The Wu-Shi-Cha formula protects against ulcerative colitis by orchestrating immunity and microbiota homeostasis.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) has become a healthy burden worldwide due to its insidious onset and repetitive relapse, with a rather complex etiology, including inappropriate immune response, dysbiosis, genetic susceptibility, and unhealthy diets. The Wu-Shi-Cha (WSC) formula is a widely utilized drug to protect against gastrointestinal disorders. AIM OF THE STUDY: The study aspired to dissect the pertinent mechanisms of the WSC to treat UC. MATERIALS AND METHODS: Network pharmacology and weighted gene co-expression network analysis (WGCNA) were performed to predict the targets of WSC in the context of UC and colorectal cancer. Dextran sodium sulfate (DSS) was used to construct murine models of experimental colitis, and the WSC was given to colitis mice for 14 days. Feces and colon samples were subjected to 16S rRNA gene sequencing combined with liquid chromatography-mass spectrometry (LC-MS) and biochemical experiments, respectively. RESULTS: Network pharmacology analysis predicted that the WSC formula could orchestrate inflammation, infection, and tumorigenesis, and WGCNA based on The Cancer Genome Atlas (TCGA) database showed a potent anti-neoplastic effect of the WSC therapy for colorectal cancer. The WSC therapy rescued bursts of pro-inflammatory cytokines and colonic epithelial collapse in DSS-induced colitis mice. Moreover, the high dose of WSC treatment facilitated the alternative activation of peritoneal macrophages (Mφs) and these Mφs were conducive to the survival of intestinal stem cells (ISCs), and the disturbed homeostasis of gut microbiota was re-established after WSC treatment, as evidenced by the decreased colonization of pathological taxa in the fecal samples. CONCLUSION: The WSC formula suppresses inflammation and re-establishes the homeostasis of gut microbiota, thereby ameliorating colitis progression.

LRRK2 deficiency mitigates colitis progression by favoring resolution of inflammation and restoring homeostasis of gut microbiota.

Leucine rich-repeat kinase 2 (LRRK2) has been considered a susceptibility gene for ulcerative colitis (UC), and its protein abundance was enhanced in the peripheral blood mononuclear cells (PBMCs) from UC cohorts as compared to healthy volunteers. In preclinical models of colitis, Lrrk2 deficiency ameliorated dextran sodium sulfate (DSS)-induced colitis progression, whereas the processes were aggravated by R1441C mutation. While intestinal macrophages (MФs) from Lrrk2 knock-out (Lrrk2-/-) mice exhibited a tendency to transit to alternatively activated MФs, R1441C MФs mutation facilitated the pro-inflammatory phenotype polarization, determined by RNA sequencing and qPCR. Moreover, we characterized their microbiota profiles and found that loss of Lrrk2 increased the bacterial richness and altered bacterial community structure, and this shift contributed to the alleviation of colitis development and progression. We proposed that Lrrk2 deficiency promotes M2 MФ transition and facilitates probiotics colonization, providing a protective role during colitis.

Pharmacological Mechanism of Pingxiao Formula against Colorectal Cancer.

Colorectal cancer (CRC) is the most common cancer worldwide and develops due to a broad range of causative factors. Pingxiao (PX) formula and Xihuang (XH) formula are two commonly used drugs to treat CRC, especially as an alternative therapy for those patients who could not suffer surgery, chemotherapy, or immunotherapy, namely, elder or advanced CRC patients. However, the pertinent pharmacological mechanisms are still elusive. The investigation was designed to explain the pharmacological mechanisms of the PX formula. A murine model of CRC was established by injecting CT26.WT cells into the caecum of 4-week-old male Balb/c mice, following PX or XH treatment for 30 days. Network pharmacology analysis combined with weighted gene coexpression network analysis (WGCNA) predicted the pharmacological mechanisms and therapeutic value. High-throughput 16S rRNA sequencing determined the alterations in the gut microbiota communities. Western blotting, immunofluorescence, and flow cytometry examined the influence of PX on the tumor microenvironment (TME). Injection of CT26.WT-induced CRC in Balb/c mice was markedly attenuated by PX treatment. Compared with XH administration, PX exhibited a stronger antitumor effect, such as smaller tumor volume, lower interleukin 17 (IL-17), IL-6 and tumor necrosis factor-alpha (TNFα) serum levels, and higher interferon-gamma (IFN-γ) concentration. Network pharmacology analysis demonstrated that both PX and XH targets were enriched in cancers and inflammatory responses. RNA sequencing confirmed that PX treatment induced cancer cell apoptosis and inhibited inflammatory reactions within the tumor. Moreover, the PX formula considerably restored homeostasis of the gut microbiota, which was not observed in the XH group. PX targets, those associated with the survival probability of CRC patients, correlated with macrophage (Mφ) infiltration, which presented an independent risk factor for the CRC outcome. PX treatment promoted the transition of alternatively activated Mφs (M2 Mφs) to classically activated Mφs (M1 Mφs). Moreover, the peritoneal Mφs from the PX group inhibited the migration of CW26.WT cells, as evidenced by the wound healing experiment and transwell assay, which was consistent with the decreased expression of the vascular endothelial growth factor (VEGF). Furthermore, the coculturing system confirmed that PX-treated Mφs suppressed colorectal tumor-derived organoid proliferation. PX formula exhibits a potential antitumor effect against CRC by suppressing the colonization of pathological microorganisms, reshaping Mφ effector functions and hence inhibiting cancer cell proliferation.

Orai2 deficiency attenutates experimental colitis by facilitating the colonization of Akkermansia muciniphila.

Orai2 is a component of store-operated Calcium channels (SOCCs) and exerts a pivotal role in immunity. In intestinal macrophages (Mφs), Orai2 deficiency influenced linoleic acid (LA)-arachidonic acid (ARA) derivatives by regulating Pla2g6 and Alox5. 16S rRNA sequencing showed that deleting Orai2 facilitated the prevalence of Akkermansia muciniphila, and untargeted metabolomics confirmed the suppressed level of leukotriene A. Moreover, Orai2 deficiency ameliorated the progression of experimental murine colitis, as shown by attenuated structural collapse of colon and pro-inflammatory cytokine concentrations, and rescued dysbiosis. The administration of a Pla2g6 inhibitor (Bromoenol lactone) not only inhibited the relative abundance of A. muciniphila in the feces of Orai2 knockout (Orai2-/-) mice, but also abolished the increased activity of Calcium-released activated Calcium channel (CRAC) in Orai2-/- intestinal Mφs, corroborating the involvement of Pla2g6 in Orai2 signaling. In conclusion, Orai2 deficiency increases Pla2g6 and hence facilitating A. muciniphila colonization, which might be a potential strategy to combat colitis.

Pharmacological mechanism of Shenlingbaizhu formula against experimental colitis.

BACKGROUND: Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) characterized by an overactive immune response and destruction of the colorectal epithelium with intricate pathological factors. Shenlingbaizhu (SLBZ) formula, included in the Chinese Pharmacopoeia 2020, has been widely utilized to treat UC. PURPOSE: The present study was designed to uncover the underlying molecular mechanisms of SLBZ formula against UC. METHODS: A murine model of experimental colitis was established by orally feeding 2% dextran sodium sulfate (DSS) to mice for 7 days, followed by SLBA treatment for the next 15 days. Network pharmacology analysis was performed to predict the pharmacological mechanisms. High-throughput 16S rRNA sequencing integrated with liquid chromatography-mass spectrometry (LC-MS) was conducted on mouse stool in order to determine alterations in the composition of the intestinal microbiota and metabolites. Western blotting, immunofluorescence, and flow cytometry were performed to examine the anti-inflammatory role of SLBZ. RESULTS: DSS treatment induced experimental colitis, and this induction was alleviated by SLBZ treatment, as evidenced by rescued pathological symptoms in the experimental colitis mouse groups. Network pharmacology analysis showed that SLBZ-target genes were enriched in pathogen-induced infectious and inflammatory pathways, as well as neoplastic processes. SLBZ administration also modulated the gut microbiota composition and metabolic profiles of experimental colitis mice and alleviated the progression of experimental colitis. We further showed via in-vitro experiments that SLBZ suppressed macrophage (Mφ) transition to pro-inflammatory phenotype (M1), rescued tumor necrosis factor-α (TNFα)-induced pyroptosis of intestinal organoids (IOs), and decreased the recruitment of Mφs by epithelial cells. CONCLUSION: SLBZ formula is an effective treatment for murine colitis and showed a stronger therapeutic capacity than melasazine. The pharmacological mechanisms of SLBZ involve the re-establishment of an anti-inflammatory milieu and healthy microbiome, which favors mucosal healing.

High ORAI3 expression correlates with good prognosis in human muscle-invasive bladder cancer.

The involvement of store-operated calcium channels (SOCCs) in tumor initiation and metastatic dissemination has been extensively studied, but how its member ORAI3 influences tumor progression is still elusive. The present study aimed to evaluate the prognostic value of ORAI3 expression and examine the correlation between ORAI3 expression and immune cell infiltration within the tumor microenvironment (TME) in human muscle-invasive bladder cancer (MIBC). We examined the expression profile of ORAI3 in MIBC using data from two databases; analyzed the correlation between ORAI3 expression and patient survival; explored cellular pathways related to ORAI3 expression by Gene Set Enrichment Analysis (GSEA); and predicted potential drugs using Connectivity Map (CMap). ORAI3 was significantly lower expressed in tumor mass compared to normal samples in MIBC, with a higher level of methylation at the promoter region in tumor than in normal tissue, indicating that ORAI3 is suppressed during cancer progression. Survival analysis showed that higher expression of ORAI3 correlated with good prognosis in MIBC. GSEA demonstrated that ORAI3 expression inversely correlated with cell differentiation, development and gene silencing, with differential expression of genes involved in epidermal and keratinocyte differentiation pathways and inflammatory responses. RNA sequencing of an ORAI3-silenced human bladder cancer cell line (T24 cells) corroborated enhancement of pro-neoplastic pathways in absence of ORAI3. Western blottingMoreover, ORAI3 facilitated the recruitment of Th17 cells and natural killer cells, whereas hampered Th2 and macrophage infiltration. Our results revealed 4 molecules with potential to be beneficial as adjuvant drugs in MIBC treatment. We concluded that high ORAI3 expression correlates with increased survival in human MIBC.

The Pharmacological Mechanism of Guchangzhixie Capsule Against Experimental Colitis.

Ulcerative colitis (UC) is the major type of inflammatory bowel disease (IBD) characterized by an overactive immune response and destruction of colorectal epithelium with intricate pathological factors. Guchangzhixie (GCZX) capsule, included in the Chinese Pharmacopoeia 2020, has been widely utilized against UC. However, the underlying molecular mechanisms have not been elucidated. In the present study, a murine model of experimental colitis was established by orally feeding 4% dextran sodium sulfate (DSS) for 5 days and subsequently subjecting to GCZX treatment for another 15 days. Network pharmacology analysis was performed to predict the pertinent mechanisms of GCZX capsule. Cellular experiments examining the functional changes of intestinal organoids (IOs), macrophages (Mφs), and human colon epithelial cell cells (NCM460 cell line) after GCZX therapy were performed. Sequencing of 16S rRNA was conducted on the stools from the mouse model. Liquid chromatography-mass spectrometry (LC-MS) was utilized to detect serum metabolites. As a result, DSS induced experimental colitis, and this induction was alleviated by GCZX treatment, as evidenced by rescued pathological symptoms in UC mouse models, such as rectal bleeding stopping, decreased levels of albumin, interleukin-17, as well as chemokine (C-X-C motif) ligand 1 (CXCL1), and reduction in colon length. Network pharmacology analysis showed that GCZX-target genes were enriched in pathogen-induced infections, inflammatory pathways, as well as neoplastic processes. DSS treatment decreased microbial diversity and led to the accumulation of pathological bacterial, which was reversed by GCZX capsule. PICRUSt2 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) based on profiles of microbiota composition demonstrated a decreased incidence of infectious disease and cancers after GCZX therapy. In full accordance with these data, GCZX administration suppressed Mφ transition to pro-inflammatory phenotype, alleviated tumor necrosis factor-α (TNFα)-compromised IOs functions, and decreased the recruitment of Mφs by epithelial cells. We conclude that GCZX capsule is an effective drug for UC and its pharmacological mechanisms involve re-establishing an anti-inflammatory milieu and favoring mucosal healing.