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. 2017 Jan;21(1):4-12.
doi: 10.1111/jcmm.12915. Epub 2016 Oct 26.

Naoxintong attenuates Ischaemia/reperfusion Injury through inhibiting NLRP3 inflammasome activation

Yaqiong Wang  1   2 , Xiaoxiang Yan  3 , Shouling Mi  1 , Zhang Li  4   5 , Yuexiang Wang  4   5 , Hong Zhu  1 , Xiaolei Sun  1 , Buchang Zhao  6 , Chao Zhao  6 , Yunzeng Zou  1   7 , Kai Hu  1 , Xiaoqiang Ding  2 , Aijun Sun  1   7 , Junbo Ge  1   7
Affiliations

Naoxintong attenuates Ischaemia/reperfusion Injury through inhibiting NLRP3 inflammasome activation

Yaqiong Wang et al. J Cell Mol Med. 2017 Jan.

Abstract

Naoxintong (NXT) is a Chinese Materia Medica standardized product extracted from 16 various kinds of Chinese traditional herbal medicines including Salvia miltiorrhiza, Angelica sinensis, Astragali Radix. Naoxintong is clinically effective in treating ischaemia heart disease. Nucleotide-binding oligomerization domain-Like Receptor with a Pyrin domain 3 (NLRP3) inflammasome has been critically involved in myocardial ischaemia/reperfusion (I/R) injury. Here, we have been suggested that NXT might attenuate myocardial I/R injury via suppression of NLRP3 inflammasome activation. Male C57BL6 mice were subjected to myocardial I/R injury via 45 min. coronary ligation and release for the indicated times. Naoxintong (0.7 g/kg/day) and PBS were orally administrated for 2 weeks before surgery. Cardiac function assessed by echocardiography was significantly improved in the NXT group compared to PBS group at day 2 after myocardial I/R. NLRP3 inflammasome activation is crucially involved in the initial inflammatory response after myocardial I/R injury, leading to cleaved caspase-1, mature interleukin (IL)-1β production, accompanying by macrophage and neutrophil infiltration. The cardioprotective effect of NXT was associated with a diminished NLRP3 inflammasome activation, decreased pro-inflammatory macrophage (M1 macrophages) and neutrophil infiltration after myocardial I/R injury. In addition, serum levels of IL-1β, indicators of NLRP3 inflammasome activation, were also significantly suppressed in the NXT treated group after I/R injury. Naoxintong exerts cardioprotive effects at least partly by suppression of NLRP3 inflammasome activation in this I/R injury model.

Keywords: NLRP3 inflammasome; inflammation; ischaemia reperfusion injury; macrophage polarity; naoxintong.

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Figures

Figure 1
Figure 1
NXT improved ventricular function after myocardial I/R injury. (A) Representative M‐mode of echocardiographic images of the heart at day 2 after myocardial I/R injury in four treatment groups. (B) Echocardiographic analysis of ejection fraction (EF) and fractional shortening (FS) after I/R or sham operation (n = 7–9). (C) Echocardiographic analysis of ejection fraction (EF) and fractional shortening (FS) after I/R or sham operation between C57BL/6 and NLRP3 KO mice (n = 6). NS, not significant; **P < 0.01. Statistical analysis was performed by ANOVA with bonferroni post hoc analysis.
Figure 2
Figure 2
NXT decreased infarct size after myocardial ischaemia reperfusion (I/R) injury. (A and C) At 48 hrs after I/R, hearts were perfused with Evans blue and stained with 2,3,5‐triphenyltetrazolium chloride (TTC) for the measurement of infarct area. Blue area is non‐ischaemia zone, viable parts of the heart appear red and the infarct area white. Area at risk (AAR) includes the red and white parts. A is C57BL/6 mice and C is NLRP3 KO mice. (B and D) Quantification of the infarct area/AAR shows that infarct size was reduced in the NTX treatment group, whereas AAR/LV was comparable between two groups (n = 7). B is C57BL/6 mice and D is NLRP3 KO mice. Statistical analysis was performed by t‐test.
Figure 3
Figure 3
NXT suppressed NLRP3 inflammasome activation in the heart after myocardial I/R injury. The protein levels of NLRP3, pro‐caspase‐1 (Pro‐Casp‐1), cleaved caspase‐1 (Casp‐1 P20) and mature IL‐1β (IL‐1β P17) were determined by western blot in the heart at 24 hrs post I/R. Each bands were quantified. Data represent three independent experiments. A is C57BL/6 mice and B is NLRP3 KO mice. NS, not significant, *P < 0.05, **P < 0.01. Data were analysed by anova with bonferroni post hoc analysis.
Figure 4
Figure 4
NXT inhibited mature IL‐1β expression. (A) IL‐1β immunostaining of heart tissue on day 1 post I/R (n = 5). (B) Serum levels of IL‐1β were determined with ELISA kit at 24 hrs after I/R (n = 6–9). NS, not significant, *P < 0.05, **P < 0.01. Data were analysed by t‐test (A) and anova with bonferroni post hoc analysis (B).
Figure 5
Figure 5
Gating strategy for infiltrating myeloid cells in the ischemic heart. Single cells suspension prepared from the heart at day 2 after I/R was stained for CD11b, Gr‐1 and F4/80, allowing the identification of 2 different populations: CD11b+Gr‐1+ neutrophils and CD11b+F4/80+ macrophages. Macrophages were further divided into M1 and M2 macrophages based on the expression levels of CD11b and F4/80.
Figure 6
Figure 6
Macrophage and nuetriphil infiltration in the heart were reduced by NXT treatment. (A and B) Flow cytometric analysis of infiltrating macrophages, neutrophils (A) and M1 and M2 macrophages (B) in the heart on day 2 post I/R (n = 4–6 each). (C) F4/80 immunostaining of heart tissue on day 1 post I/R (n = 5). NS, not significant, *P < 0.05, **P < 0.01. Data were analysed by anova with bonferroni post hoc analysis (A and B) and t‐test (C).
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