 expression is closely associated with T cell infiltration in CRC. A co-expression analysis showed that USP20 expression was positively correlated with several immune checkpoint genes including ADORA2A, CD160, CD27 and TNFRSF25 genes and positively associated with multiple multi-drug resistance genes such as MRP1, MRP3, and MRP5 genes. USP20 expression positively correlated with the sensitivity of cells to multiple anticancer drugs. Overexpression of USP20 enhanced the migration and invasive ability of CRC cells. Enrichment pathway analyses showed the USP20 may play a role via the Notch pathway, Hedgehog pathway and beta-catenin pathway. Conclusion: USP20 is downregulated in CRC and associated with prognosis in CRC. USP20 enhances CRC cells metastasis and is associated with immune infiltration, immune checkpoints, and chemotherapy resistance.

The E3 ubiquitin ligase RNF31 mediates the development of ulcerative colitis by regulating NLRP3 inflammasome activation.

Ulcerative colitis (UC) is characterized by dysregulated inflammation and disruption of the intestinal barrier. The NLRP3 inflammasome, which is composed of NLRP3, ASC, and caspase-1, plays a crucial role in UC pathogenesis by triggering the production of proinflammatory cytokines. In this study, we investigated the regulatory role of RNF31 in NLRP3 inflammasome activation during UC development. Through comprehensive analysis of ulcerative colitis tissues using the GEO database and immunohistochemistry, we found that RNF31 expression was elevated in UC tissues, which prompted further investigation into its function. We constructed an RNF31 knockdown cell model and observed a significant reduction in NLRP3 inflammasome activation, indicating the involvement of RNF31 in regulating NLRP3. Mechanistically, RNF31 could interact with NLRP3 through the RBR structural domain, leading to increased K63-linked ubiquitination of NLRP3 and consequent stabilization. Coimmunoprecipitation experiments revealed a mutual interaction between RNF31 and NLRP3, substantiating their functional association. Finally, an in vivo mouse model with RNF31 knockdown showed a notable reduction in NLRP3 expression, which was accompanied by a decrease in the proinflammatory cytokines IL-18 and IL-1ß. The successful attenuation of DSS-induced tissue inflammation by this treatment confirmed the physiological relevance of RNF31-mediated regulation of NLRP3. This study unveils a novel regulatory pathway by which RNF31 affects NLRP3 inflammasome activation, providing new insights into UC pathogenesis and potential therapeutic targets for UC treatment.

MiR-22-3p regulates the proliferation, migration and invasion of colorectal cancer cells by directly targeting KDM3A through the Hippo pathway.

Colorectal cancer (CRC) has one of the highest incidences and mortality rates of all malignancies worldwide. microRNAs (miRNAs) have been reported to be involved in many biological processes of diseases. MiR-22-3p is considered to be involved in cancer progression, but its role in CRC remains unclear. In this study, we detected that in CRC, the level of miR-22-3p is downregulated. MiR-22-3p has antitumor effects in CRC. miR-22-3p can reduce the proliferative, invasive and migrative capacity of CRC cells. Luciferase reporter analyses confirmed that KDM3A was a target of miR-22-3p, which can directly target the 3'UTR of KDM3A and decrease the expression of KDM3A in CRC cells. Our study also confirmed that KDM3A plays a role as an oncogene in CRC. KDM3A overexpression attenuated the tumor suppressor effects of miR-22-3p in CRC cells, demonstrating that miR-22-3p exerts antitumor effects by targeting KDM3A. Overexpression of miR-22-3p in CRC reduced YAP1 expression, whereas overexpression of KDM3A restored the expression of YAP1. In summary, miR-22-3p might inhibit the progression of CRC by targeting KDM3A to regulate the HIPPO signaling pathway, which may provide an opportunity for the treatment of CRC.

Molecular mechanisms of Huanglian jiedu decoction on ulcerative colitis based on network pharmacology and molecular docking.

Huanglian jiedu decoction (HLJDD) is a heat-clearing and detoxifying agent composed of four kinds of Chinese herbal medicine. Previous studies have shown that HLJDD can improve the inflammatory response of ulcerative colitis (UC) and maintain intestinal barrier function. However, its molecular mechanism is not completely clear. In this study, we verified the bioactive components (BCI) and potential targets of HLJDD in the treatment of UC using network pharmacology and molecular docking, and constructed the pharmacological network and PPI network. Then the core genes were enriched by GO and KEGG. Finally, the bioactive components were docked with the key targets to verify the binding ability between them. A total of 54 active components related to UC were identified. Ten genes are very important to the PPI network. Functional analysis showed that these target genes were mainly involved in the regulation of cell response to different stimuli, IL-17 signal pathway and TNF signal pathway. The results of molecular docking showed that the active components of HLJDD had a good binding ability with the Hub gene. This study systematically elucidates the "multi-component, multi-target, multi-pathway" mechanism of anti-UC with HLJDD for the first time, suggesting that HLJDD or its active components may be candidate drugs for the treatment of ulcerative colitis.

Group II metabotropic glutamate receptor agonist promotes retinal ganglion cell survival by reducing neuronal excitotoxicity in a rat chronic ocular hypertension model.

Glaucoma, the second leading cause of irreversible blindness worldwide, is characterized by the selective death of retinal ganglion cells (RGCs). The group II metabotropic glutamate receptor (mGluR II) activation has been linked to RGC survival, however, the mechanism by which it promotes neuronal survival remains poorly defined. In the present work, we show that extracellular application of LY341495, an mGluR II antagonist could increase the RGC firing frequency, suggesting that activation of mGluR II by endogenously released glutamate could modulate RGC excitability. LY354740, an mGluR II agonist, significantly decreased RGC excitability and the reduced presynaptic excitatory inputs and post-synaptic Ca2+-permeable currents mediated the LY354740-induced effects. By using a well-characterized in vivo male Sprague-Dawley rat glaucoma model, we further demonstrate that in the early stage of experimental glaucoma, the expression of mGluR II dimer-formed protein was significantly reduced, and pre-activation of mGluR II by intravitreal injection of LY354740 before establishment of the glaucoma model could effectively reduce excitatory inputs, thereby reversing hyperexcitability induced by elevated intraocular pressure. Furthermore, LY354740 could increase the expression level of brain-derived neurotrophic factor in the glaucomatous retinas, further protecting RGCs. Our study indicates that the abnormal expression of mGluR II may accelerate RGC apoptosis in glaucoma, and demonstrates that mGluR II agonist LY354740 can be used as a novel method to counter RGC apoptosis in glaucoma.