Total glucosides of paeony alleviates cGAS-STING-mediated diseases by blocking the STING-IRF3 interaction.

In the realm of autoimmune and inflammatory diseases, the cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) signaling pathway has been thoroughly investigated and established. Despite this, the clinical approval of drugs targeting the cGAS-STING pathway has been limited. The Total glucosides of paeony (TGP) is highly anti-inflammatory and is commonly used in the treatment of rheumatoid arthritis (RA), emerged as a subject of our study. We found that the TGP markedly reduced the activation of the cGAS-STING signaling pathway, triggered by various cGAS-STING agonists, in mouse bone marrow-derived macrophages (BMDMs) and Tohoku Hospital Pediatrics-1 (THP-1) cells. This inhibition was noted alongside the suppression of interferon regulatory factor 3 (IRF3) phosphorylation and the expression of interferon-beta (IFN-ß), C-X-C motif chemokine ligand 10 (CXCL10), and inflammatory mediators such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). The mechanism of action appeared to involve the TGP's attenuation of the STING-IRF3 interaction, without affecting STING oligomerization, thereby inhibiting the activation of downstream signaling pathways. In vivo, the TGP hindered the initiation of the cGAS-STING pathway by the STING agonist dimethylxanthenone-4-acetic acid (DMXAA) and exhibited promising therapeutic effects in a model of acute liver injury induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN). Our findings underscore the potential of the TGP as an effective inhibitor of the cGAS-STING pathway, offering a new treatment avenue for inflammatory and autoimmune diseases mediated by this pathway.

[Mechanism of total glucosides of White Paeony Capsules in ameliorating acute lung injury based on NLRP3 inflammasome].

This study deciphered the ameliorating effect and molecular mechanism of the total glucosides of White Paeony Capsules(TGP) in the treatment of mice model with acute lung injury(ALI) via NOD-like receptor thermal protein domain associated protein 3(NLRP3) signaling pathway of the inflammasome. The study established an inflammasome activation model of primed bone marrow-derived macrophages(BMDMs), and its molecular mechanism was investigated by Western blot(WB), immunofluorescence staining, enzyme-linked immunosorbent assay(ELISA), and flow cytometry. C57BL/6J mice were randomly divided into a blank control group, a TGP group, a model group(LPS group), LPS+low-and high-dose TGP groups, LPS+MCC950 group, and LPS+MCC950+TGP group, with eight mice per group. The ALI model was induced in mice. Finally, bronchoalveolar lavage fluid(BALF) and lung tissue were collected. Lung index and lung weight wet-to-dry ratio were determined for each group of mice. The pathological changes in lung tissue were observed through hematoxylin-eosin(HE) staining. The number of neutrophils in the BALF of each group was detected using flow cytometry. The levels of interleukin(IL)-1ß, IL-6, and tumor necrosis factor(TNF)-α in the BALF were determined by ELISA. The expressions of IL-1ß, IL-18, IL-6, and TNF-α in the lung tissue were determined by real-time quantitative PCR(RT-qPCR). This study demonstrated that TGP dramatically blocked the activation of the NLRP3 inflammasome by inhibiting the production of upstream mitochondrial reactive oxygen species(mtROS) and the subsequent oligomerization of apoptosis-associated specks(ASC). Additionally, in the ALI mice model, compared with the blank control group, the model group showed alveolar structure rupture, thic-kening of alveolar septa, and dramatically increased lung index, lung weight wet-to-dry ratio in lung tissue, neutrophil count, and inflammatory factor levels. Compared with the model group, the pathological morphology of lung tissue was significantly ameliorated in the TGP and MCC950 groups, and the lung index and lung weight wet-to-dry ratio were significantly reduced. Neutrophil counts were reduced, and levels of inflammatory factors were significantly downregulated. Notably, compared with the MCC950 group, there was no significant difference in effect in the MCC950+TGP group. Collectively, the study reveals that TGP may ameliorate ALI in mice by inhibiting the activation of NLRP3 inflammasome, providing a safe and effective drug candidate for the prevention or treatment of ALI/ARDS.

The combination of Schisandrin C and Luteolin synergistically attenuates hepatitis B virus infection via repressing HBV replication and promoting cGAS-STING pathway activation in macrophages.

BACKGROUND: HBV infection can result in severe liver diseases and is one of the primary causes of liver cell carcinoma-related mortality. Liuwei Wuling tablet (LWWL) is a traditional Chinese medicine formula, with a protecting liver and decreasing enzyme activity, usually used to treat chronic hepatitis B with NAs in clinic. However, its main active ingredients and mechanism of action have not been fully investigated. Hence, we aimed to screen the active ingredient and effective ingredient combinations from Liuwei Wuling tablet to explore the anti-herpatitis B virus activity and mechanism. METHODS: Analysis and screening of effective antiviral components in LWWL by network pharmacology, luteolin (Lut) may be a compound with significant antiviral activity. The mechanism of antiviral action of Lut was also found by real-time PCR detection and western blotting. Meanwhile, we established a co-culture model to investigate the antiviral mechanism of Schisandrin C (SC), one of the main active components of Schisandra chinensis fructus (the sovereign drug of LWWL). Next, HBV-infected mice were established by tail vein injection of pAAV-HBV1.2 plasmid and administered continuously for 20 days. And their antiviral capacity was evaluated by checking serum levels of HBsAg, HBeAg, levels of HBV DNA, and liver levels of HBcAg. RESULTS: In this study, we conducted network pharmacology analysis on LWWL, and through in vitro experimental validation and data analysis, we found that luteolin (Lut) possessed obviously anti-HBV activity, inhibiting HBV replication by downregulating hepatocyte nuclear factor 4α (HNF4α) via the ERK pathway. Additionally, we established a co-culture system and proved that SC promoted activation of cGAS-STINIG pathway and IFN-ß production in THP-1 cells to inhibit HBV replication in HepG2.2.15 cells. Moreover, we found the combination of SC and Lut shows a greater effect in inhibiting HBV compared to SC or Lut alone in HBV-infected mice. CONCLUSION: Taken together, our study suggests that combination of SC and Lut may be potential candidate drug for the prevention and treatment of chronic hepatitis B.

Flavonoid extracted from Epimedium attenuate cGAS-STING-mediated diseases by targeting the formation of functional STING signalosome.

Hyperactivation of the cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling pathway has been shown to be associated with the development of a variety of inflammatory diseases, and the discovery of an inhibitor of the cGAS-STING signalling pathway holds great promise in the therapeutic interventions. Epimedium flavonoid (EF), a major active ingredient isolated from the medicinal plant Epimedium, has been reported to have good anti-inflammatory activity, but its exact mechanism of action remains unclear. In the present study, we found that EF in mouse bone marrow-derived macrophages (BMDMs), THP-1 (Tohoku Hospital Pediatrics-1) as well as in human peripheral blood mononuclear cells (hPBMC) inhibited the activation of the cGAS-STING signalling pathway, which subsequently led to a decrease in the expression of type I interferon (IFN-ß, CXCL10 and ISG15) and pro-inflammatory cytokines (IL-6 and TNF-α). Mechanistically, EF does not affect STING oligomerization, but inhibits the formation of functional STING signalosome by attenuating the interaction of interferon regulatory factor 3 (IRF3) with STING and TANK-binding kinase 1 (TBK1). Importantly, in vivo experiments, EF has shown promising therapeutic effects on inflammatory diseases mediated by the cGAS-STING pathway, which include the agonist model induced by DMXAA stimulation, the autoimmune inflammatory disease model induced by three prime repair exonuclease 1 (Trex1) deficiency, and the non-alcoholic steatohepatitis (NASH) model induced by a pathogenic amino acid and choline deficiency diet (MCD). To summarize, our study suggests that EF is a potent potential inhibitor component of the cGAS-STING signalling pathway for the treatment of inflammatory diseases mediated by the cGAS-STING signalling pathway.

Liuweiwuling Tablet relieves the inflammatory transformation of hepatocellular carcinoma by inhibiting the PI3K/AKT/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Liuweiwuling Tablet (LWWL) is a patented Chinese medicine approved by the Chinese National Medical Products Administration (NMPA). Clinically, it is used to treat a range of liver diseases that precede hepatocellular carcinoma (HCC), including hepatitis, liver fibrosis and cirrhosis. LWWL is hypothesized to inhibit the inflammatory transformation of HCC, which may have a positive impact on the prevention and treatment of HCC. However, its exact mechanism of action remains unknown. AIM OF THE STUDY: To investigate how LWWL is effective in the treatment of HCC and to validate the pathways involved in this process. MATERIALS AND METHODS: An in vivo model of HCC induced by diethylnitrosamine (DEN) was established to study the effect of LWWL on the development of HCC. The rat serum was analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transpeptidase (γ-GT). The rat liver tissues were stained with hematoxylin and eosin (HE) and Masson's trichrome for pathological analysis. Rat liver tissue was subjected to transcriptome sequencing. Expression of inflammatory and liver fibrosis-related factors in bone marrow-derived macrophages (BMDMs) and LX-2 cells was detected by QRT-PCR, ELISA and Western blot (WB). The expression of apoptosis and stemness genes in HepG2 and Huh7 cells was assessed through flow cytometry and QRT-PCR. Transcriptomics, network pharmacology, WB, and QRT-PCR were employed to validate the mechanisms associated with the amelioration of HCC development by LWWL. RESULTS: LWWL significantly reduced the severity of hepatitis and liver fibrosis, the expression of tumor stemness genes, and the incidence of HCC. In addition, LWWL inhibited the release of inflammatory substances and nuclear accumulation of P65 protein in BMDMs as well as the conversion of LX-2 cells to fibroblasts. LWWL inhibited the proliferation of HepG2 and Huh7 cells, including the initiation of apoptosis and the reduction of stemness gene expression. Importantly, LWWL regulates the PI3K/AKT/NF-κB pathway, which affects hepatic inflammation and cancer progression. CONCLUSION: LWWL inhibited the occurrence and development of HCC by modulating the severity of hepatitis and liver fibrosis, indicating the potential clinical relevance of LWWL in preventing and treating HCC.

Papaya CpERF9 acts as a transcriptional repressor of cell-wall-modifying genes CpPME1/2 and CpPG5 involved in fruit ripening.

KEY MESSAGE: CpERF9 controls papaya fruit ripening through transcriptional repression of cell-wall-modifying genes CpPME1/2 and CpPG5 by directly binding to their promoters. Papaya fruit ripening is an intricate and highly coordinated developmental process which is controlled by the action of ethylene and expression of numerous ethylene-responsive genes. Ethylene response factors (ERFs) representing the last regulators of ethylene-signaling pathway determine the specificities of ethylene response. However, knowledge concerning the transcriptional controlling mechanism of ERF-mediated papaya fruit ripening is limited. In the present work, a gene-encoding AP2/ERF protein with two ERF-associated amphiphilic repression (EAR) motifs, named CpERF9, was characterized from papaya fruit. CpERF9 was found to localize in nucleus, and possess transcriptional repression ability. CpERF9 expression steadily decreased during papaya fruit ripening, while several genes encoding pectin methylesterases (PMEs) and polygalacturonases (PGs), such as CpPME1/2 and CpPG5, were gradually increased, paralleling the decline of fruit firmness. Electrophoretic mobility shift assay (EMSA) demonstrated a specific binding of CpERF9 to promoters of CpPME1/2 and CpPG5, via the GCC-box motif. Transient expression of CpERF9 in tobacco repressed CpPME1/2 and CpPG5 promoter activities, which was depended on two EAR motifs of CpERF9 protein. Taken together, these findings suggest that papaya CpERF9 may act as a transcriptional repressor of several cell-wall modifying genes, such as CpPME1/2 and CpPG5, via directly binding to their promoters.

Splicing proofreading at 5' splice sites by ATPase Prp28p.

Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5' splice site (SS) selection is attained is not fully clear. Through a series of yeast genetic screens, we isolated alleles of prp28 that improve splicing of suboptimal 5'SS substrates, demonstrating that WT-Prp28p proofreads, and consequently rejects, poor 5'SS. Prp28p is thought to facilitate the disruption of 5'SS-U1 snRNA pairing to allow for 5'SS-U6 snRNA pairing in the catalytic spliceosome; unexpectedly, 5'SS proofreading by Prp28p is dependent on competition with the stability of the 5'SS:U6 duplex, but not the 5'SS:U1 duplex. E404K, the strongest prp28 allele containing a mutation located in the linker region between adenosine triphosphatase (ATPase) subdomains, exhibited lower RNA-binding activity and enhanced splicing of suboptimal substrates before first-step catalysis, suggesting that decreased Prp28p activity allows longer time for suboptimal 5'SS substrates to pair with U6 snRNA and thereby reduces splicing fidelity. Residue E404 is critical for providing high splicing activity, demonstrated here in both yeast and Drosophila cells. Thus, the subdomain linker in Prp28p plays important roles both in splicing efficiency across species and in proofreading of 5'SS.