Identifying the Chemical Pro le and Pharmacological Mechanism of Xinbao Pill against Myocardial Ischemia-Reperfusion Injury Using Ultra- Fast Liquid Chromatography-Quadrupole-Time-Of- Flight Tandem Mass Spectrometry Coupled with Network Pharmacology-Based Investigation

Ying Yang Guangzhou University of Chinese Medicine Ting Chen Guangdong Xinbao Pharm-tech Co., Itd Jiaming Liu Guangdong Pharmaceutical University Sixuan Chen Guangzhou University of Chinese Medicine Rongqing Cai Guangdong Xinbao Pharm-tech Co., Itd Liqiong Wu Guangdong Xinbao Pharm-tech Co. Itd Jiexiong Hu Guangdong Xinbao Pharm-tech Co., Itd Qiongying Lin Guangdong Xinbao Pharm-tech Co., Itd Xiaoxiao Qi Guangzhou University of Chinese Medicine Zhongqiu Liu Guangzhou University of Chinese Medicine Yuanyuan Cheng (  chengyuanyuan@gzucm.edu.cn ) Guangzhou University of Chinese Medicine https://orcid.org/0000-0003-1105-0495

Borneolum syntheticum (Bingpian) and Chinese toad (Bufo gargarizans, Chansu). Xinbao pill has the effects of warming and tonifying heart and kidney; replenishing Qi and assisting Yang; promoting blood circulation to remove obstructions from meridians. In TCM clinic, Xinbao pill is used to treat chronic cardiac insu ciency caused by heart and kidney Yang de ciency; heart pulse stasis, bradycardia and sinus syndrome caused by sinus insu ciency; angina pectoris caused by ischemic heart disease and ischemic changes of electrocardiogram (4). Pharmacological studies showed that XBW and its components have a de nite cardioprotection. For example, XBW suppressed cardiac hypertrophy via regulation of PI3K/Akt/GSK3β signaling pathway (4). XBW also attenuated chronic heart failure in a rat model (5,6). Ginsenoside Rg1, Rb1 (7), Rg3 (8), Rd (9) signi cantly reduced myocardial infarct size and improved cardiac function in I/R injured model through suppressing oxidative stress, apoptosis and in ammation. Notoginsenside R1 pretreatment ameliorated myocardial injury induced by I/R via inhibiting ROCK and promoting mitochondrial ATP synthase δ-subunits (10). However, as a Chinese traditional formula, only few studies have illustrated the cardioprotection of XBW against MI/RI. Therefore, it is meaningful to clarify the underlying mechanism of XBW against MIRI.
In this study, we rst used the ultra-fast liquid chromatography-quadrupole-time-of-ight tandem mass spectrometry (UPLC-Q-TOF-MS) to identify the chemical pro le of XBW, and used the database to nd out the predicted target of the chemicals, then built a compound-target-disease network. Finally, we systematically investigated the cardioprotective effect of XBW against MI/RI in vitro and in vivo model and veri ed predicted pathway by pharmacological assays (Fig. 1).

Cells and treatment
Rat cardiomyocyte H9c2 cells were purchased from the American Type Culture Collection (ATCC, USA).
H9c2 cells were cultured in DMEM including 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS) at 37°C, 5% CO2. For treatment, H9c2 cells were treated with indicated drugs (XBW (0, 10, 60, 240, 720 µg/mL), diazoxide (100 µM)) and then subjected to OGD condition (After medium was washed by PBS and replaced with DMEM without glucose and FBS, cells were put into a chamber saturated with 95% N2 and 5% CO2) for 6 h and reperfusion for another 18 h. For drug preparation, powdered XBW was extracted with ethanol by ultrasonic extraction for 30 min twice. After concentration, extracts were dissolved in DMEM containing 0.05% ethanol and ltered by 0.22 µm to prepare different doses given drugs.

Cell Viability
The cell viability was measured by MTT assay. In brief, after drug treatment, 0.5 mg/mL MTT solution was added to 96-well plate and incubated at 37 ℃ for 4 h. When removal of the supernatant, 150 µL of DMSO was used to dissolve the formazan products. The absorption was determined by a microplate reader at 490 nm (Thermo Fisher Scienti c, Waltham, CA, USA).

Western blot analysis
Brie y, H9c2 cells and cardiac tissue were extracted by 1 X RIPA buffer (Solarbio Life Sciences, Beijing, China) containing protease and phosphatase inhibitors. The proteins were loaded to 10% SDSpolyacrylamide gels for electrophoresis and transferred to a polyvinylidenedi uoride (PVDF) membrane. After blotting in 5% nonfat milk for 1.0 h, the membranes were incubated with speci c primary antibodies (Bcl2, Caspase-3, Bax, Baclin-1, LC3II, BIP, GAPDH, β-tubulin, β-actin) overnight and goat anti-rabbit or goat anti-mouse IgG-horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT. After washing with 1 X TBST, the bands were detected with enhanced chemiluminescence (ECL) detection reagents from Absin Bioscience Inc (Shanghai, China).

Animal experiments
The experimental procedures and protocols were approved by the Committee on Ethical USE of Animals of Guangzhou University of Chinese Medicine (No.IITCM-20180306). SD male rats (weighting 250-280 g) were obtained from animal laboratory center of Southern Medical University and divided into three groups: Sham group (n = 6), MI/R model group (MI/R + Saline, n = 6), XBW group (MI/R + XBW, n = 6). In brief, SD rats were anesthetized by intraperitoneal injection of 40 mg/kg 2% pentobarbital sodium. After anesthesia, rats were xed and plugged into a ventilator. After opening the chest between the 3rd and 4th rib, the left anterior descending (LAD) artery was ligated with a 6 − 0 silk suture and PE10 tube for 30 min, and then reperfusion for 24 h. Sham group only received left thoracotomy without ligation. The MI/R + XBW group received 180 mg/kg XBW by intragastric administration at 1 h before MI/R. Powdered XBW was dissolved in 0.9% saline, and prepared the given solution according to the weight of rats. After 24 h of reperfusion, heart tissues were cut into 2 mm sections, and incubated in 1% TTC solution at 37 ℃ for 15 min, then xed in 4% PFA overnight. The images of these sections were captured, and the infarct size was analyzed and calculated by image J.

H&E staining
Hearts were xed in the 4% PFA solution, dehydrated by gradient alcohol, and embedded with para n. 5 µm of para n slices were rehydrated by gradient alcohol, and stained with hematoxylin and eosin. Then, the slices were washed with water, dehydrated by 80%, 90% and 100% ethanol, and sealed with neutral gum. Finally, the slices were observed and photographed under light microscope.

Predicting targets of compounds in XBW
The mol2 format les of UPLC-Q-TOF-MS-identi ed chemicals were downloaded from Pubchem database and uploaded to TargetNet webserver (http://targetnet.scbdd.com). In practice, predict protein targets with the score of > 0.90 were selected. In addition, the targets of chemicals reported in literatures were also collected.

Collecting MI/RI-associated targets
The targets related to MI/RI were selected from OMIM database (https://omim.org/) and the literatures using "myocardial ischemia-reperfusion injury" as the keywords.
Gene Ontology (GO) and pathway enrichment of potential targets The Gene Ontology (GO) biological process (BP), molecular function (MF) and cellular component (CC), Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway were analyzed by using the String Database (https://string-db.org/cgi/input.pl). Only the false discovery rate (FDR) ≤ 0.05 were selected.

Statistics analysis
The results were expressed as means ± SD or means ± SEM from no less than three independent experiments. Statistical analysis was performed by one-way ANOVA with GraphPad Prism software (La Jolla, CA, USA). And A p-value < 0.05 was considered as statistical signi cance.
Effect of XBW against myocardial ischemia-reperfusion injury in rat model in vivo and oxygen glucose deprivation-reperfusion (OGD/R) cell model in vitro LAD ligation-induced MI/RI rat model was used to investigate the cardioprotective effect of XBW. As shown in Figure 3A and 3B, XBW administration signi cantly reduced infarct size. CK-MB is one of biomarker in serum for MI/RI, and XBW administration decreased the level of CK-MB induced by MI/RI ( Figure 3C). Echocardiography in Figure 3D exhibited that XBW improved cardiac function. H&E staining showed that XBW administration attenuated in ammatory cell in ltration, and disordered myocardial ber induced by MI/RI ( Figure 3E). In vitro study, XBW protected H9c2 cell against OGD/R injury in a dosedependent manner ( Figure 4A-B). These results suggested that XBW ameliorated MI/RI.

Target Identi cation and Network Analysis
Using TargetNet database and literatures reported targets, we obtained 246 targets of 37 compounds in XBW ( Figure 5A). The network contained 283 nodes and 462 edges, and its average number of neighbors was 3.216. After crossing with MI/RI targets, 50 targets were identi ed and a compound-target network was constructed in Figure 5B and 5C.
After GO enrichment analysis of the targets by the String database, the top 15 enrichment results listed in biological processes (BP), molecular functions (MF) and cellular components (CC) are shown in Figure  6A, which indicated that XBW may regulate the apoptosis and stress response of cardiomycytes via protein binding, enzyme binding, transcription factor binding, protein kinase binding, extracellular space, CHOP-ATF4 complex, etc to attenuate MI/RI. To clarify the underlying pathways of XBW on MI/RI, KEGG pathway analysis was performed in Figure 6B, which exhibited the top 20 related signaling pathways excluding the speci c cancer related pathways. HIF-1 signaling pathway, PI3K-Akt signaling pathway, autophagy, FoxO signaling pathway, apoptosis, etc. Based on the protein-protein interactions (PPI) analysis ( Figure 6C), CASP3, MTOR (apamycin), SIRT1, HIF1A, ATF4, GRP78 (BIP, glucose regulated protein 78) and ATG7 were identi ed with high-degree targets, which played important roles in apoptosis, autophagy and endoplasmic reticulum (ER) stress.

Experimental validations of the molecular mechanisms of XBW against MIRI.
Cardiomyocyte apoptosis is a key factor in the pathological process of MI/RI. As shown in Figure 7A, after 24 h reperfusion, TUNEL staining showed the percentage of apoptosis-positive cells in MI/RI group increased, while decreased in XBW treatment group. In addition, the apoptosis related proteins were detected. XBW increased the ratio of Bcl2/Bax expression and decreased caspase 3 expression ( Figure  7B-D).
During MI/RI, autophagy was also activated following MI/RI in cardiomyocytes to involve in the apoptosis of myocardial cells (26, 27). Bclin-1 and LC3II expressions were detected ( Figure 8A-C). The expressions of Bclin-1 and LC3II in cardiac tissue were markedly increased in MI/RI group, while signi cantly decreased in XBW administration group. In vitro study, XBW could also decrease Beclin-1 expression induced by OGD/R in H9c2 cells ( Figure 8D and 8E). Cardiomyocytes open the unfolded protein response triggered by ER stress as a defensive mechanism at early stage of MI/RI, however, excessive ER stress induced cell apoptosis or even death. As shown in Figure 8F and 8G, the expression of BIP, a marker of ER stress, was signi cantly increased in MI/RI group compared with that in the Sham group, however, XBW administration reduced the elevation of BIP expression. Taken together, these data demonstrated that the cardioprotective effect of XBW against MI/RI was associated with the attenuation of ER stress and autophagy.

Discussion
Myocardial ischemia-reperfusion injury is a di cult clinical problem myocardial infarction therapy, however, current medications for treating MIRI are not ideal (2). The traditional Chinese medicine exhibits unique advantages in the treatment of cardiovascular diseases, based on multiple components and multiple targets (28). XBW is a patented traditional Chinese herbal formula, which has been listed in China for more than 30 years. It is used for treating ischemic heart disease and chronic heart failure (4). However, there is a lack of evidences for the material basis and underlying mechanism of XBW against MI/RI. In the current study, we integrated chemical pro le, network pharmacology, pharmacology and molecular cell biology to investigate the cardioprotective effect and mechanism of XBW against MI/IR. XBW has been used to treating coronary heart disease and chronic heart failure (5). In our study, an in vivo MI/RI rat model was used by performing LAD. The results showed that XBW administration remarkably decreased MI/RI-induced myocardial infarct size and improved cardiac LV function. Moreover, the in vitro results revealed that XBW could also reduce OGD/R-induced cell injury. XBW is composed of nine Chinese medicine. We used UPLC-Q-TOF-MS/MS method to identify 37 chemical constitutes in Table 1, which provided the information of material basis. Importantly, most of these components were from Panax ginseng C.A.Mey and Fuzi, which are monarch drugs in XBW. For example, Panax ginseng has the effects of invigorating Qi, promoting tissue regeneration and enhancing human body resistances (29). Fuzi possesses the effects of causing restoration from collapse, reinforcing re and Yang, and is used to treat acute myocardial infarction and chronic heart failure (30). Consistently, reports have also showed that Panax ginseng, ginsenoside Rg3 and notoginsenoside R1 can alleviate MI/RI by suppressing oxidative stress, apoptosis, in ammation and regulating myocardial energy metabolism (8, 10,31). Fuzi and its alkaloids can improve inotropic effect, left ventricular diastolic function (32), and energy metabolism (33), scavenge hydroxyl radicals and suppress lipid peroxidationto show the cardioprotective effects (34). Moreover, network pharmacological analysis of XBW identi ed that ginsenoside Rg3, Rg1, Rb3, arenobufagin, and notoginsenoside R1 had high degrees, which may be the active compounds of XBW. Thus, the components of XBW are complicated and their pharmacological effects need to be veri ed in the future work.
After screening with MI/RI-related proteins, 50 putative targets of XBW were collected. Among them, CASP3 (caspase 3) activation is a biochemical hallmark of apoptosis (35); BCL2 (B cell lymphoma-2) plays an important role in the negative regulation of apoptosis (36); GRP78 (BIP) is an ER homeostasis marker and upregulated during MI/IR (37); Eukaryotic initiation factor 2 alpha (eIF2α) and activating transcription factor-4 (ATF4) can mediate myocardial ER stress (38). mTOR and Beclin-1 are two key autophagy-related proteins in MI/R injury (39). The results illustrated that XBW might regulate above proteins to show cardioprotective effect. GO enrichment analysis showed that XBW can treating MI/RI by regulation of cell death, apoptosis process, and response to stress. KEGG enrichment analysis demonstrated that apoptosis, autophagy, HIF-1 signaling pathway, PI3K/Akt signaling pathway, and FoxO signaling pathway were involved in XBW for treating MI/RI. To further validate the prediction and analysis, we investigated the key potential mechanism of XBW against MI/RI in vitro and in vivo.
Reducing cardiomyocyte death and infarct size is necessary to MI/RI. Myocardial apoptosis is a key factor for the most of cell death during cardiac pathological processes of MI/RI, while blocking the apoptosis-related signaling pathways helps prevent myocardial injury (40,41). Anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax are involved in the stage of apoptosis. In the present study, XBW suppressed myocardial apoptosis with the decreased TUNEL positive cells and the increased ratio of Bcl2/Bax expression. Therefore, XBW has the potential effects for attenuating myocardial apoptosis for the patients with MI/RI. An emerging number of evidences have indicated that ER stress is involved in the development and pathogenesis of MI/RI (42). During MI/RI, the balance of the homeostasis for the endoplasmic reticulum is broken, subsequently unfolded or misfolded proteins are accumulating in myocardial cells, and eventually triggering ER stress (43). At the early stage, a certain degree of ER stress helps self-repair injured cells, however, if ER stress is excessive, it will provoke the apoptotic signaling pathway activation (44). GRP78 (BIP) is a calcium ion-binding molecular chaperone in the endoplasmic reticulum. When undergoing ER stress, GRP78 and endoplasmic reticulum cross to activate the downstream CHOPassociated apoptotic signaling pathways (45). In our study, XBW treatment decreased the expression of BIP induced by MI/RI in vivo or OGD/R injury in vitro. Autophagy has a dual function in MI/RI (46). Several studies showed that reduction of autophagy clearance in myocardial cells during MI/RI threatens cell survival (47,48). Promoting autophagy moderately may protect cell and mitochondrial injury in MI/RI (26,27). However, at the late stage of MI/RI, it induces excessive activation of autophagy, resulting in cytotoxic cell death (49)(50)(51). Thus, prevention of excessive autophagy activated during MI/RI may be bene t to reduce cardiomyocyte death and improve cardiac function. In present study, XBW treatment inhibited MI/RI-induced beclin-1 and LC3II expressions to inactivate excessive autophagy. These results fully demonstrated that XBW protected heart against MI/RI through the multicomponent, multitarget and multipathway. The pathogenesis of MI/RI is complex, and XBW has potential clinical application value for the prevention and treatment of multiple pathways.
However, there are still several limitations in our study to solve in the future work. By using UPLC-Q-TOF-MS/MS method, 37 major compounds from six medicinal materials, but the volatile constituents in the left three ones including Cinnamomum cassia (L.) J.Presl (Rougui), moschus (Shexiang) and Borneolum syntheticum (Bingpian) had not been detected, which needs QC-MS analysis in our later work to rich material basis of XBW. In addition, the present study only evaluated the overall e cacy and mechanism of XBW, buy the effects and underlying mechanism of the identi ed active compounds, such as ginsenoside Rg3, Rg1, Rb3, etc, have to be further veri ed.

Conclusion
In the present study, we revealed the therapeutic effect and underlying mechanism of XBW against MI/IR based on chemical pro le, network pharmacology and experimental support. 37 chemical constituents in XBW were identi ed, 50 potential MI/RI targets, and 5 signi cant pathways were achieved by network pharmacology analysis. Collectively, our results demonstrated that XBW ameliorated the apoptosis of cardiomyocytes in MI/RI by suppressing autophagy and ER stress (Fig. 9).       cells were treated with indicated doses of XBW (0, 30, 60, 240 μg/mL), and followed by 6 h ODG condition and 18 h reperfusion. Protein of H9c2 cells were isolated and subjected to Western blotting. n = 3, ## p<0.01, ### p<0.001, Ctrl vs OGD/R; *p<0.05,**p<0.001, OGD/R+XBW vs OGD/R. (F and G) The expression of BIP in heart tissue. After MI/RI, the heart tissues in each group were collected and subjected to Western blotting analysis. n = 5, ## p<0.01, Sham vs MI/R group; *P<0.05, MI/R+XBW vs MI/R group.

Figure 9
Overview of potential mechanism of XBW against myocardial ischemia-reperfusion injury.

Supplementary Files
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