Identification of marker genes associated with N6-methyladenosine and autophagy in ulcerative colitis

BACKGROUND Both N6-methyladenosine(m6A)methylation and autophagy are considered relevant to the pathogenesis of ulcerative colitis(UC).However,a systematic exploration of the role of the com-bination of m6A methylation and autophagy in UC remains to be performed.AIM To elucidate the autophagy-related genes of m6A with a diagnostic value for UC.METHODS The correlation between m6A-related genes and autophagy-related genes(ARGs)was analyzed.Finally,gene set enrichment analysis(GSEA)was performed on the characteristic genes.Additionally,the expression levels of four characteristic genes were verified in dextran sulfate sodium(DSS)-induced colitis in mice.RESULTS GSEA indicated that BAG3,P4HB and TP53INP2 were involved in the inflammatory response and TNF-αsignalling via nuclear factor kappa-B.Furthermore,polymerase chain reaction results showed significantly higher mRNA levels of BAG3 and P4HB and lower mRNA levels of FMR1 and TP53INP2 in the DSS group compared to the control group.CONCLUSION This study identified four m6A-ARGs that predict the occurrence of UC,thus providing a scientific reference for further studies on the pathogenesis of UC.

Gut microbiota contributes to the distinction between two traditional Chinese medicine syndromes of ulcerative colitis

BACKGROUND Ulcerative colitis(UC)is considered to be closely associated with alteration of intestinal microorganisms.According to the traditional Chinese medicine(TCM)theory,UC can be divided into two disease syndromes called Pi-Xu-Shi-Yun(PXSY)and Da-Chang-Shi-Re(DCSR).The relationships among gut microbiota,TCM syndromes,and UC pathogenesis have not been well investigated.AIM To investigate the role of gut microbiota in UC and the distinction of microbiota dysbiosis between PXSY and DCSR syndromes.METHODS From May 2015 to February 2016,UC patients presenting to LongHua Hospital who met the established inclusion and exclusion criteria were enrolled in this retrospective study.Fresh stool specimens of UC patients with PXSY or DCSR were collected.The feces of the control group came from the health examination population of Longhua Hospital.The composition of gut bacterial communities in stool samples was determined by the pyrosequencing of 16S ribosomal RNA.The high-throughput sequencing reads were processed with QIIME,and biological functions were predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States.RESULTS The composition of gut bacterial communities in 93 stool samples(30 healthy controls,32 patients with PXSY syndrome,and 31 patients with DCSR syndrome)was determined by the pyrosequencing of 16S ribosomal RNA.Beta diversity showed that the composition of the microbiota was different among the three groups.At the family level,Porphyromonadaceae,Rikeneliaceae,and Lachnospiraceae significantly decreased while Enterococcus,Streptococcus,and other potential pathogens significantly increased in UC patients compared to healthy subjects.At the genus level,Parabacteroides,Dorea,and Ruminococcus decreased while Faeca-libacterium showed increased abundance in UC compared to healthy controls.Five differential taxa were identified between PXSY and DCSR syndromes.At the genus level,a significantly increased abundance of Streptococcus was observed in DCSR patients,while Lachnoclostridium increased in PXSY patients.The differential functional pathways of the gut microbiome between the PXSY and DCSR groups mainly included lipid metabolism,immunity,and the metabolism of polypeptides.CONCLUSION Our study suggests that the gut microbiota contributes to the distinction between the two TCM syndromes of UC.

Systems pharmacology approach reveals protective mechanisms of Jian-Pi Qing-Chang decoction on ulcerative colitis

BACKGROUND Given the complex pathogenesis of ulcerative colitis (UC), the conventional therapeutic methods are not fully curative. As a sort of systematic complementary and alternative medicine, traditional Chinese medicine (TCM) provides new options for the standard therapy. Nevertheless, there are still numerous problems with the promotion of TCM attributed to its complexity, and consequently, new research approaches are urgently needed. Thus, we explored the protective effects of Jian-Pi Qing-Chang (JPQC) decoction on UC based on systems pharmacology approach, which might fill the current innovation gap in drug discovery and clinical practice pertaining to TCM. AIM To investigate the protective mechanisms of JPQC decoction on UC based on systems pharmacology approach. METHODS We performed systems pharmacology to predict the active ingredients, the matched targets, and the potential pharmacological mechanism of JPQC on UC. In vivo, we explored the effects of JPQC in a colitis model induced by dextran sulfate sodium. In vitro, we adopted the bone marrow-derived macrophages (BMDMs) as well as BMDMs co-cultured with Caco2 cells to verify the underlying mechanisms and effects of JPQC on UC under TNF-α stimulation. RESULTS Systems pharmacology revealed 170 targets for the 107 active ingredients of JPQC and 112 candidate targets of UC. Protein-protein interaction networks were established to identify the underlying therapeutic targets of JPQC on UC. Based on enrichment analyses, we proposed our hypothesis that JPQC might have a protective effect on UC via the NF-κB/HIF-1α signalling pathway. Subsequent experimental validation revealed that treatment with TNFα activated the NF-κB/HIF-1α signalling pathway in BMDMs, thereby damaging the epithelial barrier permeability in co-cultured Caco2 cells, while JPQC rescued this situation. The findings were also confirmed in a dextran sulfate sodium-induced colitis model. CONCLUSION JPQC could improve the mucosal inflammatory response and intestinal epithelial barrier function via the NF-κB/HIF-1α signalling pathway, which provides new perspectives on the pharmaceutical development and clinical practice of TCM.

Systems pharmacology approach reveals the antiinflammatory effects of Ampelopsis grossedentata on dextran sodium sulfate-induced colitis

AIM To investigate the protective effects of Ampelopsis grossedentata(AMP) on dextran sulfate sodium(DSS)-induced colitis in mice based on systems pharmacology approach.METHODS Systems pharmacology approach was used to predict the active ingredients, candidate targets and the efficacy of AMP on ulcerative colitis(UC) using a holistic process of active compound screening, target fishing, network construction and analysis. A DSSinduced colitis model in C57 BL/6 mice(n = 10/group) was constructed and treated with 5-aminosalicylic acid(100 mg/kg/d) and AMP(400 mg/kg/d) to confirm the underlying mechanisms and effects of AMP on UC with western blot analyses, polymerase chain reaction, histological staining and immunohistochemistry.RESULTS The therapeutic effects of AMP against DSS-induced colitis were determined in the beginning, and the results showed that AMP significantly improved the disease in general observations and histopathology analysis. Subsequent systems pharmacology predicted 89 corresponding targets for the four candidate compounds of AMP, as well as 123 candidate targets of UC, and protein-protein interaction networks were constructed for the interaction of putative targets of AMP against UC. Enrichment analyses on TNF-α and RANKL/RANK, a receptor activator of NF-κB signaling pathways, were then carried out. Experimental validation revealed that inflammation-related signaling pathways were activated in the DSS group, and AMP significantly suppressed DSS-induced high expression of IRAK1, TRAF6, IκB and NF-κB, and inhibited the elevated expression levels of TNF-α, IL-1β, IL-6 and IL-8.CONCLUSION AMP could exert protective effects on UC via suppressing the IRAK1/TRAF6/NF-κB-mediated inflammatory signaling pathways.

Jianpi Qingchang Decoction-containing serum regulates the autophagy ofinterstitial cells

Objective： To investigate the effects of Jianpi Qingchang Decoction-containing serum （JQD-CS） on interstitial cells ofCajal （ICCs） autophagy and cell cycle arrest in vitro. Methods： ICCs were collected from the small intestines of miceand analyzed using an anti-c-Kit antibody. ICCs were divided into five groups： the blank group, the rapamycin group （anautophagy inducer）, the 5% （rapamycin ＋ 5% JQD-CS）, the 20% （rapamycin ＋ 20% JQD-CS） JQD-CS groups, and the3-Methyladenine （3-MA） group （rapamycin ＋ 3-MA; positive control）. Transmission electron microscopy was used toobserve the ultrastructure of ICCs. Western blotting was used to detect the expression of microtubule-associated protein1 light chain 3 （LC3-II）, Beclin-1, phosphatidylinositol 3-kinase （PI3K）, p-PI3K, protein kinase B （AKt）, p-AKt,mammalian target of rapamycin （mTOR）, and p-mTOR. Ca2＋ current was examined by patch-clamp experiments. Cellcycle was detected by flow cytometry. Results： Unlike in the rapamycin group, the ICC structures were more integratedand a lower number of autophagic vacuoles were observed in the 20% JQD-CS group. Moreover, the expression ofLC3-II and Beclin-1 decreased, the expression of c-Kit, p-PI3K, p-AKt increased, the maximum current density valuedecreased, and the number of cells in the G1 phase increased while those in the G2/M phase decreased, with the additionof 20% JQD-CS. Conclusion： JQD-CS can antagonize rapamycin-induced autophagy in ICCs in vitro by promoting thephosphorylation of PI3K/AKt pathway, inhibiting Ca2 ＋ inflow, and regulating the cell cycle.