Citri Reticulatae Pericarpium alleviates postmyocardial infarction heart failure by upregulating PPARγ expression

Heart failure after myocardial infarction (MI) is the leading cause of death worldwide. Citri Reticulatae Pericarpium (CRP) is a traditional Chinese herbal medicine that has been used in the clinic for centuries. In this study, we aimed to investigate the roles of CRP in cardiac remodelling and heart failure after MI, as well as the molecular mechanisms involved. Male C57BL/6 mice aged 8 weeks were subjected to coronary artery ligation to mimic the clinical situation in vivo. Echocardiography was used to assess the systolic function of the mouse heart. Masson trichrome staining and Wheat germ agglutinin (WGA) staining were utilised to determine the fibrotic area and cross‐sectional area of the mouse heart, respectively. Cardiomyocytes and fibroblasts were isolated from neonatal rats aged 0–3 days in vitro using enzyme digestion. TUNEL staining and EdU staining were performed to evaluate apoptosis and proliferation, respectively. Gene expression changes were analysed by qRT–PCR, and protein expression changes were assessed by Western blotting. Our findings revealed that CRP attenuated cardiac hypertrophy, fibrosis and apoptosis and alleviated heart failure after MI in vivo. Furthermore, CRP mitigated cardiomyocyte apoptosis and fibroblast proliferation and differentiation into myofibroblasts. In addition, the PPARγ inhibitor T0070907 completely abolished the abovementioned beneficial effects of CRP, and the PPARγ activator rosiglitazone failed to further ameliorate cardiac apoptosis and fibrosis in vitro. CRP alleviates cardiac hypertrophy, fibrosis, and apoptosis and can ameliorate heart failure after MI via activation of PPARγ.

occur after cardiac ischaemia. For example, due to a lack of blood supply, cardiomyocytes undergo necrosis and apoptosis, while myofibroblasts are activated to preserve ventricular shape. In addition, the noninfarcted myocardium undergoes eccentric hypertrophy, ultimately leading to heart dilation. 2 Post-MI adverse cardiac remodelling, especially cardiac fibrosis, can develop into advanced heart failure. 3 Currently, there are a number of pharmacological treatments and preventive strategies for improving the prognosis of patients with post-MI heart failure, such as angiotensin II receptor antagonists, betablockers, acetyl choline esterase inhibition and Entresto. [4][5][6] However, the incidence of heart failure after MI is increasing year to year, ranging from 14% to 36% according to different studies, 7 which places a heavy burden on society and individuals. Therefore, identifying novel therapeutics for post-MI heart failure is of great significance.
PPARs are nuclear hormone receptor superfamily transcription factors activated by ligands. PPARs, or peroxisome proliferator-activated receptors, were discovered in 1990, 8 and mainly regulate fatty acid oxidation in fat and other tissues and glucose metabolism. 9 There are three PPAR isoforms: PPARα, PPARβ/δ, and PPARγ. 10 Accumulating evidence indicates that PPARγ plays key roles in controlling cardiac metabolism. [11][12][13] Previous studies have revealed that activation of PPARγ can significantly ameliorate cardiac hypertrophy and cardiac fibrosis in mice with pressure overload-induced, 14 angiotensin II (Ang II)-induced 15 and isoproterenol (ISO)-induced 16 pathological myocardial remodelling. In addition, upregulating PPARγ expression can increase cardiac output in rats with myocardial ischaemia-reperfusion (I/R) injury. 17 Moreover, the PPARγ agonist pioglitazone can probably reduce the expression of inflammatory cytokines, including TNFα, TNFβ and MCP-1, and attenuate cardiac remodelling and heart failure in mice with MI. 18 A recently published review systematically identified PPARγ as a novel therapeutic target for cardiac fibrosis. 19 However, other studies have found that PPARγ activation can result in fluid retention, weight gain, osteoporosis and even cause heart failure. Thus, identifying potential PPARγ regulators that do not cause side effects may provide options for the clinical treatment of post-MI heart failure.
Traditional Chinese medicine (TCM) plays important roles in the clinical treatment of cardiovascular diseases. 20 Citri Reticulatae Pericarpium (CRP) is a famous TCM that is used for the treatment of multiple diseases, such as cardiovascular diseases, digestive diseases, respiratory diseases and even tumours. 21,22 CRP is the dried fruit peel of Citrus reticulata Blanco. The southeastern region of China is the main production area of CRP, such as Fujian, Guangdong and Zhejiang provinces. Phytochemical studies have identified more than one hundred chemical components of CRP, and its abundant bioactive compounds, including flavonoids, phenolic acids and limonoids, can exert positive effects on health. 22 CRP has been described as a qi regulator for centuries and was registered in the first edition of the Chinese Pharmacopoeia in 1953. CRP has attracted increasing attention from researchers because it has multiple pharmacologic effects and rich resources with low toxicity and costs. 23 Modern pharmacological investigations have revealed that CRP can fight against inflammation, oxidative stress, atherosclerosis, thrombus, liver injury and tumours. 22,[24][25][26][27] Our prior studies showed that CRP can alleviate cardiac hypertrophy and fibrosis induced by Ang II and ISO via activating PPARγ. 15,16 However, whether CRP has a protective effect on post-MI heart failure is unclear.
In the current research, we demonstrated that CRP can protect against post-MI heart failure by activating PPARγ, suggesting the potential of CRP in the clinical treatment of heart failure after MI.

| CRP attenuates cardiac injury after MI in vivo
Since our previous studies demonstrated that CRP protects against Ang II-induced and ISO-induced cardiac dysfunction, 15,16 we further investigated the role of CRP in post-MI heart failure in the present study. C57BL/6 mice aged 8 weeks were subjected to LAD and then given CRP for 3 weeks. To determine the optimal dosage of CRP, we set three concentrations for treatment. As shown in Figure S2, the dosage of 0.5 and 1.0 g/kg/day could both improve cardiac systolic function after MI. We choose the administration of 0.5 g/kg/day for further investigation because the 1.0 g/kg/day dosage did not perform better.
Then the results of echocardiography suggested again that CRP significantly increased the LVEF and LVFS ( Figure 1A). In addition, WGA staining and qRT-PCR showed that CRP reduced cardiac hypertrophy ( Figure 1B) and decreased the activation of fetal genes (Anp and Bnp) in the hearts of mice with MI ( Figure 1C). Cardiac fibrosis is a pivotal hallmark of cardiac remodelling and heart failure after MI. Our data revealed that compared with vehicle, CRP obviously attenuated collagen deposition in the heart post-MI (Figure 2A-C). Cardiac apoptosis is another feature of cardiac remodelling and heart failure after MI. We found that the expression of the antiapoptotic protein Bcl2 was increased and that the expression of the proapoptotic protein Bax was decreased by CRP treatment ( Figure 2D). Taken together, our results demonstrate that CRP can alleviate cardiac hypertrophy and cardiac fibrosis as well as cardiac apoptosis after MI injury and thus plays important roles in post-MI heart failure.

| CRP ameliorates OGD-induced cardiomyocyte apoptosis and TGFβ-induced cardiac fibroblast activation in vitro
To further investigate the protective effects and underlying mechanisms of CRP in vitro, neonatal rat cardiomyocytes (CMs) and cardiac fibroblasts (CFs) were obtained by enzyme digestion. Similarly, three different concentrations were set to find the most suitable dosage of CRP in vitro. It was found that 0.5 and 1.0 μg/mL CRP administration could both ameliorate the cell apoptosis induced by oxygen-glucose deprivation (OGD), but there was no significant difference between the two concentrations ( Figure S3). Therefore, we choose the 0.5 μg/mL dosage as the optimal concentration for further study in vitro. As shown in Figure 3A, B, CRP decreased the number of TUNEL-positive nuclei and reduced the Bax/Bcl2 ratio in OGD-treated CMs, providing evidence for the antiapoptotic function of CRP. In addition, prior to administering TGFβ to activate CFs, CRP was mixed in the culture medium for 24 h.
Immunofluorescence analysis indicated that TGFβ enhanced CF proliferation and differentiation into myofibroblasts and that CRP administration reversed these effects ( Figure 3C). We further examined the mRNA levels of Col1a1, Col3a1 and α-SMA by qRT-PCR, and the results illustrated that CRP may reduce the increase in the expression of fibrotic genes in TGFβ-stimulated CFs ( Figure 3D). Collectively, these results showed that CRP can alleviate OGD-induced cardiomyocyte apoptosis and TGFβ-induced cardiac fibroblast activation in vitro.

| PPARγ is activated by CRP treatment both in vivo and in vitro
Metabolic disturbance is a crucial feature of cardiac remodelling and heart failure after MI, and PPARγ is a well-known regulator F I G U R E 1 Citri Reticulatae Pericarpium (CRP) alleviates cardiac dysfunction and hypertrophy after myocardial infarction (MI). (A) Parameters measured by echocardiography showed that CRP-treated mice with MI showed preservation of the ejection fraction (EF) and fractional shortening (FS) (n = 8,7,6,7). (B) WGA staining indicated that CRP reduced the increase in the cross-sectional area in mice with MI (n = 6). (C) qRT-PCR showed that activation of Anp and Bnp expression in post-MI heart failure was reversed by CRP treatment (n = 6). The data are presented as the mean ± SD. *p <0.05; **p <0.01; ***p <0.001. Scale bar = 20 μm of metabolism. [28][29][30][31] Our Western blotting results showed that These data suggest that PPARγ activation contributes to the beneficial effects of CRP in protecting against post-MI heart failure.

| CRP alleviates OGD-induced cardiomyocyte apoptosis and TGFβ-induced cardiac fibroblast activation by upregulating PPARγ expression
To study whether PPARγ is a pivotal downstream effector of CRP, rosiglitazone and T0070907 were applied. We found that T0070907 inhibited the beneficial effects of CRP on OGDinduced cardiomyocyte apoptosis, while rosiglitazone failed to F I G U R E 2 Citri Reticulatae Pericarpium (CRP) decreases myocardial infarction (MI)-induced cardiac fibrosis and apoptosis. (A) Masson trichrome staining (n = 8,7,6,7), (B) qRT-PCR (n = 6) and (C) Western blotting (n = 6) revealed that CRP decreased the fibrotic area and downregulated the expression of fibrotic molecules at both the mRNA (Col1a1, Col3a1 and α-SMA) and protein levels (collagen type I and α-SMA). (D) The Bax/Bcl2 ratio was increased in the MI group but decreased upon CRP treatment (n = 6). The data are presented as the mean ± SD. *p <0.05; ***p <0.001. Scale bar = 500 μm provide additional protection in the presence of CRP ( Figure 5A  intragastrically administered CRP. According to the echocardiography results, the LVEF and LVFS were increased by CRP treatment, while both were significantly decreased by T0070907 injection ( Figure 6A). F I G U R E 3 Citri Reticulatae Pericarpium (CRP) ameliorates OGD-induced cardiomyocyte apoptosis and TGFβ-induced cardiac fibroblast activation. (A) Immunofluorescence for α-actinin was used to identify CMs (red). TUNEL staining was applied to label apoptotic nuclei (green). DAPI was utilised to stain the nuclei (blue) (n = 6). (B) The Bax/Bcl2 ratio was determined by Western blotting (n = 6). (C) Immunofluorescence for α-SMA was used to assess the extent of CF differentiation (green). EdU staining was performed to label proliferating nuclei (red). DAPI was utilised to stain nuclei (blue) (n = 6). (D) qRT-PCR analysis was used to determine expression changes in fibrotic genes (n = 6). The data are presented as the mean ± SD. ***p <0.001. Scale bar = 50 μm The cardioprotective effect of CRP in cardiac hypertrophy after MI was also blocked by a PPARγ inhibitor ( Figure 6B), as was the expression of fetal genes (ANP and BNP) ( Figure 6C). Regarding cardiac fibrosis, the CRP-mediated reduction in the fibrotic area and downregulation of fibrotic molecule expression were reversed by T0070907 ( Figure 7A-C). As shown in Figure 7D, T0070907 inhibited the ability of CRP to alleviate cardiac apoptosis after MI. Consequently, these results indicated that CRP exerts positive effects on post-MI heart failure by upregulating PPARγ expression.

| DISCUSSION
Reversing cardiac remodelling after MI is a major challenge worldwide. 32 The efforts of scientists worldwide have led to a decrease in the death rate in the acute phase after MI. However, chronic injury after MI, pathological cardiac remodelling and heart failure result in high morbidity and mortality in MI patients. 33 Currently, there are limited pharmacological therapies for heart failure after MI. Novel strategies are urgently needed.
CRP, a traditional Chinese herbal medicine used in the clinic for centuries, has anti-inflammatory, antioxidant, anticancer and other beneficial properties according to modern pharmacological studies. 22,24,34 In addition, CRP may be effective for diseases affecting multiple systems, such as the digestive system, the respiratory system and particularly the cardiovascular system. 22 Cardiac remodelling and heart failure after MI involve metabolic dysfunction, in which PPARγ is a pivotal regulator. 37 Accumulating evidence showed that hesperidin has positive effects on various diseases including acute liver injury, 44 acute lung injury, 45 tumours 46 and diabetes. 47 Moreover, hesperidin could protect against myocardial ischaemia, cardiac hypertrophy and myocardial toxicity by upregulating PPARγ expression, 25,48,49 which is consistent with our findings (Figures S4 and S5). As shown in Figure S4B, C, hesperidin, nobiletin and tangeretin could alleviate CMs apoptosis induced and increase the expression of PPARγ. Furthermore, our results showed that hesperidin, nobiletin and tangeretin could preserve cardiac systolic function after MI and activate PPARγ ( Figure S5A, B). Besides, one of our previous studies suggested that nobiletin attenuated pathological cardiac remodelling after MI via activating PPARγ, 50 which is identical to our findings here. As to tangeretin, several studies indicated that it played an important role in lipid metabolism by increasing PPARγ

F I G U R E 4 PPARγ expression is upregulated by Citri Reticulatae Pericarpium (CRP) both in vivo and in vitro. (A-C) Western blotting indicated that PPARγ expression was reduced in the hearts of mice
with MI, OGD-treated CMs and TGFβ-stimulated CFs but dramatically upregulated upon CRP treatment in the abovementioned pathological models (n = 6). The data are presented as the mean ± SD. *p <0.05; ***P <0.001 expression. 51,52 However, it is the first time that we identified that tangeretin could mitigate CMs apoptosis and heart failure after MI. Meanwhile, tangeretin could activate PPARγ both in vitro ( Figure S4C) and in vivo ( Figure S5B). Therefore, like hesperidin and nobiletin, tangeretin also contributed to cardiac benefits of CRP. Interestingly, HMF could obviously promote CMs proliferation ( Figure S4A), but showed no effects on CMs apoptosis ( Figure S4B). In a word, we believe that among the bioactive flavonoids in CRP, hesperidin, nobiletin and tangeretin play important roles in protection against CMs apoptosis and heart failure after MI. It is possible that these ingredients have cooperative effects with each other. Combination experiments of the three effective candidates are urgently needed in future studies.
In conclusion, we demonstrated that CRP attenuated cardiac injury after MI by activating PPARγ and provided a potential therapeutic way for patients who suffered from heart failure after MI. F I G U R E 5 Citri Reticulatae Pericarpium (CRP) decreases cardiomyocyte apoptosis and cardiac fibroblast activation by increasing PPARγ expression. (A) TUNEL staining showed that T0070907 abolished the protective effects of CRP on OGD-induced CM apoptosis, but rosiglitazone did not induce further improvements in the presence of CRP (n = 6). (B) The same conclusion was drawn by measuring Bax and Bcl2 expression using Western blotting (n = 6). (C) EdU staining and immunofluorescence for α-SMA indicated that T0070907 blocked CRP's inhibitory effects on TGFβ-stimulated CF proliferation and differentiation into myofibroblasts, but rosiglitazone failed to further suppress the CF activation induced by TGFβ in combination with CRP (n = 6). (D)qRT-PCR showed that upon TGFβ stimulation, the expression of fibrotic genes (Col1a1, Col3a1 and α-SMA) that was downregulated by CRP treatment was upregulated by T0070907 but not further downregulated by rosiglitazone (n = 6). The data presented as the mean ± SD. *p <0.05; **p <0.01; ***p <0.001. Scale bar = 50 μm

| Phytochemical analysis
To determine whether CRP used in our study contains bioactive flavo-

| Animal models
Adult male C57BL/6 mice were purchased from the Experimental Animal Center of Nanjing Medical University (Nanjing, China). The mice were randomly divided into groups. MI was established by ligation of the left anterior descending coronary artery (LAD) utilising a 7/0 silk thread. The sham group mice underwent the same process except F I G U R E 6 Citri Reticulatae Pericarpium (CRP) protects against post-myocardial infarction (MI) cardiac dysfunction and hypertrophy by activating PPARγ. (A) Echocardiography indicated that the MI-induced reductions in LVEF and LVFS were reversed by CRP and completely inhibited by T0070907 (n = 8,7,6,7). (B) WGA staining showed that enlargement of the heart cross-sectional area in mice with MI was reduced by CRP and abolished by T0070907 (n = 6). (C) qRT-PCR indicated that activation of Anp and Bnp expression in the hearts tissues of mice with MI were be reversed by CRP treatment and blocked by T0070907 administration (n = 6). The data are presented as the mean ± SD. *p <0.05; **p <0.01; ***p <0.001. Scale bar = 20 μm F I G U R E 7 Citri Reticulatae Pericarpium (CRP) attenuates cardiac fibrosis and apoptosis by upregulating PPARγ expression. (A) Masson trichrome staining (n = 8,7,6,7), (B) qRT-PCR (n = 6) and (C) Western blotting (n = 6) revealed that the increase in the fibrotic area, fibrotic gene expression and fibrotic protein expression in mice with myocardial infarction (MI) was attenuated by CRP administration, but the protective effects were reversed by T0070907. (D) Western blotting analysis showed that the increase in the Bax/Bcl2 ratio in the MI group declined after CRP treatment, but T0070907 inhibited the cardioprotective effect of CRP (n = 6). The data are presented as the mean ± SD. *p <0.05; **p <0.01; ***p <0.001. Scale bar = 500 μm LAD ligation. CRP was obtained from Shijiazhuang Yiling Pharmaceutical Co., Ltd. (Shijiazhuang, Hebei, China). Hesperidin, nobiletin, tangeretin, HMF and PMF were purchased from Nanjing Ben Cao Co. Ltd. (Nanjing, China), and their purities were > 98%, as determined by HPLC. The animals were intragastrically administered CRP (0.25, 0.5 or 1.0 g/kg/day), hesperidin (0.3 g/kg/day), nobiletin (0.05 g/kg/ day) or tangeretin (0.1 g/kg/day) for 3 weeks after MI. A PPARγ inhibitor (T0070907) was used to investigate the mechanism by which CRP protects against post-MI heart failure. CRP was administered after MI, and T0070907 was intraperitoneally injected (1 mg/kg/day) concurrently for 21 days.

| Cell treatment
To mimic the lack of blood and oxygen in mice with MI, CMs were maintained in glucose-free Dulbecco's modified Eagle's medium  4.12 | α-Actinin and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) costaining Paraformaldehyde (4%) and Triton X-100 (0.5%) were used to fix and permeabilise CMs, respectively, for 20 min. Then, 10% goat serum in PBS was applied to block nonspecific protein sites for 1 h. All of these processes were performed at room temperature. CMs were incubated with an α-actinin primary antibody (

| Statistical analysis
All data were expressed as the mean ± SD. One-way ANOVA followed by Bonferroni's post hoc test was applied for multiple-group comparisons. A p value<0.05 was considered statistically significant.
All analyses were performed using GraphPad Prism 8 software.