Sishen Pill Ameliorates Dextran Sulfate Sodium (DSS)-Induced Colitis with Spleen-Kidney Yang Deficiency Syndromes: Role of Gut Microbiota, Fecal Metabolites, Inflammatory Dendritic Cells, and TLR4/NF-κB Pathway

Abstract

Sishen pill (SSP) is an old Chinese medicine used to treat colitis with spleen-kidney-yang deficiency (SKYD) syndromes. However, its exact mechanism of action has not yet been fully elucidated. The aim of this study was to evaluate the effects and potential mechanisms of SSP on colitis with SKYD syndromes in mice. Colitis with SKYD syndromes was induced by rhubarb, hydrocortisone, and dextran sulfate sodium (DSS), and treatment was provided with SSP. Flow cytometry was performed to examine the inflammatory dendritic cell (infDC) regulations of SSP. The changes in the gut microbiota (GM) and fecal metabolites post-SSP treatment were investigated using the combination of 16S rRNA sequencing and untargeted metabolomics. Additionally, we also examined whether SSPs could regulate the infDCs by modifying TLR4/NF-κB signaling pathways. Compared with the DSS group, the disease activity index, colonic weight, index of colonic weight, and colonic injury scores, as well as the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-12p70 decreased significantly in the DSS + SSP group, while free triiodothyronine (FT3), free tetraiodothyronine (FT4), testosterone (TESTO), body weight change, colonic length, and the levels of IL-10 increased. Also, SSP decreased the amounts of CD103+CD11c+iNOS+, CD103+CD11c+TNF-α+, CD11c+CD103+CD324+, CD103+CD11c+MHC-II+, and CD103+CD11c+CD115+. Interestingly, 16S rRNA sequencing and untargeted metabolomics showed that SSP treatment restored the dysbiosis of GM and improved the dysfunction in fecal metabolism in colitis mice with SKYD syndromes. Correlation analysis indicated that the modulatory effects of SSP on FT3, FT4, IL-10, colonic weight index, CD103+CD11c+TNF-α+, CD103+CD11c+MHC-II+, and 13 common differential metabolites were related to alterations in the abundance of Parvibacter, Aerococcus, norank_f_Lachnospiraceae, Lachnospiraceae_UCG-006, Akkermansia, and Rhodococcus in the GM. In addition, SSP markedly inhibited the activation of the TLR4, MyD88, TRAF6, TAB2, and NF-κBp65 proteins and activated IκB. These results indicate that SSP can effectively alleviate colitis mice with SKYD syndrome by regulating infDCs, GM, fecal metabolites, and TLR4/NF-κB signaling pathways.

Sishen Pill and its active phytochemicals in treating inflammatory bowel disease and colon cancer: an overview

“…Many mechanistic studies on Sishen Pills focus on the regulatory effects of the NF-κB pathway ( Wang et al, 2019d ; Ge et al, 2022 ; Zhaohua et al, 2022 ). NF-κB is a classical key inflammatory modulator.…”

“…Also, the genotoxin produced by Escherichia-Shigella can penetrate the colon cell membrane and migrate to the nucleus, causing DNA double strand breaks, cell cycle arrest, chromosomal aberrations, intestinal epithelial damage, and eventually leading to cancer ( Fan et al, 2021b ). The above studies show that Sishen Pill can increase the relative abundance of AKK in the intestine ( Chen et al, 2020b ; Ge et al, 2022 ; Jin et al, 2023 ) and its effective metabolites, evodiamine, and corylin, leading to significant inhibition of Escherichia-Shigella the proliferation ( Zhu et al, 2021 ; Wang et al, 2023f ) thereby exerting anti-inflammatory and anticancer pharmacological activities.…”

“…Briefly, several studies focused on the inhibitory effects of Sishen Pill on the toll-like receptor (TLR): Huang ( Huang et al, 2021 ) and Zhao ( Zhaohua et al, 2022 ) found that the formula could inhibit expression levels of myeloid differentiation factor 88 (MyD88), interleukin-1 receptor associated kinase 4 (Irak4), and nuclear factor-kappa B(NF-κB) by down-regulating the activation of TLR2. Wang ( Wang et al, 2019a ) and Ge ( Ge et al, 2022 ) confirmed that TLR4 was the key target, and downregulating TLR4 could inhibit the occurrence of subsequent inflammatory responses through MyD88-dependent and MyD88-independent pathways. In addition, Zhang ( Zhang et al, 2021c ), Wang ( Wang et al, 2022a ) and Zhao ( Zhao et al, 2013 ) explored the molecular mechanism of the Sishen Pill in inhibiting the inflammatory response and promoting intestinal mucosal repair via phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt), Janus kinase (JAK)/signal transducer and activator of transcription 5 (STAT5), and mitogen activated protein kinase (MAPK) signal pathways.…”

“…Moreover, the regulation of intestinal immune cells by Sishen Pill mainly manifests in different subsets of T lymphocytes and regulatory T cells (Treg) ( Liu et al, 2016 ), helper T cells (Th) ( Liu et al, 2016 ), follicular helper T cells (Tfh) ( Liu et al, 2020 ), follicular regulatory T cells (Tfr) ( Huang et al, 2022 ; Kang et al, 2022 ), memory T cells (TM) ( Ge et al, 2020 ), and dendritic cells ( Liu et al, 2022 ). For regulating the gut microbiota, Sishen Pill has been shown to increase the relative abundance of beneficial bacteria, such as Lactobacillus and Akkermansia , and to promote an increase in intestinal butyrate content ( Chen et al, 2020b ; Wang et al, 2022b ; Ge et al, 2022 ). In summary, the above studies have elucidated the mechanism of action of Sishen Pill in treating IBD from different perspectives, but there is still a lack of deeper exploration on the key targets of action, and the application of molecular inhibitors/activators or gene knockout animal model and other experimental methods is necessary and anticipated in future research.…”

Sishen Pill inhibits intestinal inflammation in diarrhea mice via regulating kidney-intestinal bacteria-metabolic pathway

“…Baill, Ziziphus jujuba Mill, Zingiber officinale Roscoe, which have the function of warming the kidney and spleen, consolidating the gut and relieving diarrhea ( Li, 2012 ). It is often used clinically in the treatment of irritable bowel syndrome, ulcerative colitis, functional diarrhea, etc ( Zhu et al, 2016 ; Chen et al, 2020 ; Liu et al, 2020 ; Li et al, 2022c ; Li et al, 2022d ; Ge et al, 2022 ; Li et al, 2023 ). Zhou et al pointed out that SSP markedly increase the level of sIgA in diarrhea rats with DKYS, promoted the repair of gut mucosa, maintained the integrity of gut mucosa and other effects ( Zhou, 2007 ).…”