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

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.

NRF-2/HO-1 Pathway-Mediated SHOX2 Activation Is a Key Switch for Heart Rate Acceleration by Yixin-Fumai Granules.

Population aging has led to increased sick sinus syndrome (SSS) incidence; however, no effective and safe medical therapy has been reported thus far. Yixin-Fumai granules (YXFMs), a Chinese medicine granule designed for bradyarrhythmia treatment, can effectively increase SSS patients' heart rate. Senescence-induced sinoatrial node (SAN) degeneration is an important part of SSS pathogenesis, and older people often show high levels of oxidative stress; reactive oxygen species (ROS) accumulation in the SAN causes abnormal SAN pacing or conduction functions. The current study observed the protective effects of YXFMs on senescent SAN and explored the relationship between the NRF-2/HO-1 pathway, SHOX2, and T-type calcium channels. We selected naturally senescent C57BL/6 mice with bradycardia to simulate SSS; electrocardiography, Masson's trichrome staining, and DHE staining were used to assess SAN function and tissue damage. Immunofluorescence staining and Western blotting were used to assay related proteins. In vitro, we treated human-induced pluripotent stem cell-derived atrial myocytes (hiPSC-AMs) and mouse atrial myocyte-derived cell line HL-1 with D-galactose to simulate senescent SAN-pacemaker cells. CardioExcyte96 was used to evaluate the pulsatile function of the hiPSC-AMs, and the mechanism was verified by DCFH-DA, immunofluorescence staining, RT-qPCR, and Western blotting. The results demonstrated that YXFMs effectively inhibited senescence-induced SAN hypofunction, and this effect possibly originated from scavenging of ROS and promotion of NRF-2, SHOX2, and T-type calcium channel expression. In vitro experiment results indicated that ML385, si-SHOX2, LDN193189, and Mibefradil reversed YXFMs' effects. Moreover, we, for the first time, found that ROS accumulation may hinder SHOX2 expression; YXFMs can activate SHOX2 through the NRF-2/HO-1 pathway-mediated ROS scavenging and then regulate CACNA1G through the SHOX2/BMP4/GATA4/NKX2-5 axis, improve T-type calcium channel function, and ameliorate the SAN dysfunction. Finally, through network pharmacology and molecular docking, we screened for the most stable YXFMs compound that docks to NRF-2, laying the foundation for future studies.

Yixin-Fumai granules improve sick sinus syndrome in aging mice through Nrf-2/HO-1 pathway: A new target for sick sinus syndrome.

ETHNOPHARMACOLOGICAL RELEVANCE: Yixin-Fumai granules (YXFMs)-composed of Ginseng quinquefolium (L.) Alph. Wood, Ophiopogon japonicus (Thunb.) Ker Gawl, Schisandra arisanensis Hayata, Astragalus aaronsohnianus Eig, Salvia cryptantha Montbret & Aucher ex Benth, and Ligusticum striatum DC-are compound granules used in traditional Chinese medicine to increase heart rate and thus treat bradyarrhythmia. It may be effective in treating sick sinus syndrome (SSS). AIM: To observe the effect of YXFMs on aging-induced SSS in mice and explore whether this effect is related to the Nrf-2/HO-1 signaling pathway. MATERIALS AND METHODS: Mice with a significant decrease in the heart rate due to natural aging were selected to construct an SSS model. After the mice were administered YXFMs, the damage to their sinoartrial node (SAN) was assessed through electrocardiography, Masson's trichrome staining, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Dihydroethidium staining and immunofluorescence staining were used to assay reactive oxygen species (ROS) content and HCN4, respectively. Moreover, to observe the effects of YXFMs in vitro, the HL-1 cell line, derived from mouse atrial myocytes, was used to simulate SAN pacemaker cells, with H2O2 used as the cellular oxidative stress (OS) inducer. 2,7-Dichlorodihydrofluorescein diacetate staining was used to assay ROS content, whereas immunofluorescence staining and Western blotting were used to elucidate the related protein expression. Finally, mice were injected the Nrf-2 inhibitor ML385 to reversely verify the effects of YXFMs. RESULTS: In our in vivo experiments, YXFMs significantly inhibited aging-induced SSS, shortened the R-R interval, increased heart rate, alleviated fibrosis, reduced apoptosis rate and ROS content, and promote HCN4 expression in the SAN. In our in vitro experiments, YXFMs significantly inhibited H2O2-induced cell peroxidation damage, promoted Nrf-2 activation and nuclear metastasis, increased HO-1 expression- thereby inhibiting ROS accumulation-and finally, upregulated HCN4 expression through the inhibition of histone deacetylase 4 (HDAC4) expression and its nuclear metastasis. Finally, injection of the Nrf-2 inhibitor ML385 after YXFMs administration inhibited their protective effect in the mice. CONCLUSION: Here, we elaborated on the relationship between aging-induced SSS and the Nrf-2/HO-1 pathway for the first time and proposed that YXFMs improve SSS via the Nrf-2/HO-1 axis. Specifically, YXFMs promoted Nrf-2 activation and plasma-nuclear transfer to enhance HO-1 expression via the Nrf-2/HO-1 axis. This inhibited OS and reduced ROS accumulation in the SAN, and then, through the ROS/HDAC4 axis, reduced HDAC4 expression and plasma-nuclear transfer. Thereby, the OS-induced HCN4 loss in the SAN was inhibited-improving the function of If channel and thus producing SAN protection effect against SSS and improving the heart rate and R-R interval. In the future, we plan to use bioinformatics analysis technology to execute the next step of our research, namely to determine the effect of isolated, purified components of YXFMs in SSS, to increase its efficiency and reduce the toxicity of YXFMs.

Shenxian-Shengmai Oral Liquid Improves Sinoatrial Node Dysfunction through the PKC/NOX-2 Signaling Pathway.

Sick sinus syndrome (SSS) is one of the common causes of cardiac syncope and sudden death; the occurrence of SSS is associated with the accumulation of ROS in the sinoatrial node (SAN). Shenxian-shengmai (SXSM) is a traditional Chinese medicine available as oral liquid that causes a significant increase in heart rate. The objective of this study is to observe the improvement of SXSM on SAN function in SSS mice and explore its potential mechanism. In the current study, SSS was simulated in mice by inducing SAN dysfunction using a micro-osmotic pump to inject angiotensin II (Ang II). The mouse model with SSS was used to determine the effect of SXSM on SAN function and to explore its potential mechanism. Furthermore, the HL-1 cell line, derived from mouse atrial myocytes, was used to simulate SAN pacemaker cells. Our results indicated that SXSM significantly increased the heart rate of SSS mice by reducing the AngII-induced accumulation of ROS in the SAN and by inhibiting the expression of HDAC4, thereby reducing the loss of HCN4, a critical component of the cardiac conduction system. MASSON staining revealed a reduction of SAN damage in SSS mice that were treated with SXSM compared with controls. In vitro experiments showed that AngII treatment caused an upregulation of the PKC/NOX-2 signaling pathway in HL-1 cells which could be prevented by pretreatment with SXSM. The protective effect of SXSM was attenuated upon treatment with the PCK agonist PMA. In conclusion, SXSM reduced the AngII-induced accumulation of ROS in the SAN through the PKC/NOX2 signaling pathway, improving the functioning of the SAN and preventing the decrease of heart rate in SSS mice.