Dihydromyricetin improving myocardial function in the mice induced by CCl4

Abstract

Objective: To study the role and underlying mechanisms of dihydromyricetin on the myocardial function in mice induced by CCl₄.

Methods: Eighteen C57BL/6 mice (6-8 W, female) were randomly divided into the following three groups: control group, CCl₄-induced positive group (CCl₄ group), dihydromyricetin group, six mice/group. NLRP3-deficient (NLRP3^-/-) C57BL/6 mice used the same age, gender, and modeling method. The HL-1 cells were used for in vitro experiments. The HL-1 cells were treated with PBS, CCl₄, and CCl₄ + DMY respectively.

Results: The RT-qPCR results showed that compared to the mice induced by CCl₄, the dihydromyricetin increased the Arg-1 mRNA level in the mouse myocardial tissues. The mRNA expressions of the iNOS, IL-33, and ST2 were reduced by the dihydromyricetin. The results of immunohistochemistry showed that dihydromyricetin decreased IL-33 protein expressions in the myocardial tissues. Western blot results also showed that compared with the control group, the activation of NLRP3 inflammasomes in the myocardial tissues of mice injured by CCl₄ was increased, and dihydromyricetin can reduce NLRP3 inflammasomes activation in the myocardial tissues induced by CCl₄. The results of ELISA showed that dihydromyricetin could reduce the IL-1β level in the serum of the mice induced by CCl_4. Consistent with the in vivo results, compared with the control group, the NLRP3 inflammasome activation and IL-33/ST2 expression were increased in the CCl₄-treated HL-1 cells, while DMY significantly weakened this effect. Interestingly, NLRP3 deficiency enhanced the protective effect of DMY on myocardial function in mice.

Conclusions: IL-33/ST2 signaling pathways and NLRP3 inflammasome activation may be involved in dihydromyricetin improving the myocardial function of the mice induced by CCl₄.

Keywords: IL-33; NLRP3 inflammasome; ST2; dihydromyricetin; myocardial function.

Figure 1.

The schematic diagram of in vivo and in vitro experimental procedures. In vivo protocol, CCl₄ administration, the mice received intraperitoneal injections of CCl₄ (10%) from the initial modeling day, twice weekly for 8 W. Dihydromyricetin (DMY) treatment, the dihydromyricetin (10 mg/ml) was given intragastrically to the mice every day for 7 days prior to CCl₄ exposure, continuing throughout the experimental period. In vitro protocol, the HL-1 cells were inoculated into six-well plates at 1 × 10⁶/ml, using complete DMEM medium for 24 h. The HL-1 cells were treated with PBS, CCl₄, and CCl₄ + DMY respectively. The cells were treated with CCl₄ (40 mmol/l) for 1 h and DMY (50 μmol/l) for 24 h.

Figure 2.

Body weight change curve of the mice in different groups. Eighteen female C57BL/6 mice were randomly divided into the three groups: the control group (given vehicle only), the CCl₄ group (given CCl₄ with vehicle) and the dihydromyricetin group (given dihydromyricetin 7 days prior to CCl₄ administration), with six mice in each group. The body weights were monitored every day, and the weight change curve was drawn in weeks (n = 18). The DMY group mice were compared with the control group and the CCl₄ group mice, ^##p < 0.01, ***p < 0.001, ^&&&p < 0.001.

Figure 3.

DMY reduced the increase of heart weight caused by CCl₄. (a) The weight of the three group mice was weighed and compared before with the dissection (n = 18). (b) Take out the mouse hearts, weigh and compare the weight of the mouse hearts among the three groups (n = 18). (c) The heart indexes were calculated. The heart indexes = heart weight/body weight of mice before with the dissection (n = 18). The values represent mean ± SD (n = 6). The DMY group mice were compared with the CCl₄ group mice, *p < 0.05, ***p < 0.001.

Figure 4.

The effect of dihydromyricetin on histomorphology of the mouse heart. The myocardial tissues in the mice were extracted, embedded in the paraffin and stained with HE staining. The myocardial tissue morphology was observed under the microscope (100×). The arrow indicates the main site of the lesion.

Figure 5.

DMY alleviated the cardiac inflammation caused by CCl₄. (a) The iNOS mRNA expressions in the myocardium tissues of the mice were detected by the RT-qPCR (n = 18). (b) The mRNA expressions of Arg-1 in the myocardium tissues were determined by the RT-qPCR (n = 18). The values represent mean ± SD (n = 6). The dihydromyricetin group mice were compared with the CCl₄ group mice, ^#p < 0.05. The CCl₄ group mice were compared with the blank control group mice, **p < 0.01, ***p < 0.001. iNOS: inducible NO synthase; Arg-1: Arginase-1.

Figure 6.

DMY inhibited the NLRP3 inflammasome activation induced by CCl₄. Eighteen female C57BL/6 mice were randomly divided into the three groups: the control group (given CCl₄ and dihydromyricetin solvent), the CCl₄ group (given CCl₄) and the dihydromyricetin group (given dihydromyricetin 7 days before CCl₄ administration), with 6 mice in each group. (a–d) The protein levels of the NLRP3, IL-1β, pro-IL-1β, Caspase-1, pro-caspase-1, and GAPDH in the myocardial tissues were measured by Western Blot (n = 18). (e) The expression of IL-1β in myocardial tissue was determinated by the RT-qPCR. (f) The IL-1β levels in the serum were detected by the ELISA (n = 18). The HL-1 cells were treated with PBS (Control), CCl₄, and CCl₄ +DMY respectively. (g–j) The protein levels of the NLRP3, IL-1β, pro-IL-1β, Caspase-1, pro-caspase-1, and GAPDH in the HL-1 cells were measured by Western Blot (n = 3). (k) The expression of IL-1β in the HL-1 cells was determinated by the RT-qPCR (n = 3). (l) The IL-1β levels in the HL-1 cell supernatant were detected by the ELISA (n = 3). The DMY group mice were compared with the CCl₄ group mice, *p < 0.05. NLRP3, NOD-like receptor protein 3; Caspase-1, cysteinyl aspartate specific proteinase 1; pro-caspase-1, the precursor of cysteinyl aspartate specific proteinase 1; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase.

Figure 7.

DMY decreased the expressions of IL-33 and ST2 induced by CCl₄. (a and b) The mRNA and protein expressions of IL-33 and ST2 in the myocardium tissues of the mice were detected by the RT-qPCR and Western Blot, respectively (n = 18). (c) The immunohistochemistry showed the IL-33 expression (scale = 50 μm). (d) The expressions of IL-33 and ST2 in the HL-1 cells were detected by RT-qPCR (n = 3). (e) The expressions of IL-33 and ST2 in the HL-1 cells were also detected by Western Blot (n = 3). The DMY group mice were compared with the CCl₄ group mice, *p < 0.05, **p <0.01, ***p < 0.001. ST2, suppression of tumorigenicity 2.

Figure 8.

NLRP3 deficiency enhanced the protective effect of DMY on myocardial function in mice. NLRP3-deficient (NLRP3^-/-) C57BL/6 were selected and performed in the same grouping and modeling methods as WT mice. Taking the mouse tails, RT-qPCR (a) and western blot (b) detected the NLRP3 expression in the tails of NLRP3-deficient (NLRP3^-/-) C57BL/6 (n = 18). (c–f) The changes of left ventricular function (LVIDs, LVIDd, LVEF, and LVFS) were detected by echocardiography (n = 18). The values represent mean ± SD (n = 6). The NLRP3^-/- group mice were compared with the control group mice, ^&p < 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.