Background

Shaoyao decoction (SYD), a traditional Chinese medicine prescription that originated in the Jin-Yuan Dynasty, has shown effects in treating ulcerative colitis. However, the underlying mechanism is unclear. We combined network pharmacology with molecular biology technology to detect the mechanism underlying the effect of SYD on ulcerative colitis. We combined network pharmacology with molecular biology technology to detected the further mechanism in SYD effect on ulcerative colitis.

Purpose

In this study, we investigated the mechanism by which SYD exerts a protective effect against ulcerative colitis in vivo and in vitro.

Study design and methods

We focused on two aspects of the mechanism by which SYD relieves ulcerative colitis, regulation of the MAPK cascade and the NF-κB signaling pathway, through analysis of the “active ingredient-target-disease” network followed by GO enrichment and KEGG pathway analysis according to network pharmacology. Mice with ulcerative colitis underwent 5% dextran sulfate sodium (DSS), and the RAW 264.7 cell model was used to identify important targets.

Results

We found that after 5% DSS treatment, the inflammation indexes and the expression of NLRP3-related proteins were increased concomitant with the loss of mucins and occludin. Treatment with SYD (2.25 g/kg, BW) significantly improved the expression of mucins and occludin after DSS at the protein and transcriptional levels. Furthermore, SYD treatment significantly reduced NF-κB P65 and P38 expression, thus exerting a great antinecrotic effect, as revealed by TUNEL staining and Western blotting. The beneficial effects of SYD were almost canceled by NSC 95397 (an inhibitor of mitogen-activated protein kinase phosphatase-1 (MKP1)) after DSS treatment in vivo or LPS treatment in vitro. In addition, treatment with SYD reduced caspase-1 activity and rescued the release of ASC and GSDMD, thus inhibiting the assembly of NLRP3 and maintaining the integrity of the intestinal barrier. We also conducted in vitro experiments in the LPS-induced RAW 264.7 cell model and found that cells incubated with 1 mg/ml SYD for 24 h possessed the highest cell viability. Next, we incubated 1 mg/ml SYD for 24 h after treatment with 1 µg/ml LPS for 6 h. We showed that 1 mg/ml SYD displayed anti-inflammatory and anti-necrotic effects through the NLRP3, NF-κB P65 and P38 pathways, and the effects of SYD were also inhibited by 10 nM NSC 95397.

Conclusion

These results demonstrate that SYD has protective effects against ulcerative colitis and alleviates pyroptosis by inhibiting the MKP1/NF-κB/NLRP3 pathway.

中文翻译：

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芍药汤通过MKP1/NF-κB通路减弱DSS诱导的溃疡性结肠炎、巨噬细胞和NLRP3炎性体激活

背景

芍药汤是一种起源于晋元的中药方剂，在治疗溃疡性结肠炎方面已见成效。然而，潜在的机制尚不清楚。我们将网络药理学与分子生物学技术相结合，检测SYD对溃疡性结肠炎影响的潜在机制。我们将网络药理学与分子生物学技术相结合，检测SYD对溃疡性结肠炎作用的进一步机制。

目的

在这项研究中，我们研究了 SYD 在体内和体外对溃疡性结肠炎发挥保护作用的机制。

研究设计和方法

我们通过对“活性成分-靶点-疾病”网络的分析，然后进行 GO 富集和 KEGG 通路分析，重点关注 SYD 缓解溃疡性结肠炎机制、MAPK 级联调节和 NF-κB 信号通路两个方面。到网络药理学。溃疡性结肠炎小鼠接受 5% 葡聚糖硫酸钠 (DSS) 治疗，并使用 RAW 264.7 细胞模型确定重要靶点。

结果

我们发现，在 5% DSS 处理后，炎症指标和 NLRP3 相关蛋白的表达增加，同时黏蛋白和 occludin 的丢失。SYD (2.25 g/kg, BW) 处理显着改善了 DSS 后粘蛋白和 occludin 在蛋白质和转录水平上的表达。此外，TUNEL 染色和蛋白质印迹表明，SYD 处理显着降低了 NF-κB P65 和 P38 的表达，从而发挥了很好的抗坏死作用。在体内DSS治疗或体外LPS治疗后，SYD的有益作用几乎被NSC 95397（丝裂原活化蛋白激酶磷酸酶-1（MKP1）的抑制剂）抵消. 此外，SYD 治疗降低了 caspase-1 活性并挽救了 ASC 和 GSDMD 的释放，从而抑制了 NLRP3 的组装并保持了肠道屏障的完整性。我们还在 LPS 诱导的 RAW 264.7 细胞模型中进行了体外实验，发现用 1 mg/ml SYD 孵育 24 小时的细胞具有最高的细胞活力。接下来，我们在用 1 µg/ml LPS 处理 6 小时后，将 1 mg/ml SYD 孵育 24 小时。我们发现 1 mg/ml SYD 通过 NLRP3、NF-κB P65 和 P38 通路显示出抗炎和抗坏死作用，并且 SYD 的作用也被 10 nM NSC 95397 抑制。

结论

这些结果表明，SYD 对溃疡性结肠炎具有保护作用，并通过抑制 MKP1/NF-κB/NLRP3 通路减轻细胞焦亡。