Matrine protects colon mucosal epithelial cells against inflammation and apoptosis via the Janus kinase 2 /signal transducer and activator of transcription 3 pathway

Abstract

Ulcerative colitis (UC) is a type of chronic disease of inflammation, and matrine has anti-inflammatory activity. However, it is unclear that whether matrine can alleviate UC. This study aimed to evaluate the effect of matrine on DSS-induced intestinal epithelial cell injury. Cell viability was performed by MTT assay. Then cell apoptosis was analyzed using the TUNEL assay and flow cytometry. The levels of interleukin (IL)-2, IL-6, TNF-α, and IL-1β were evaluated using qRT-PCR. Myeloperoxidase (MPO) activity was detected using ELISA assay. Nitric oxide (NO) production was detected by the Griess reagent. Bax, cleaved caspase-3, Bcl-2, JAK2, p-JAK2, STAT3, p-STAT3, STAT5, p-STAT5 levels were measured by Western blot. Bax (6A7) was asses using immunoprecipitation and immunofluorescence assays. The results illustrated that cell viability was inhibited as the concentration of DSS increased. Matrine did not affect cell viability at the concentration of 0-2 mg/ml but inhibited cell viability in a time-independent manner. Matrine suppressed the levels of pro-inflammatory factors, MPO activity, NO production, and apoptosis of DSS-stimulated cells. Furthermore, we found that matrine inhibited the levels of p-JAK2/JAK2 and p-STAT3/STAT3 but did not affect p-STAT5/STAT5. AG490 treatment further enhanced the effect of matrine on the apoptosis and pro-inflammatory factor levels in DSS-induced cells. In summary, matrine protected NCM460 cell against injury by inactivating the JAK2/STAT3 pathway. These data suggested for the first time that matrine may effective in treating UC.

Keywords: JAK2; STAT3 pathway; Ulcerative colitis; apoptosis; matrine.

Figure 1.

Effects of matrine on cell viability of NCM460 cells. (a) Chemical structure of matrine. (b) Cell viability was measured using MTT assay after NCM460 cells treating with 0, 0.5, 1, 2, and 4% DSS for 24 h. (c) Cell viability was assessed using MTT assay after treating with 0, 0.25, 0.5, 1, 2, and 4 mg/ml matrine for 24 h. (d) Cell viability was assessed using MTT assay after the cells treating with 2 mg/ml matrine for 0, 12, 24, 48 and 72 h. (e) Matrine IC50 was calculated. *P < 0.05. **P < 0.01. ***P < 0.001.

Figure 2.

Matrine reduced the release of inflammatory factors in NCM460 cells stimulated by DSS. After DSS-stimulated NCM460 cells treating with 1, 2, and 3 mg/ml matrine, the expression of (a) TNF-α, (b) IL-1β, (c) IL-2, and (d) IL-6 was detected using qRT-PCR, (e) MPO activity was detected using ELISA assay, and (f) NO production was detected using Griess reagent. **P < 0.01 vs. the control group. &P < 0.05 and &&P < 0.01 vs. the DSS group. ns: no significant difference.

Figure 3.

Matrine suppressed DSS-induced apoptosis of NCM460 cells. Cell apoptosis was assessed using (a) the TUNEL assay, and (b) flow cytometry. (c) The Bcl-2, Bax (6A7), Bax, and cleaved caspase-3 expression was measured by Western blot. (d) Bax (6A7) was examined using IF assay. **P < 0.01 vs. the control group. &P < 0.05 and &&P < 0.01 vs. the DSS group.

Figure 4.

Effect of matrine on JAK2/STAT3 pathway. The JAK2, p-JAK2, STAT3, p-STAT3, STAT5, and p-STAT5 expression was tested using Western blot. **P < 0.01 vs. the control group. &&P < 0.01 vs. the DSS group.

Figure 5.

Matrine suppresses DSS-induced NCM460 cell apoptosis via the JAK2/STAT3 pathway. (a) Cell viability was measured using MTT assay after NCM460 cells treating with 0, 1, 2.5, 5, 10, 20, and 40 μM for 24 h. Cell apoptosis was assessed using (b) the TUNEL assay, and (c) flow cytometry. (d) Western blot was conducted to analyze the expression of apoptotic related factors including Bax (6A7), Bcl-2, Bax, cleaved caspase-3, and PARP. (e) Bax (6A7) was examined using IF assay. **P < 0.01 vs. the control group. &P < 0.05 and &&P < 0.01 vs. the DSS group. #P < 0.05 and ##P < 0.01 vs. the DSS + Matrine group.

Figure 6.

Matrine alleviates DSS-induced inflammation in NCM460 cells through the JAK2/STAT3 pathway. After DSS-induced NCM460 cells treating with matrine and AG490, the expression of (a) TNF-α, (b) IL-1β, (c) IL-2, and (d) IL-6 was detected using qRT-PCR, (e) MPO activity was detected using ELISA assay, and (f) NO production was detected using Griess reagent. **P < 0.01 vs. the control group. &P < 0.05 and &&P < 0.01 vs. the DSS group. #P < 0.05 and ##P < 0.01 vs. the DSS + Matrine group.