Korean J Physiol Pharmacol.  2021 Nov;25(6):533-543. 10.4196/kjpp.2021.25.6.533.

Triptolide improves myocardial fibrosis in rats through inhibition of nuclear factor kappa B and NLR family pyrin domain containing 3 inflammasome pathway

• Shen J
• Ma H
• Wang C

• KMID: 2521479
• DOI: http://doi.org/10.4196/kjpp.2021.25.6.533

Abstract

Myocardial fibrosis (MF) is the result of persistent and repeated aggravation of myocardial ischemia and hypoxia, leading to the gradual development of heart failure of chronic ischemic heart disease. Triptolide (TPL) is identified to be involved in the treatment for MF. This study aims to explore the mechanism of TPL in the treatment of MF. The MF rat model was established, subcutaneously injected with isoproterenol and treated by subcutaneous injection of TPL. The cardiac function of each group was evaluated, including LVEF, LVFS, LVES, and LVED. The expressions of ANP, BNP, inflammatory related factors (IL-1β, IL-18, TNF-α, MCP-1, VCAM-1), NLRP3 inflammasome factors (NLRP3, ASC) and fibrosis related factors (TGF-β1, COL1, and COL3) in rats were dete cted. H&E staining and Masson staining were used to observe myocardial cell inflammation and fibrosis of rats. Western blot was used to detect the p-P65 and t-P65 levels in nucleoprotein of rat myocardial tissues. LVED and LVES of MF group were significantly upregulated, LVEF and LVFS were significantly downregulated, while TPL treatment reversed these trends; TPL treatment downregulated the tissue injury and improved the pathological damage of MF rats. TPL treatment downregulated the levels of inflammatory factors and fibrosis factors, and inhibited the activation of NLRP3 inflammasome. Activation of NLRP3 inflammasome or NF-κB pathway reversed the effect of TPL on MF. Collectively, TPL inhibited the activation of NLRP3 inflammasome by inhibiting NF-κB pathway, and improved MF in MF rats.

Keyword

Endomyocardial fibrosis; Heart function tests; NF-κB pathway; NLRP3 inflammasome; Triptolide

Figure

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  Fig. 1 Chemical structure of TPL. TPL, triptolide.

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  Fig. 2 TPL improved cardiac dysfunction in MF rats. Myocardial fibrosis model was established by subcutaneous injection of ISO, and TPL was injected subcutaneously for 7 days. (A–D) The LVEF, LVFS, LVES and LVED were measured by the small animal ultrasonograph; (E) RT-qPCR was used to detect the ANP and BNP levels in myocardial tissues of rats in each group; n = 6. Three independent repeated tests were performed and the data were expressed as mean ± SD; one-way was used for variance analysis; Tukey’s multiple comparisons test was used for post-hoc test. TPL, triptolide; MF, myocardial fibrosis; DMSO, dimethyl sulfoxide; ISO, isoproterenol; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVES, left ventricular end systolic diameter; LVED, left ventricular end diastolic diameter; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide. Compared with sham group, *p < 0.05, **p < 0.01; compared with MF group, #p < 0.05, ##p < 0.01.

• 

  Fig. 3 TPL downregulated the degree of MF and the expression of fibrosis related factors in MF rats. (A) H&E staining and Masson staining were used to observe the pathological morphology and MF of rats in each group (×200); (B) Masson staining results were quantitatively analyzed by measuring collagen volume fraction; (C) RT-qPCR was used to detect the expressions of TGF-β1, COL1, and COL3; (D) The expressions of TGF-β1, COL1, and COL3 were detected by immunohistochemistry; n = 6. Three independent repeated tests were performed and the data were expressed as mean ± SD; one-way was used for variance analysis; Tukey’s multiple comparisons test was used for post-hoc test. TPL, triptolide; MF, myocardial fibrosis; DMSO, dimethyl sulfoxide. Compared with sham group, **p < 0.01; compared with MF group, #p < 0.05, ##p < 0.01.

• 

  Fig. 4 TPL downregulated the level of inflammatory factors in MF rats. (A, B) RT-qPCR and WB were used to respectively detect the mRNA and protein levels of inflammatory factors IL-1β, IL-18 and TNF-α in the myocardium of rats; (C) RT-qPCR was used to detect the mRNA expression of MCP-1 and VCAM-1 in myocardium of rats in each group; n = 6. Three independent repeated tests were performed and the data were expressed as mean ± SD; one-way ANOVA was used for variance analysis; Tukey’s multiple comparisons test was used for post-hoc test. TPL, triptolide; MF, myocardial fibrosis; WB, Western blot; MCP-1, monocyte chemoattractant protein 1; VCAM-1, vascular cell adhesion molecule 1; DMSO, dimethyl sulfoxide. Compared with sham group, **p < 0.01; compared with MF group, ##p < 0.01.

• 

  Fig. 5 TPL inhibited the activation of NLRP3 inflammasome in MF rats. (A–D) RT-PCR and WB were used to detect the mRNA and protein levels of NLRP3 and ASC in MF rats; (E) The expressions of NLRP3 and ASC were detected by immunohistochemistry (×200); n = 6. Three independent repeated tests were performed and the data were expressed as mean ± SD; one-way ANOVA was used for variance analysis; Tukey’s multiple comparisons test was used for post-hoc test. TPL, triptolide; MF, myocardial fibrosis; WB, Western blot; DMSO, dimethyl sulfoxide. Compared with sham group, **p < 0.01; compared with MF group, ##p < 0.01.

• 

  Fig. 6 TPL improved the degree of MF in MF rats by inhibiting the activation of NLRP3 inflammasome pathway. (A) RT-qPCR was used to detect the mRNA expressions of inflammatory factors IL-1β, IL-18, and TNF-α in myocardial tissues; (B) RT-qPCR was used to detect the mRNA expressions of TGF-β1, COL1, and COL3; (C) The expressions of TGF-β1, COL1, and COL3 were detected by immunohistochemistry (×200); n = 6. Three independent repeated tests were performed and the data were expressed as mean ± SD; independent t-test was used for comparisons between 2 groups. TPL, triptolide; MF, myocardial fibrosis; PBS, phosphate buffer saline. Compared with TPL + PBS group, **p < 0.01.

• 

  Fig. 7 TPL inhibited the activation of NLRP3 inflammasome by inhibiting NF-κB pathway and improved the degree of MF. (A) WB was used to detect the expressions of p-P65 and t-P65 in myocardium nucleoprotein of rats in each group; (B) RT-qPCR was used to detect the mRNA expressions of inflammatory factors IL-1β, IL-18, and TNF-α in myocardial tissues of rats in each group; (C) RT-qPCR was used to detect the mRNA expressions of fibrosis related factors TGF-β1, COL1, and COL3 in myocardium; n = 6. Three independent repeated tests were performed and the data were expressed as mean ± SD; one-way was used for variance analysis; Tukey’s multiple comparisons test was used for post-hoc test. TPL, triptolide; MF, myocardial fibrosis; WB, Western blot; DMSO, dimethyl sulfoxide; PBS, phosphate buffer saline. Compared with TPL + PBS group, **p < 0.01. Compared with the MF group, ##p < 0.01.

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