The involvement of Sting in exacerbating acute lung injury in sepsis via the PARP-1/NLRP3 signaling pathway.

BACKGROUND: Interferon gene stimulator (Sting) is an indispensable adaptor protein that plays a crucial role in acute lung injury (ALI) induced by sepsis, and the PARP-1/NLRP3 signaling pathway may be an integral component of the inflammatory response mediated by Sting. However, the regulatory role of Sting in the PARP-1/NLRP3 pathway in ALI remains insufficiently elucidated. METHODS: Using lipopolysaccharide (LPS) to induce ALI in C57BL/6 mice and HUVEC cells, an in vivo and in vitro model was established. In vivo, Sting agonists and inhibitors were administered, while in vitro, Sting was knocked down using siRNA. ELISA was employed to quantify the levels of IL-1ß, IL-6, and TNF-α. TUNEL staining was conducted to assess cellular apoptosis, while co-immunoprecipitation was utilized to investigate the interaction between Sting and NLRP3. Expression levels of Sting, NLRP3, PARP-1, among others, were assessed via Western blotting and RT-qPCR. Lung HE staining and lung wet/dry ratio were evaluated in the in vivo mouse model. To validate the role of the PARP-1/NLRP3 signaling pathway, PARP-1 inhibitors were employed both in vivo and in vitro. RESULTS: In vitro experiments revealed that the Sting agonist group exacerbated LPS-induced pulmonary pathological damage, pulmonary edema, inflammatory response (increased levels of IL-6, TNF-α, and IL-1ß), and cellular injury, whereas the Sting inhibitor group significantly ameliorated the aforementioned injuries, with further improvement observed in the combination therapy of Sting inhibitor and PARP-1 inhibitor. Western blotting and RT-qPCR results demonstrated significant suppression of ICAM-1, VCAM-1, NLRP3, and PARP-1 expression in the Sting inhibitor group, with this reduction further enhanced in the Sting inhibitor + PARP-1 inhibitor treatment group, exhibiting opposite outcomes to the agonist. Furthermore, in vitro experiments using HUVEC cell lines validated these findings. CONCLUSIONS: Our study provides new insights into the roles of Sting and the PARP-1/NLRP3 signaling pathway in inflammatory responses, offering novel targets for the development of therapeutic interventions against inflammatory reactions.

Deciphering the pharmacological mechanisms of Shenlingbaizhu formula in antibiotic-associated diarrhea treatment: Network pharmacological analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Shenlingbaizhu (SLBZ) formula, a classical traditional Chinese medicinal (TCM) formula, has been widely used for treating antibiotic-associated diarrhea (AAD). However, the underlying pharmacological mechanisms have not yet been investigated thoroughly. AIM OF THE STUDY: To explore the remission mechanism of SLBZ in the treatment of AAD, we conducted network pharmacological analysis and experimental validation in vitro and in vivo. MATERIALS AND METHODS: In this study, the main compounds of SLBZ were identified by ultra-high-performance liquid chromatography-mass spectroscopy (UHPLC-MS) and online databases. The targets of the active components and AAD-related targets were predicted by network pharmacology, and the potential targets of SLBZ against AAD were obtained. Then the core targets were recognized after Protein-Protein Interaction (PPI) analysis. Based on these, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analyses were conducted, and the key pathway was screened. Subsequently, molecular docking was performed using Auto Dock Vina to find the key components that played a crucial role in that pathway. Molecular dynamics simulation was performed by Gromacs software to detect the binding mode. Finally, the results were confirmed by in vitro and in vivo experiments. RESULTS: A total of 66 active ingredients of SLBZ were detected by UHPLC-MS, and 128 active ingredients were screened out by network pharmacological analysis. Additionally, 935 drug targets and 1686 AAD-related targets were obtained. Seventy-eight intersected genes were selected as potential therapeutic targets and 19 genes were excavated as core targets. Enrichment analysis revealed PI3K-AKT signaling pathway was the key pathway in SLBZ against AAD. Topological analysis further revealed that JAK2, MTOR, TLR4, and SYK were the key targets affected by SLBZ on the PI3K-AKT pathway, and 52 components of SLBZ were associated with them. Molecular docking and dynamics simulation revealed strong binding affinities between MTOR and diosgenin. Subsequently, after SLBZ treatment, the expression levels of JAK2, MTOR, TLR4, and SYK were found significantly upregulated in the AAD model rats (p < 0.05). The cell experiment further validated the good binding ability between MTOR and diosgenin. CONCLUSION: We demonstrate that the therapeutic effect of SLBZ on AAD was achieved in part by inhibiting the PI3K-AKT pathway.

Electroacupuncture promotes macrophage/microglial M2 polarization and suppresses inflammatory pain through AMPK.

Inflammatory pain, the most prevalent disease globally, remains challenging to manage. Electroacupuncture emerges as an effective therapy, yet its underlying mechanisms are not fully understood. This study investigates whether adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-regulated silent information regulator 1 (SIRT1) contributes to electroacupuncture's antinociceptive effects by modulating macrophage/microglial polarization in the spinal dorsal horn of a mouse model of inflammatory pain. In this study, mice, introduced to inflammatory pain through subcutaneous injections of complete freund's adjuvant (CFA) in the plantar area, underwent electroacupuncture therapy every alternate day for 30-min sessions. The assessment of mechanical allodynia and thermal hyperalgesia in these subjects was carried out using paw withdrawal frequency and paw withdrawal latency measurements, respectively. Western blot analysis measured levels of AMPK, phosphorylation-adenosine 5'-monophosphate (AMP)-activated protein kinase, SIRT1, inducible nitric oxide synthase, cluster of differentiation 86, arginase 1, and interleukin 10. In contrast to the group treated solely with CFA, the cohort receiving both CFA and electroacupuncture demonstrated notable decreases in both thermal hyperalgesia and mechanical allodynia. This was accompanied by a marked enhancement in AMPK phosphorylation levels. AMPK knockdown reversed electroacupuncture's analgesic effects and reduced M2 macrophage/microglial polarization enhancement. Additionally, AMPK knockdown significantly weakened electroacupuncture-induced SIRT1 upregulation, and EX-527 injection attenuated electroacupuncture's facilitation of M2 macrophage/microglial polarization without affecting AMPK phosphorylation levels. Furthermore, combining electroacupuncture with SRT1720 enhanced the analgesic effect of SRT1720. Our findings suggest that AMPK regulation of SIRT1 plays a critical role in electroacupuncture's antinociceptive effect through the promotion of M2 macrophage/microglial polarization.

Tuneable Schottky contact of MoSi2N4/TaS2 van der Waals heterostructure.

The two-dimensional MoSi2N4 monolayer is an emerging semiconductor material that offers considerable promise due to its ultra-thin profile, tuneable mechanical properties, excellent optoelectronic properties and exceptional environmental stability. The van der Waals (vdW) heterostructure formed by stacking such two-dimensional monolayers has demonstrated superior performance across various domains. In this study, a vdW heterostructure combining the two-dimensional MoSi2N4 and TaS2 monolayers is examined using first-principles density functional theory. In its ground state, this van der Waals heterostructure establishes an ohmic contact with an exceptionally low potential barrier height. By modulating the vdW heterostructure with an applied electric field of -0.1 V/Å and under vertical stress, we discovered that MoSi2N4 and TaS2 can transition from an ohmic contact to a p-type Schottky with an ultra-low Schottky barrier height (SBH). Our observations may give valuable insights for designing reconfigurable, tuneable Schottky nano-devices with enhanced electronic and optical properties based on MoSi2N4/TaS2.

Identification and Validation of a Metabolism-Related Prognostic Signature Associated with M2 Macrophage Infiltration in Gastric Cancer.

High levels of M2 macrophage infiltration invariably contribute to poor cancer prognosis and can be manipulated by metabolic reprogramming in the tumor microenvironment. However, the metabolism-related genes (MRGs) affecting M2 macrophage infiltration and their clinical implications are not fully understood. In this study, we identified 173 MRGs associated with M2 macrophage infiltration in cases of gastric cancer (GC) using the TCGA and GEO databases. Twelve MRGs were eventually adopted as the prognostic signature to develop a risk model. In the high-risk group, the patients showed poorer survival outcomes than patients in the low-risk group. Additionally, the patients in the high-risk group were less sensitive to certain drugs, such as 5-Fluorouracil, Oxaliplatin, and Cisplatin. Risk scores were positively correlated with the infiltration of multiple immune cells, including CD8+ T cells and M2 macrophages. Furthermore, a difference was observed in the expression and distribution between the 12 signature genes in the tumor microenvironment through single-cell sequencing analysis. In vitro experiments proved that the M2 polarization of macrophages was suppressed by Sorcin-knockdown GC cells, thereby hindering the proliferation and migration of GC cells. These findings provide a valuable prognostic signature for evaluating clinical outcomes and corresponding treatment options and identifying potential targets for GC treatment.

Mifepristone inhibited tumor progression by disrupting the stability of PD-L1 by miR-127-3p/VAMP2 in ovarian cancer.

To investigate the effect of mifepristone on PD-L1 through miR-127-3p/VAMP2 axis to inhibit the malignant biological behavior of ovarian cancer cells. Western blotting was used to detect the protein expression of VAMP2, PD-L1, CyclinD1, Cl-caspase-3 and Bax; qRT-PCR was used to detect the expression of miR-127-3p; double luciferase reporter gene was used to verify the targeted binding of miR-127-3p to VAMP2. The results showed that mifepristone up-regulated the expression of miR-127-3p and mifepristone could significantly inhibit the proliferation of ovarian cancer SKOV3 cells and A2780 cells, promote apoptosis, inhibit the expression of PD-L1, down regulate the expression of CyclinD1 and up regulate the expression of cl-caspase-3 and Bax; silencing miR-127-3p could restore the effects of mifepristone on the proliferation and apoptosis of SKOV3 cells and A2780 cells, as well as the expression of PD-L1, CyclinD1, Cl-caspase-3 and Bax protein; our study confirmed that mifepristone can regulate the expression of VAMP2 and PD-L1 through miR-127-3p and VAMP2 can positively regulate the expression of PD-L1; finally, we found that mifepristone can down regulate PD-L1 through miR-127-3p/VAMP2 axis, inhibit proliferation and promote apoptosis of ovarian cancer cells. Mifepristone can down regulate PD-L1 through miR-127-3p/VAMP2 axis and inhibit the progression of ovarian cancer cells.

The anterior cingulate cortex projection to the dorsomedial striatum modulates hyperalgesia in a chronic constriction injury mouse model.

INTRODUCTION: The aim of the study was to study the role of the anterior cingulate cortex (ACC)-dorsal midbrain striatum (DMS) in neuropathic pain in mice. MATERIAL AND METHODS: Optogenetics has been increasingly used in neuroscience research to selectively and precisely control the activity of a defined group of central neurons to determine their roles in behavioral functions in animals. The most important opsins are blue-sensitive ChR2 and yellow-sensitive NpHR. Calcium-calmodulin dependent protein kinase Iiα (CaMKIIα) is mostly expressed in the pyramidal excitatory neurons. Mice were injected with AAV2/9-CamKII-ChR2-mCherry, AAV2/9-CamKII-eNpHR3.0-GFP or AAV2/9-CamKII-mCherry virus in the ACC region, and the optical fiber implantation was performed in the ACC or DMS region. Mice were then followed up for 2 to 8 weeks and behavioral tests were carried out in the presence or absence of the blue/yellow light (473 nm/589 nm). Pain behavioral tests with or without the blue/yellow light at the same time were performed on the third and the seventh day after the chronic constriction injury of sciatic nerve model (CCI) was established. The pain thresholds of left and right hind limbs of mice in all groups were measured. RESULTS: No matter whether activating the neurons in ACC or DMS, compared with normal mice in the ChR2-off-right group, and the mCherry-on-right group, the thermal pain threshold and mechanical pain threshold of the normal mice in the ChR2-on-right group were significantly lower. When inhibiting the neurons in the ACC or DMS, on day 3 and day 7 after CCI operation, the thermal pain threshold and mechanical pain threshold of the CCI mice of the NpHR-on-right group were significantly higher compared with the NpHR-off-right and mCherry-on-right groups. CONCLUSIONS: The anterior cingulate cortex-dorsal midbrain striatum may be involved in the regulation of neuropathic pain in mice.

Electroacupuncture Attenuates CFA-Induced Inflammatory Pain by Regulating CaMKII.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine kinase that is ubiquitously distributed in the central and peripheral nervous systems. Moreover, its phosphorylated protein (P-CaMKII) is involved in memory, mood, and pain regulation in the anterior cingulate cortex (ACC). Electroacupuncture (EA) is a traditional Chinese therapeutic technique that can effectively treat chronic inflammatory pain. However, the CaMKII-GluA1 role in EA analgesia in the ACC remains unclear. This study investigated the role of P-CaMKII and P-GluA1 in a mouse model of inflammatory pain induced by complete Freund's adjuvant (CFA). There were increased P-CaMKII and P-GluA1 levels in the ACC. We found that intracerebroventricular injection of KN93, a CaMKII inhibitor, as well as EA stimulation, attenuated complete Freund's adjuvant-induced pain behavior. Further, EA increased pCaMKII-PICK1 complex (abbreviated as C-P complex) levels. Our findings demonstrate that EA inhibits inflammatory pain by inhibiting CaMKII-GluA1 phosphorylation. P-CaMKII is involved in EA analgesia as the pCaMKII-PICK1 complex.

[Involvement of miR-34a and p53 protein of cerebral cortex in electroacpuncture analgesia in mice with neuropathic pain].

OBJECTIVE: To explore the involvement of miR-34a in cerebral cortex mediated anti-hyperalgesic effect of electroacupuncture (EA) in mice with neuropathic pain induced by chronic constriction injury (CCI) of sciatic nerve, so as to reveal its mechanisms underlying improvement of neuropathic pain. METHODS: A total of 75 male C57BL/6 mice were equally randomized into 3 groups: sham, CCI model and CCI+EA (n=25 in each group). Mice of the sham group received simple separation of the right sciatic nerve without ligation. The CCI model was established by liagation of the right sciatic nerve. EA (2 Hz /15 Hz, 1 mA) was applied to bilateral "Zusanli" (ST36) and "Sanyinjiao" (SP9) for 30 min, once every other day. The mechanical and thermal pain threshold of the bilateral hind-paws was detected at the 3rd, 5th and 7th day after modeling, and the expression of miR-34a of bilateral cerebral cortex tissues and that of p53 protein of the left cerebral cortex were determined by using quantitive real time PCR and Western blot, respectively. RESULTS: The mechnical paw withdrawal frequency were significantly higher and the thermal paw withdrawal latencies (PWLs) were significantly shorter at the affected hind-limb (rather than at the healthy hind limb) on day 3, 5 and 7 in the CCI model group than those in the sham group (P<0.05), and considerably reversed at the affected hind-limb (rather than at the healthy hind limb) in the EA group than in the CCI model group (P<0.05), suggesting an analgesic effect of EA intervention. After modeling, the expression levels of miR-34a and p53 on day 3, 5 and 7 were significantly up-regulated in the left cerebral cortex tissue (rather than in the right cerebral cortex) of the CCI model group in comparison with the sham group (P<0.05). After EA intervention, the up-regulated expression levels of miR-34a and p53 in the left cerebral cortex tissue (rather than in the right cerebral cortex) were obviously suppressed in the EA group relevant to the CCI model group (P<0.05). CONCLUSION: EA stimulation of ST36 and SP9 can down-regulate the expression of miR-34a and p53 in the contra-lateral cerebral cortex tissue of the CCI mice, which may contribute to its anti-hyperalgesic effect.

Spinal miR-34a regulates inflammatory pain by targeting SIRT1 in complete Freund's adjuvant mice.

Sirtuin1 (SIRT1), which is regulated by microRNA-34a (miR-34a), can modulate pathophysiology processes, including nonalcoholic fatty liver disease and intestinal ischemia/reperfusion injury. We previously reported that SIRT1, an NAD+-dependent deacetylase, plays a vital role in the development of neuropathic pain. However, the role of miR-34a/SIRT1 in complete Freund's adjuvant (CFA)-induced inflammatory pain remains unclear. In the present study, we examined miR-34a and SIRT1 in CFA mice. MiR-34a levels increased, while SIRT1 decreased in the spinal cord. Inhibiting miR-34a by intrathecal injection of miR-34a antagomir attenuated CFA-induced pain behavior. Moreover, miR-34a antagomir inhibited the CFA-induced SIRT1 decrease in the spinal cord. Furthermore, the analgesic effect of miR-34a antagomir was abrogated by the SIRT1 inhibitor EX-527. Our data provide support that the underlying mechanisms of miR-34a in promoting inflammatory pain may involve negative regulation of SIRT1.