Palmatine Alleviates Acute Myocardial Infarction Through Activating pAMPK/Nrf2 Signaling Pathway in Mouse Model

Acute myocardial infarction is one of the major leading causes for heart failure, which can lead to the irreversible loss of cardiomyocytes and impaired cardiac function. Hence, the efficient therapeutic agents are still urgent. Our study aimed to explore the role of a natural isoquinoline alkaloid, palmatine, in an acute myocardial infarction mouse model. In this study, intragastric administrated palmatine significantly enhanced left ventricle ejection fraction and left ventricle end-systolic of infarcted mice heart. Meanwhile, palmatine administration partially recovered myocardial structure and attenuated the cardiac fibrosis and infiltration of inflammatory cells. In addition, the usage of palmatine further enhanced the increased transforming growth factor (TGF)-beta1 level, reduced the elevated tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta level in the myocardium of acute myocardial infarction–induced mice, as well as elevated the reduced superoxide dismutase production and inhibited the increased malondialdehyde secretion in infarcted myocardium of mice. Meanwhile, acute myocardial infarction led the significant upregulation of Bcl-2-associated X and downregulation of B-cell lymphoma-2 in the myocardium, and palmatine administration statistically enabled to recover the expression changes of these two apoptosis-related proteins. Moreover, palmatine administration obviously elevated the expression levels of phosphorylated AMP-activated protein kinase and nuclear factor erythroid 2–related factor 2 in the myocardium of acute myocardial infarction–induced mice. In a word, our study indicated that palmatine could protect infarcted myocardium of mice from apoptosis, inflammation, and oxidative stress. Our results suggested that palmatine might be a novel therapeutic agent for acute myocardial infarction.


Introduction
Cardiovascular disease is becoming the major leading cause for adverse outcomes in the world, and the major reason for cardiovascular disease is acute myocardial infarction (AMI) (Gulati et al. 2020;Pollard 2000). Due to the higher morbidity and mortality of AMI in recent years and even 2.5 million cases annually (Benjamin et al. 2017;Fu et al. 2020), the efficient agents that potentially applied for AMI treatment are necessary. Increasing evidences demonstrate that the inflammatory cell infiltration and cardiomyocyte apoptosis often occurred in the myocardium of hearts due to ischemia and hypoxia of the infarct area (Galluzzi et al. 2018;Li et al. 2010). In addition, mitochondrial reactive oxygen species (ROS) caused by AMI in ischemic areas can lead to oxidative stress (Ramachandra et al. 2020). Hence, the identification of novel agents against inflammatory response, cardiomyocyte apoptosis, and oxidative stress in the myocardium of hearts may provide a molecular basis for targeted therapy.
Palmatine (1), a natural isoquinoline alkaloid, is extracted from rhizome of Coptis chinensis Franch., Ranunculaceae (Long et al. 2019). Increasing reports suggest that palmatine has multiple biological activities including antioxidant stress, anti-inflammatory, anti-tumor, neuroprotective, and gastric protective activities (Hui et al. 2020;Zhang et al. 2012). For example, Lee et al. reported that palmatine inhibited inflammation and apoptosis of hepatocyte during acute liver injury (Lee et al. 2010). Palmatine alleviated the injury caused by ulcerative colitis through maintaining the integrity of intestinal barrier and reducing colitis . One previous study revealed that palmatine alleviated myocardial ischemiareperfusion-induced AMI by inhibiting oxidative stress and inflammatory response in a rat model using the ligation of the left anterior descending branch of the coronary artery (Kim et al. 2009). However, it is unclear with the underlying molecular mechanisms involved in the protective effect on AMI in mice, and our study focused on it.
In the present study, we found that palmatine administration significantly reduces the levels of Bcl-2-associated X (Bax), tumor necrosis factor (TNF)-alpha (TNF-α), interleukin (IL)-1beta (IL-1β), and malondialdehyde (MDA), while elevated the levels of Bcell lymphoma-2 (Bcl-2), superoxide dismutase (SOD), and transforming growth factor (TGF)-beta1 (TGF-β1) in the myocardium of AMI mouse model. These findings suggested that palmatine holds a cardioprotective role by reducing inflammation and oxidative stress and also suppressing cardiomyocyte apoptosis in the myocardium of AMI mouse model. Moreover, the obvious activation of the phosphorylated AMP-activated protein kinase (pAMPK)/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway in the myocardium of AMI mouse after palmatine administration was observed, indicating that the cardioprotective effect of palmatine was closely correlated to the activation of the pAMPK/Nrf2 signaling pathway. Our findings here suggested that palmatine could be a new strategy for treating AMI.

Animal Experiments
Animal experiments were performed in accordance with the guidelines for the care and use of experimental animals (National Research Commission, Eighth Edition, 2011). Forty male C57 mice (8-week-old, 200-250 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Palmatine was purchased from Nanjing Plant Origin Biological Co., Ltd. (Batch No. Yz091520, Nanjing, China). Animals were randomly and equally divided into sham operation + normal saline group (sham or NC), AMI + normal saline group (MI), AMI + 20 mg/kg palmatine group (LD), and AMI + 100 mg/kg palmatine group (HD). N = 10 mice in each group.
The mice were anesthetized with i.p. injection of pentobarbital sodium (50 mg/kg). Then, the chest was opened through the fourth intercostal space, and the anterior descending branch of the left coronary artery was ligated to induce acute myocardial infarction as previously described . Sham operated mice underwent the same procedures without ligation and treated with equal volume of saline as negative control (sham or NC). Success of MI characterized as elevated ST was confirmed by ECG. Mice in 2 palmatine groups were treated with intragastric administration of palmatine for 30 days, while mice in NC and MI groups were treated with equal volume of normal saline. At the end of the experimental course, the left ventricle ejection fraction (LVEF) and left ventricle end-systolic (LVES) of mice were evaluated by using Acuson Sequoia 512 Color Doppler ultrasound (Siemens, Munich, Germany). Then mice were sacrificed and the myocardium of each mouse was harvested for further examination. And, this study was approved by the animal ethics committee of Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University (approval number: SHRM-IACUC-044). Procedures operated in this research were completed in keeping with the standards set out in the Announcement of Helsinki and laboratory guidelines of research in China.

H&E and Masson's Trichrome Staining
The pathological and morphological changes of myocardial tissues in mice were evaluated by using hematoxylin and eosin staining, and Masson's trichrome staining as previously described (Liu et al. 2021). In brief, the myocardial tissues of mice were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4-μm-thick paraffin sections. Subsequently, the sections were stained with hematoxylin for 3-5 min and eosin for 5-15 s. In addition, a Masson's trichrome staining kit (Solarbio Science & Technology Co., Ltd., Beijing, China) was used to perform Masson's trichrome staining according to instruction. The histopathological changes in the myocardium were examined by a light microscope.

Statistical Analysis
The data are expressed as mean ± standard deviation (SD) from three independent experiments. And, differences were statistically analyzed by two-tailed Student's t test (two groups) or one-way analysis of variance (ANOVA) (multiple groups). p value less than 0.05 was considered the significant threshold.

Palmatine Protected AMI Injury in Mouse Model
To verify the therapeutic effect of palmatine (1) on AMI, the mouse model was established, and Color Doppler ultrasound was performed to evaluate changes of cardiac function of mice (Fig. 1A). As shown in Fig. 1B, administration of palmatine statistically enhanced both LVEF and LVES of rats after AMI injury. Further, the role of palmatine on infarcted heart was validated using H&E and Masson trichrome staining. As shown in Fig. 2A, B, rats in the NC group exhibited intact myocardial structure, and neither fibrosis nor infiltration of inflammatory cells was observed. In the AMI group, intact myocardial structure was partly lost, and fibrosis and infiltration of inflammatory cells occurred. However, histopathological changes abovementioned were statistically weakened in AMI mice after treated with palmatine. Taken together, our findings suggested that palmatine could enable to improve cardiac functions of mice with AMI and effectively attenuated infarction caused histological damages.

Palmatine Reduced the Inflammatory Response of AMI Injury In Vivo
In order to verify the effect of palmatine on the inflammatory response caused by AMI, the levels of inflammatory cytokines in myocardial tissue were detected by ELISA. As shown in Fig. 3A-C, TGF-β1, TNF-α, and IL-1β, that were expressed in low levels in the myocardial tissue of sham operated mice, were statistically increased after AMI incubation. Mice treated with palmatine further enhanced TGF-β1 level, while reduced the levels of TNF-α and IL-1β. It suggested that palmatine enables to reduce inflammatory response in mice after AMI injury. In addition, the effect of palmatine on concentrations of SOD and MDA in the myocardium was also detected. As shown in Fig. 3D, E, the concentration of SOD was reduced, while MDA concentration was increased in the myocardium of rats after AMI injury compared to that in the NC group, and palmatine administration elevated SOD level and decreased MDA level in the myocardium. These data suggested that palmatine reduced the inflammatory response in the myocardium of AMI mice.

Palmatine Attenuated Cardiomyocyte Apoptosis in Mice with AMI
In order to explore the effect of palmatine on cardiomyocyte apoptosis in mice caused by AMI, the expression changes of Bax and Bcl-2 in the myocardium were quantified by qRT-PCR and western blot assays. Data showed that AMI injury led to the statistical upregulation of Bax and downregulation of Bcl-2 in the myocardium compared to those in sham operated mice, and palmatine administration obviously recovered the expressions of Bax and Bcl-2 (Fig. 4A, B). Meanwhile, western blot assay further confirmed the expression change of Bax and Bcl-2 affected by palmatine (Fig. 4C, D). Taken together, palmatine administration significantly reversed the elevated Bax and decreased Bcl-2 induced by AMI injury in A, TNF-α B, IL-1β C, SOD D, and MDA E in the myocardium were detected by specific ELISA kits. NC, sham operated mice received normal saline; MI, AMI mice received normal saline; LD, AMI mice received 20 mg/kg palmatine; HD, AMI mice received 100 mg/kg palmatine. Data are presented as mean ± standard deviation (SD). ** p < 0.01, *** p < 0.001, **** p < 0.0001. n = 10 rats. These data suggested that palmatine might attenuate cardiomyocyte apoptosis in mice with AMI.

Palmatine Activated pAMPK/Nrf2 Signaling Pathway
Finally, we investigated whether palmatine exerted cardioprotective effects through affecting the pAMPK/Nrf2 signaling pathway. The expression changes of pAMPK and Nrf2 proteins in the myocardium of mice were detected by western blot. As shown in Fig. 5A-C, AMI treatment resulted in statistical upregulation of pAMPK in myocardial tissues compared to that in sham operated mice. However, no obvious difference in Nrf2 expression change was observed, but also a slight increase. After being treated with palmatine, either pAMPK or nuclear Nrf2 expression in the myocardium was upregulated. Meanwhile, there was no significant change on AMPK expression between different groups (Fig. 5D, E). These findings suggested that palmatine might attenuate AMI injury through activating the pAMPK/Nrf2 signaling pathway.

Discussion
Recently, more and more attentions have been focused on the identification of efficient agents for AMI due to its heavy burden to human health (Németh et al. 2021), although previous studies have revealed that the protective role of palmatine was associated with its antioxidative stress and anti-inflammatory response (Kim et al. 2009). But the potential mechanisms have not been well studied. In the present study, we confirmed that palmatine protected AMI through reducing oxidative stress and inflammatory response. Moreover, our results also revealed that inhibition of cardiomyocyte apoptosis also accounts for the protective role of palmatine on AMI-induced cardiac dysfunction. Moreover, our findings suggested that the activation of the pAMPK/Nrf2 signaling pathway was also associated with the improved role of palmatine on AMI.
Being a natural isoquinoline alkaloid, palmatine was initially found to have considerable anti-inflammatory bioactivity and enable to reduce the release of inflammatory factors (Yasukawa et al. 1991). For example, palmatine attenuated dextran sulfate sodium (DSS)-induced colitis via promoting mitophagy- Fig. 4 Effect of palmatine on the expression of Bax and Bcl-2 in the myocardium of mice. A, B The expression levels of Bax A and Bcl-2 B in the myocardium of mice were detected by qRT-PCR. C Western blot for detecting the expression of Bax and Bcl-2. D Quantitative analysis of western blot for Bax and Bcl-2. NC, sham operated mice received normal saline; MI, AMI mice received normal saline; LD, AMI mice received 20 mg/kg palmatine; HD, AMI mice received 100 mg/kg palmatine. Data are presented as mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n = 10 mediated NLRP3 inflammasome inactivation (Mai et al. 2019). Palmatine attenuated LPS-induced inflammatory response in mouse mammary epithelial cells (Ma et al. 2021). Palmatine ameliorated Helicobacter pylori-induced chronic atrophic gastritis by inhibiting MMP-10 through ADAM17/EGFR ). In addition, there are neuroprotective effects of palmatine via enhancing the antioxidant defense and small heat shock protein expression in a β-transgenic Caenorhabditis elegans (Jia et al. 2021). Here, we revealed the improved role of palmatine on AMI-induced impaired cardiac function in mice. There were other roles of this agent in human diseases needed to be explored in the subsequent experiments.
Here, we also detected the expression of these inflammatory cytokines, and found that palmatine could obviously reduce this inflammatory cytokine production in the myocardium of AMI mice. Moreover, the expression of TGF-β1, an anti-inflammatory factor (Ling and Robinson 2002), was significantly elevated in AMI mouse model after palmatine administration. These data further confirmed the anti-inflammatory role of palmatine. In addition, palmatine also notably reduced Bax expression and increased Bcl-2 expression in AMI model. Bax and Bcl-2 are two apoptosis-related proteins, and Bax is a pro-apoptotic factor while Bcl-2 is an anti-apoptotic factor (Edlich 2018;Robertson et al. 2006). Our results determined that palmatine also had the anti-apoptosis activity except for antioxidant stress and anti-inflammatory response.
AMP-activated protein kinase (AMPK), a serine/threonine protein kinase, has been reported to be involved in the regulation of cellular stress and energy homeostasis (Rodríguez and Muñoz 2021), and also considered as a key regulator of cell apoptosis, oxidative stress, and inflammation . Nrf2 protein is a downstream molecule of AMPK, and the AMPK signaling pathway is interrelated via Nrf2 and plays a crucial role in antioxidative stress response and inhibiting cell apoptosis (Lu et al. 2019;Yu et al. 2018). Tang et al. recently found that palmatine could attenuate brain ischemia-reperfusion injury via activating the AMPK/Nrf2 pathway (Tang et al. 2021). In addition, AMPK was identified to be slightly activated characterized by increased levels of phosphorylated AMPK (pAMPK) during AMI injury (Qi Fig. 5 Effect of palmatine on the expression of pAMPK and NRF2 in the myocardium of mice. A Western blot analysis of pAMPK and NRF2 in the myocardium of mice. B and C Quantitative analysis of the expression against pAMPK B and NRF2 C. D and E Western blot analysis of AMPK D and quantitative analysis of the expression against AMPK E. NC, sham operated mice received normal saline; MI, AMI mice received normal saline; LD, AMI mice received 20 mg/kg palmatine; HD, AMI mice received 100 mg/kg palmatine. Data are presented as mean ± standard deviation (SD). * p < 0.05, *** p < 0.001, **** p < 0.0001, ns indicates no significant difference. n = 10 and Young 2015; Zhang et al. 2020). Thus, whether the protective effect of palmatine was mediated by the pAMPK/Nrf2 signaling pathway was investigated in this study. We found that AMI led a certain upregulation of pAMPK level and had no obvious change of Nrf2 level. Importantly, intragastric administration of palmatine notably upregulated the protein level of pAMPK and nuclear Nrf2 in infarcted myocardium of AMI mice, suggesting that the activation of pAMPK/Nrf2 partially participated in the protective effect of palmatine on AMI. Except for the pAMPK/Nrf2 signaling pathway, there were several other pathways that were closely involved in inflammatory response and cardiomyocyte apoptosis during AMI such as the PI3K/AKT signaling pathway , PDK-1/AKT signaling pathway (Garikipati et al. 2017), NF-κB signaling pathway , and PKC/NADPH oxidase pathway (Tsai et al. 2020), and so on. Whether these relevant signaling pathways participated in the protective mechanisms of palmatine on AMI should be explored in the future.

Conclusion
Palmatine could attenuate AMI injury in mice via inhibiting the inflammatory response and cardiomyocyte apoptosis in the myocardium, and the pAMPK/Nrf2 signaling pathway was associated with the positive effect of palmatine on AMI injury. Our findings suggested that palmatine might be a potential therapeutic drug for AMI.
Author Contribution MXH and KLJ contributed in conceiving this review, collecting and analyzing the data, and editing and writing the manuscript. All authors have read and approved the final manuscript.
Funding This work was supported by the National Natural Science Foundation of China (No. 81500198).

Declarations
Protection of Animal Subjects Protocol of this study was approved by the animal ethics committee of Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University (approval number: SHRM-IACUC-044). Procedures operated in this research were completed in keeping with the standards set out in the Announcement of Helsinki and laboratory guidelines of research in China.

Conflict of Interest The authors declare no competing interests.
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