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. 2022 Sep 28:2022:5219277.
doi: 10.1155/2022/5219277. eCollection 2022.

Shenxian-Shengmai Oral Liquid Evoke Autophagy of Fibroblast to Attenuate Sinoatrial Node Fibrosis in Sick Sinus Syndrome Mice via the AKT/mTOR Pathway

Chen Chen  1 , Heng Zhang  1 , Siyi Hou  2 , Yue Liu  1 , Lu Ren  1   2 , Miao Hao  1 , Keyan Chen  3 , Lingkang Li  1 , Xintong Cai  1 , Zisu Deng  1 , Zhuang Liu  1 , Ping Hou  1   2
Affiliations

Shenxian-Shengmai Oral Liquid Evoke Autophagy of Fibroblast to Attenuate Sinoatrial Node Fibrosis in Sick Sinus Syndrome Mice via the AKT/mTOR Pathway

Chen Chen et al. Evid Based Complement Alternat Med. .

Abstract

Sick sinus syndrome (SSS) is closely associated with cardiac syncope and sudden death, wherein sinoatrial node (SAN) fibrosis is one of the main pathological changes that occur. Shenxian-Shengmai oral liquid (SXSM) has been clinically proven to significantly improve the heart rate of SSS patients. In this study, we aimed to explore the mechanism of SXSM in reducing the SAN fibrosis by combining in vitro and in vivo experiments. Accordingly, the SSS model was constructed by slowly pumping angiotensin II (AngII) with a micro-osmotic pump. The degree of fibrosis was evaluated by Masson's trichrome staining and immunofluorescence staining of the fibrosis marker protein. In addition, NIH-3T3 mouse fibroblasts were used to simulate SAN fibroblasts to further explore the mechanism, with AngII used as the cellular fibrosis inducer. Monodansylcadaverine (MDC) staining and transmission electron microscopy were employed to assay the autophagy content, whereas immunofluorescence staining and Western blotting were employed to elucidate the related protein expression. Finally, fibroblasts were given the AKT phosphorylation agonist SC79 to reversely verify the effects of SXSM. The results showed that SXSM could significantly increase the heart rate of SSS mice by reducing the deposition of extracellular matrix (ECM) in SAN induced by AngII. According to in vivo experiments, when compared with the model group, SSS mice treated with SXSM developed less fibrosis in the SAN area. In vitro experiments revealed that AngII could restrain autophagy by activating the phosphorylation of the AKT/mTOR pathway, thereby increasing the deposition of ECM. Moreover, SXSM pretreatment prevented this upregulation. After the intervention of SC79, the protective effect of SXSM was weakened. In conclusion, SXSM activated autophagy through the AKT/mTOR pathway, which in turn reduced the deposition of the ECM in SAN induced by AngII, attenuated the fibrosis of SAN, and improved the decreased heart rate in the SSS mice.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
SXSM improved the function of the SAN as well as increased the heart rate of the SSS mice. (a)The resting heart rate of each group of mice (125 ms/Div). (b)The statistical analysis of the heart rate in each mice group. (c)The statistical analysis of the R-R interval of the electrocardiogram in each group of mice. ∗∗P < 0.01 vs. SSS. SHAM: Sham-operation group. SSS: sick sinus syndrome model group. SXSM: Shenxian-Shengmai oral liquid treatment group.
Figure 2
Figure 2
SXSM attenuates SAN fibrosis in SSS mice. (a)Masson's trichrome staining of the SAN area of mice in each group. (b)Immunofluorescence staining of fibronectin and collagen-I in the SAN area of mice in each group. (c)Statistical analysis of the collagen volume fraction in the SAN area of mice in each group. (d)The results of the statistical analysis of the average optical density of fibronectin and collagen-I in the SAN area of mice in each group. ∗∗P < 0.01 vs. SSS. SHAM: sham-operation group. SSS: sick sinus syndrome model group. SXSM: Shenxian-Shengmai oral liquid treatment group.
Figure 3
Figure 3
Experimental results of CCK-8 assay. The control group cell viability was set to 100%. This analysis showed that SXSM did not induce cytotoxicity at the 10 mL/L concentration treatment for 48 h and demonstrated the most obvious effect on the NIH-3T3 cell line at the concentration of 1 mL/L.
Figure 4
Figure 4
SXSM improved the fibrosis of NIH-3T3 cells induced by AngII, and this effect could be blocked by SC79. (a)Immunofluorescence staining of NIH-3T3 cell fibrosis marker protein. (b)Statistical analysis of collagen-I average optical density. (c)Statistical analysis of fibronectin average optical density. (d)Statistical analysis of the α-SMA average optical density. ##P < 0.01 vs. AngII; ∗∗P < 0.01 vs. SXSM. CON: control group cells. Ang II: cells treated with AngII. SXSM: cells treated with AngII and SXSM. SC79: cells treated with AngII, SXSM, and SC79. AngII: angiotensin II; SXSM: Shenxian-Shengmai oral liquid; SC79: AKT phosphorylation agonist.
Figure 5
Figure 5
SXSM attenuated the autophagy inhibition of AngII on NIH-3T3 cells; this effect could be blocked by SC79. (a)MDC staining of NIH-3T3 cells. (b)Transmission electron microscopy observation of autophagosomes of NIH-3T3 cells. Scale bar: 5 μm, 2 μm. (c)Statistical analysis of MDC staining of NIH-3T3 cells.(d)Statistical analysis the of number of autophagosomes. #P < 0.05 vs. AngII, ##P < 0.01 vs. AngII; and ∗∗P < 0.01 vs. SXSM. CON: control group cells. Ang II: cells treated with AngII. SXSM: cells treated with AngII and SXSM. SC79: cells treated with AngII, SXSM, and SC79. AngII: angiotensin II; SXSM: Shenxian-Shengmai oral liquid; SC79: AKT phosphorylation agonist.
Figure 6
Figure 6
Protein assay of the Akt/mTOR signaling pathway and autophagy in NIH-3T3 cells. (a)Western blotting was applied to detect the expression of AKT, p-AKT, mTOR, p-mTOR, Beclin-1, p62, and LC3-II/LC3-I; (b)Quantitative analysis of the Western blotting results (a) #P < 0.05 vs. AngII, ##P < 0.01 vs. AngII; ∗P < 0.05 vs. SXSM, and ∗∗P < 0.01 vs. SXSM. CON: control group cells. Ang II: cells treated with AngII. SXSM: cells treated with AngII and SXSM. SC79: cells treated with AngII, SXSM, and SC79. AngII: angiotensin II; SXSM: Shenxian-Shengmai oral liquid; SC79: AKT phosphorylation agonist.
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