Modified Pulsatillae decoction inhibits DSS-induced ulcerative colitis in vitro and in vivo via IL-6/STAT3 pathway

Abstract

Background: Ulcerative colitis (UC) is a chronic inflammatory disorder of the colon and rectum, which is positively correlated with the occurrence of IBD-related colorectal cancer (IBD-CRC). Conventional therapies based on drugs such as corticosteroids, mesalamine, and immunosuppression have serious side effects. Pulsatillae decoction (PD) served as a classical prescription for the treatment of colitis in China, has been shown to exert prominent curative effects and good safety. Based on clinical experience and our amelioration, we added an extra herb into this classical prescription, but its therapeutic effect on UC and the underlying mechanism are still unclear.

Results: We first found the curative effect of modified PD on dextran sodium sulfate (DSS)-incubated NCM460 cells. Then C57BL/6 mice were administered DSS to induce UC to evaluate the therapeutic of modified PD. The results showed that modified PD alleviated the inflammatory injury, manifested in body weight, colon length, and disease activity index, with histological analysis of colon injury. Transcriptomic sequencing indicated that modified PD treatment downregulated the IL-6/STAT3 signaling pathway, and reduced the levels of p-NF-κB, IL-1β and NLRP3, which were confirmed by western blot.

Conclusions: Collectively, our results indict that modified PD could efficiently relieve clinical signs and inflammatory mediators of UC, providing evidence of the anti-colitis effect of modified PD, which might provide novel strategies for therapeutic intervention in UC, which may be applied to the prevention of IBD-CRC.

Keywords: DSS; IL-6/STAT3 pathway; Modified Pulsatillae decoction; NCM460; Ulcerative colitis.

Fig. 1

Modified PD ameliorated DSS-induced cell injury on cell viability (A) NCM460 cells were incubated with various concentrations of DSS for 24 and 48 h and their viability were determined by MTT assay. (B) Cell viability of NCM460 treated with different dosages of modified PD and DSS was analyzed by MTT assay. *: p < 0.05; ***: p < 0.001. Data are expressed as mean ± SEM.

Fig. 2

Modified PD administration alleviated symptoms of DSS-induced UC (A) Mice were divided into negative control group, model group (DSS-treated) and three modified PD groups (DSS-treated with modified PD treatment): low dose (3.185 mg/kg), medium dose (6.37 mg/kg) and high dose (12.74 mg/kg). (B) Loss of basal body weight, (C & D) colon length, (E) Pathological score, and (F) HE staining was performed to evaluate the therapeutic effect of modified PD. *: p < 0.05, **: p < 0.01, ***: p < 0.001, Data are expressed as mean ± SEM.

Fig. 3

High-throughput transcriptomic sequencing of negative control group, model group and modified PD group (n = 3 in every group) (A) The MA plot and volcano plot were used for showing variance in gene expression with respect to fold change (FC) and significance (p-value). Each dot represents an individual gene: The red point in the plot represented significantly upregulated or downregulated RNA (fold change> 2, P < 0.05) and the black point demonstrated RNA with no statistical differences (fold change< 2, P > 0.05) (Left: negative control group vs. model group; Middle: model group vs. modified PD group; Right: negative control group vs. modified PD group). (B) Microarray heat map demonstrates clustering of colon tissues (Left: negative control group vs. model group; Middle: model group vs. modified PD group; Right: negative control group vs. modified PD group). (C) GO classification of target genes. The 30 most significantly enriched GO terms for colon tissues between model group and modified PD group. (D) KEGG pathway classification of target genes. The Y-axis showed the name of pathway and the X-axis represented the rich factor. The point size indicated the number of differentially expressed genes in one pathway, and the color of the point corresponded to the range of the Q value. The 20 most significant up-regulated pathways (model group vs. modified PD group).

Fig. 4

Marker proteins expression of IL-6/STAT3 pathway (A) Western blot analysis of NLRP3, STAT3, p-STAT3, NF-κB, p-NF-κB, p-p65, IL-1β, IL-6 and TNF-α of expression in NCM460 cell. (B) The quantitative analysis for western blot. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Data are expressed as mean ± SEM.

Fig. 5

Modified PD mitigated inflammation in DSS-induced UC in vivo. Colon tissues of negative control group, model group (DSS-treated) and modified PD group (DSS-treated with modified PD treatment) were collected. (A) qRT-PCR results indicating modified PD treatment reduced mRNA level of IL-6, VEGF and TNF-α (Modified PD represent high dose modified PD treatment mice herein). (B) Effect of modified PD on the protein expression of NLRP3, STAT3, p-STAT3, NF-κB, p-NF-κB, p-p65, IL-1β, IL-6 and TNF-α. Protein expression levels were analyzed by Western Blot. (C) The quantitative analysis for western blot. *: p < 0.05, **: p < 0.01, ***: p < 0.001. Data are expressed as mean ± SEM.

Fig. 6

Modified PD inhibits DSS-induced ulcerative colitis in mice through IL-6/STAT3 pathway