PKM2 regulated by liquiritin improves ischemia and reperfusion induced endothelial injury and promotes angiogenesis through STAT3 pathway

PKM2 regulated by liquiritin improves ischemia and reperfusion induced endothelial injury and promotes angiogenesis through STAT3 pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article PKM2 regulated by liquiritin improves ischemia and reperfusion induced endothelial injury and promotes angiogenesis through STAT3 pathway Bin Zeng, Hanyang Sun, Yan Zhang, Xiaoting Liao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7283996/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Alleviating ischemia/reperfusion (I/R) induced cardiac vascular endothelial dysfunction may effectively prevent myocardial I/R injury. Studies showed that PKM2 are associated with a reduction in cardiomyocyte apoptosis and ischemic insult. Liquiritin (LIQ) has attracted attention for its cardioprotective effects. However, whether LIQ can reduce endothelial injury induced by I/R has not been fully explored and the mechanism of PKM2 in this process remains to be elucidated. In our study, we identified that LIQ preconditioning significantly improved cardiac dysfunction and increased angiogenesis after I/R injury, but inhibition of PKM2 markedly deteriorated cardiac function and angiogenesis in vivo. In vitro, experiments have revealed that LIQ significantly promoted angiogenesis, inhibited oxidative stress, apoptosis and inflammation, and then effectively prevented H 2 O 2 -induced umbilical vein endothelial cell (HUVECs) damage. LIQ up-regulated PKM2 expression, promoted PKM2 phosphorylation, increased the ratio of p-PKM2 to PKM2, and significantly increased the expression of p-STAT3. However, suppression of PKM2 by shikonin significantly abolished these protective effects. Additionally, it is worth noting that a significant decline in the beneficial effect of LIQ was observed in models that inhibit STAT3. In conclusion, LIQ upregulates PKM2, promotes PKM2 phosphorylation, increases the ratio of p-PKM2 to PKM2, and activates STAT3 pathway to effectively alleviate ECs dysfunction and stimulate angiogenesis, which may be a potential treatment for myocardial I/R injury. Health sciences/Cardiology Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Molecular biology Biological sciences/Physiology Liquiritin PKM2 STAT3 pathway endothelial dysfunction angiogenesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Revascularization is a common method to save most patients with myocardial infarction, but it can lead to ischemia/reperfusion (I/R) injury, cause irreversible myocardial necrosis, and lead to pathological cardiac remodeling and heart failure, and eventually death. The excessive production of reactive oxygen species (ROS) during reperfusion disrupts the body's antioxidant defense system, leading to oxidative stress, dysfunction of endothelial cells (ECs), and damage to vascular wall cells [ 1 , 2 ] . Vascular endothelial dysfunction is the initial stage of vascular injury and also the first critical step in the progression of myocardial I/R injury [ 3 , 4 ] , which can cause an increase in infarct size, thereby increasing the perioperative mortality rate of patients [ 5 ] . Therefore, improving vascular function and preventing endothelial injury may be an effective means of preventing and treating myocardial I/R injury. Pyruvate Kinase M2 (PKM2), a critical isozyme of pyruvate kinase (PK), maintains glycolytic functions under normoxic conditions while exhibiting hypoxia-enhanced catalytic activity. Under oxygen deprivation, PKM2 dynamically modulates glucose flux through both glycolytic and alternative hexose metabolic pathways, coordinating ATP production with biosynthetic precursor synthesis. Research has shown that PKM2 expression is low in adult cardiomyocytes, but the expression of PKM subtypes in ischemic myocardium shows significant changes, with a significant increase in PKM2 expression after myocardial ischemic injury [ 6 ] . PKM2 has been shown to play an effective role in cardiovascular function and disease, which can regulate the postnatal cardiomyocyte cycle and protect cell survival from ischemic damage [ 7 ] . Cardiomyocytes lacking PKM2 are manifested as reduced cardiac energy, stress response, angiogenesis and survival pathways [ 8 ] . In addition, ECs mainly express PKM2, which is essential for the growth of ECs and the maintenance of vascular integrity [ 9 ] . PKM2 knockout mice showed that the proliferation, migration and tubular ability of ECs were severely inhibited, and the innate immune signal of ECs was triggered [ 10 ] . Signal transducers and activators of transcription3 (STAT3), is expressed in cardiomyocytes, ECs and other cardiac cell types, and can be activated by ischemia, hypoxia and ROS, which plays a key role in cardiovascular disease [ 11 ] . In addition, STAT3 produced by ECs can improve cardiac function after I/R injury by maintaining vascular integrity and inhibiting cardiomyocytes apoptosis [ 12 ] . Research has shown that PKM2 is related to the expression of STAT3, PKM2 overexpress can activate STAT3, while PKM2 knockout inhibits STAT3 expression [ 13 ] . As the upstream kinase of STAT3, PKM2 can promote angiogenesis and nerve regeneration through phosphorylation of STAT3, thereby restoring brain function in ischemic stroke mice, inhibiting apoptosis and regulating post-ischemic inflammation of peripheral neutrophils [ 14 , 15 ] . Licorice also known as "Glycyrrhizae Radix et Rhizoma" is the dried root or rhizome of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., and Glycyrrhiza glabra L. according to the Pharmacopoeia of the People's Republic of China, which is widely used as a traditional Chinese medicine [ 14 ] . Liquiritin (LIQ) is a flavonoid compound (C 21 H 22 O 9 , molecular weight = 418.39, as shown in the figure). It is an important monomeric active component in licorice, which has a variety of pharmacological effects such as antidepressant, anti-tumor, protection of the nervous and heart system, antioxidant and anti-inflammatory [ 17 ] . Previous studies have shown that LIQ exerts a neuroprotective effect on cerebral I/R-induced injury in mice through antioxidant and anti-apoptotic mechanisms [ 18 ] , and can reduce the expression of inflammatory factors in lipopolysaccharide (LPS) -induced mice cardiomyocytes, inhibits cardiac oxidative stress and apoptosis [ 19 ] . LIQ can also reduce Adriamycin-induced cardiotoxicity [ 20 ] . Studies have shown that PKM2 can be regulated by various traditional Chinese medicines [ 21 ] . Given that PKM2 not only regulates energy metabolism but also directly modulates oxidative stress and inflammatory pathways, we hypothesize that LIQ may protect against I/R injury by targeting PKM2 to activate the STAT3 signaling pathway. In this study, we investigated the role of LIQ with a focus on the function of PKM2. We found that the expression of PKM2 and PKM2 phosphorylation increased, and the ratio of p-PKM2 to PKM2 increased after LIQ pretreatment. LIQ pretreatment prevented endothelial cell injury, promoted angiogenesis, and mitigated I/R injury in a PKM2-dependent manner, and the activation of the STAT3 signaling pathway played a crucial role in this process. 2. Materials and methods 2.1 Materials LIQ (C 21 H 22 O 9 , PubChem CID: 503737, purity > 98%) was purchased from Shanghai Yuanye Bio Technology. Shikonin (C 16 H 16 O 5 , PubChem CID: 479503) as an effective PKM2 activation inhibitor [ 22 ] was purchased from Shanghai Yuanye Bio Technology, STAT3 activation inhibitor Stattic and H 2 O 2 were both purchased from Sigma. 2.2 Animal I/R model and study protocols C57BL/6 mice aged 8–10 weeks were purchased from Skobes Biotechnology Co., Ltd. (Henan, China). The animal protocol used in this experiment was approved by the Animal Care and Use Committee of Wuhan University People's Hospital and complies with the Guidelines for the Care and Use of Experimental Animals issued by the National Institutes of Health in the United States and ARRIVE guidelines of National Centre for the Replacement, Refinement & Reduction of Animals in Research Randomly divide the mice into 5 groups: (1) Sham group, silk was placed underneath the LAD without ligated; (2) I/R group, LAD was ligated for 30 min and received 2 hours of reperfusion; (3) I/R + LIQ group, LIQ(20mg/kg);༈4༉ I/R + LIQ + Shikonin group, LIQ༈20mg/kg༉and Shikonin༈40mg/kg༉;(5) I/R + LIQ + Stattic group, LIQ༈20mg/kg༉and Stattic༈15mg/kg༉. The drug was administered intraperitoneally daily for 3 days before surgery, and each mouse was given the same total dose daily by adjusting the dose of saline. Mice were anesthetized with 80mg/kg 3% pentobarbital sodium, fixed in supine position, and breathing was controlled by a small ventilator. Perform left thoracotomy between the 3rd and 4th ribs of the left chest, the anterior descending coronary artery (LAD) was ligation for 30 min, and then the ligation line was cut and blood perfusion was restored for 2 h. In the blank group, the left anterior descending coronary artery was separated and only threaded without ligature, and the ligature was pulled out 30 minutes later. 2.3 Echocardiogram Mice were lightly anesthetized with 2% isoflurane and transthoracic echocardiography was performed with MyLab30CV ultrasound (Biosound Esaote Inc Indianapolis) to assess cardiac function on day 7 post-reperfusion in mice. 2.4 Immunohistochemical detection of myocardial neovascularization density After function measurements were recorded, mice were euthanized with 3% sodium pentobarbital and the hearts were removed immediately, the excised hearts were placed in 10% KCl solution to induce cardiac arrest during diastole.To obtain cardiac pathological samples, the atriums above the ligation of the heart were removed, the remaining tissues were fixed immediately after dissection in 4% paraformaldehyde for 30–60 min. Tissues were embedded in OCT compound (Sigma), then quickly frozen in liquid nitrogen and stored at -80°C. Cryostat sections were cut into 5 µm thin sections. Perform endothelial cell specific marker CD31 staining on each group of specimens, and staining the cytoplasm of endothelial cells with a brown color indicates positive expression. Five visual fields were randomly selected to calculate the number of CD31 positive microvessels, and Image J was used to detect mean optical density (MOD) of positive cells, which was used as a quantitative index to evaluate neovascularization density. 2.5 Cell Culture HUVECs (from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured by DMEM, which contained 10% FBS and 1% penicillin/streptomycin. According to pre experiments, 400µmol/L H 2 O 2 was used as the concentration of subsequent experiments. HUVECs were separated into five groups: (a) Control group: Cells cultured in normal medium without any treatment; (b) H 2 O 2 group: Cells induced by H 2 O 2 (400µmol/L) for 12h; (c) LIQ group (H 2 O 2 + LIQ): Cells were cultured by LIQ (1µmol/L) for 24h before H 2 O 2 treatment; (d) Shikonin group (H 2 O 2 + LIQ + Shikonin): Cells were co-cultured by LIQ and Shikonin (1µmol/L) for 24h before H 2 O 2 treatment; (e) Stattic group (H 2 O 2 + LIQ + Stattic): Cells were co-cultured by LIQ and Stattic (2µmol/L) for 24h before H 2 O 2 treatment. 2.6 Cell wound healing assay A wound-healing assay was performed as described previously [ 23 ] . HUVECs were seeded on 6-well plates (5×10 5 cells/well), after overnight, cells were treated in groups. A straight line was drawn along the bottom of the plate with a 200 µL pipette and the detached cells were washed with PBS, then replaced with serum-free basic medium, and observe the healing of scratches and take photos (at 0, 12, 24, 36 and 48 h). Measure the scratch area using Image J, and the scratch healing rate is (scratch area of 0h ~ scratch area of 24h)/0h scratch area × 100%. 2.7 Tube formation in HUVEC Tube formation assay was performed as described before [ 24 , 25 ] . Chilled liquid Matrigel (356234, Corning) was dispensed into 96-well plates (50 µl/well) and incubated at 37℃ for 1 h for solidification. Then inoculate each group of HUVECs onto a Matrigel bed (2×10 4 cells/well) and incubate for 6 hours. Use ImageJ software to image the formation of the tube and quantify the length of the tube. 2.8 Measurement of ROS in HUVEC The intracellular ROS production was measured by the ROS Assay Kit (Beyotime Biotechnology, China). In brief, cells were collected and resuspended in DMEM medium after treatment, and then measured based on the fluorescence intensity of 2’,7’-dichlorofluorescin-diacetate (DCFH-DA). The accumulation of ROS was visualized using a fluorescence microscope. 2.9 Western blot analysis The cells were harvested and lysed in RIPA buffer after being treated in groups, and the concentration was determined with the BCA kit (Beyotime, Beijing, China). After protein samples were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes, the membranes were blocked into 5% of skimmed milk for 30min and then incubated overnight at 4℃ with antibodies against PKM2(1:2000 dilution), p-PKM2 (1:2000 dilution), STAT3 (1:2000 dilution), p-STAT3 (1:10000 dilution), VEGF (1:2000 dilution), FGF2 (1:2000 dilution), IL-1β(1:2000 dilution), Bax (1:20000 dilution), Bcl-2 (1:4000 dilution), Nrf2 (1:1000 dilution), and HO-1 (1:20000 dilution), TNF-α (1:10000 dilution), β-actin (1:20000 dilution). In addition, rabbit anti-PKM2(4053), STAT3 (9139), and p-STAT3 (Tyr705, 9145) were procured from Cell Signaling Technology (CST, Danvers, MA, USA) and rabbit anti-p- PKM2 (Ser37) (ab205678) was obtained from Abcam (Cambridge, UK). After the membranes washing, the secondary antibodies were incubated at room temperature for 60min. The Image J software was used to quantitatively analyze the intensity of each protein band. 2.10 Statistical analysis GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA) is used for statistical analysis. All the results were presented as mean ± SD. Unpaired t-test was used to compare two independent samples, and one-way analysis of variance (ANOVA) was used to compare multiple groups. Values of P < 0.05 were considered statistically. 3. Results 3.1 LIQ improved cardiac function and enhanced angiogenesis after I/R in a PKM2-dependent manner To evaluate cardiac function, we performed echocardiography 7 days after surgery. The results showed that left ventricular function was impaired in mice after I/R injury, and LVEF and FS were significantly reduced (P < 0.05), while LIQ pretreatment significantly reversed this result and improved cardiac function (p < 0.05, Fig. 1 A). CD31 is an important marker of angiogenesis that is highly expressed on the surface of ECs. The vascular density in the surrounding area of left ventricular infarction was analyzed by CD31 staining to determine endothelial cell proliferation or angiogenesis. Our results showed an increase in CD31 expression in the I/R group, while LIQ pretreatment further increased the expression of CD31(p < 0.05), which indicated some improvement in angiogenesis and microcirculation (Fig. 1 B-C). It is worth noting that after inhibiting PKM2, the beneficial effect of LIQ on the heart was significantly weakened (Fig. 1 ). 3.2 LIQ promoted H 2 O 2 -induced HUVECs migration and tube formation in a PKM2-dependent manner To evaluate the effect of LIQ on the migration ability of H 2 O 2 -induced HUVECs injury and the role of PKM2, wound healing assay was examined for healing rate. Compared with the control group, the migration capacity of HUVECs reduced after H 2 O 2 treatment, while LIQ could significantly reverse this phenomenon, but this phenomenon was significantly weakened after using Shikonin to inhibit PKM2. In addition, compared to the LIQ group, the migration area of HUVECs significantly decreased after the treatment of Stattic (Fig. 2 A-B). Moreover, to assess the effect of LIQ on tube formation of the H 2 O 2 -induced injury on HUVECs, Matrigel assay was used to simulate the angiogenesis experiment. In the control group robust and well-formed capillary-like tubes could be detected. However, the tube formation was significantly inhibited after treatment of H 2 O 2 (P < 0.001), while compared with the H 2 O 2 group, LIQ treatment obviously encouraged tube formation and the tube length was increased, and this protective effect was obviously abolished by cotreatment with Shikonin (P < 0.01). Additionally, the use of Stattic significantly inhibited tube formation compared to the LIQ group (P < 0.001) (Fig. 2 C-D). 3.3 LIQ enhanced the production of pro-angiogenic molecules in H 2 O 2 -induced HUVECs in a PKM2-dependent manner Western blot was used to detect the protein expression of pro-angiogenic molecules VEGF and FGF2 in each group to investigate the effect of LIQ on angiogenesis after H 2 O 2 -induced HUVECs injury. The results showed that the decreased VEGF and FGF2 in the H 2 O 2 group could be reversed through LIQ treatment, however, this beneficial effect was significantly eliminated by PKM2 inhibitor Shikonin (P < 0.01, P < 0.001). Stattic prominently inhibited the increase of VEGF and FGF2 (P < 0.001) (Fig. 3 ). These results suggest that PKM2 is required by LIQ to promote angiogenesis. 3.4 LIQ inhibited H 2 O 2 -induced oxidative stress in HUVECs by up-regulating PKM2 DCFH-DA was used to evaluate the intracellular ROS levels in each group, and ROS production was quantified by measuring cell fluorescence intensity to detect the levels of oxidative stress in each group. Compared with the control group, the red fluorescence of HUVECs induced by H 2 O 2 was significantly enhanced, which indicated that the ROS generation increased after H 2 O 2 treatment (P < 0.001). In addition, LIQ pretreatment significantly reduced fluorescence intensity, but inhibition of PKM2 prominently reversed this change (P < 0.001) (Fig. 4 A-B). Furthermore, we observed the expression levels of antioxidant proteins Nrf2 and HO-1 in each group. After H 2 O 2 treatment the Nrf2 and HO-1 protein expression levels had significantly increased compared with the control group (P < 0.001). However, LIQ pretreatment remarkably enhanced the Nrf2 and HO-1 expression levels compared to those in the H 2 O 2 group (P < 0.001, P < 0.05), while Shikonin can inhibit this effect. In addition, Stattic significantly inhibited the anti-oxidative stress effect of LIQ (Fig. 4 C-E). 3.5 LIQ prevented H 2 O 2 -induced apoptosis of HUVECs by up-regulating PKM2 To evaluate the apoptosis of HUVECs, Western blot was used to analyze the protein expression of BCL-2 and Bax. The results showed that the decline in the Bcl-2/Bax ratio in the H 2 O 2 group could be reversed by LIQ treatment, and this beneficial effect was significantly mitigated after PKM2 inhibition (P < 0.01, P < 0.001). Moreover, Stattic prominently inhibited the increase of the Bcl-2/Bax ratio (P < 0.001) (Fig. 5 ). 3.6 LIQ reduced H 2 O 2 -induced inflammation of HUVECs induced by up-regulating PKM2 In order to detect cellular inflammation, Western blot was used to detect the levels of pro-inflammatory cytokines TNF-α and IL-1β. IL-1β and TNF-α expression was significantly upregulated in H 2 O 2 -induced HUVECs compared to the control group (P < 0.001). While LIQ obviously mitigated H 2 O 2 -induced increases in TNF-α and IL-1β (P < 0.001), however, these effects were significantly reversed after treatment with Shikonin or Stattic (P < 0.001) (Fig. 6 ) 3.7 LIQ up-regulated the ratio of p-PKM2 to PKM2 and activated STAT3 signaling pathway Western blot analysis of the expression of PKM2 protein in each group showed that LIQ pretreatment significantly increased the expression level of PKM2 protein, the level of phosphorylated PKM2, and the ratio of p-PKM2 to PKM2 (P < 0.001). PKM2 inhibitor Shikonin significantly reversed this result (P < 0.05)(Fig. 7 ). Since PKM2 is known as a STAT3 transcriptional coactivator, we also evaluated the role of STAT3 in LIQ-induced PKM2 protein accumulation in HUVECs. The results showed that LIQ pretreatment significantly increased STAT3 phosphorylation and the ratio of p-STAT3 to STAT3, but inhibition of PKM2 by shikonin significantly reduced LIQ-induced p-STAT3 activation and the ratio of p-STAT3 to STAT3 (P < 0.001) (Fig. 8 ). In addition, there was no significant difference in the expression of p-PKM2 compared with the LIQ group after Stattic inhibited STAT3, but the beneficial effect of LIQ remarkably decreased. These data indicate that STAT3 is the key downstream mediator of PKM2. Discussion ECs are the most abundant cell types in the cardiac [ 26 ] , the blood vessels constituted by them cover the cardiac and lymphatic, which participate in the pathophysiological process of the cardiac. I/R can cause endothelial damage through various mechanisms, so reducing endothelial damage and promoting angiogenesis may be an effective treatment strategy for myocardial I/R injury [ 27 ] . PK isoenzyme PKM2 is highly expressed in ECs and plays an important role in endothelial growth and vascular integrity. Studies have shown that PKM2 loss alters endothelial metabolism and inhibits endothelial growth and angiogenesis [ 10 ] . LIQ, as an important monomer active component in licorice, has anti-inflammatory, anti-oxidant, neuroprotective and other pharmacological effects. This study first confirmed the regulatory effects of LIQ on PKM2 by establishing in vivo I/R injury models and in vitro H 2 O 2 induced HUVECs injury models. LIQ can upregulate PKM2 expression, promote PKM2 phosphorylation, increase the ratio of p-PKM2 to PKM2 and activate the STAT3 signaling pathway to rescue I/R-induced ECs injury. This study used echocardiography to evaluate whether LIQ pretreatment can protect cardiac function in vivo after I/R injury. The results showed that the LVEF and FS values of LIQ pretreated mice were significantly increased, indicating that LIQ pre-treatment significantly improved left ventricular function in mice after I/R injury. After inhibiting PKM2, this protective effect of LIQ is partially eliminated. Angiogenesis is a crucial step in tissue repair and regeneration after ischemia. The reconstruction of blood flow in the infarcted area can save the infarcted myocardium by inhibiting apoptosis, removing scar tissue and recruiting progenitor cells [ 28 ] . CD31 is a specific marker of endothelial cells, which can reflect angiogenesis. Our results indicate that LIQ pretreatment significantly increased the CD31 in the infarct boundary zone of mice after I/R, indicating that LIQ can significantly promote angiogenesis in the ischemic zone to improve impaired cardiac function. However, this beneficial effect was significantly weakened after inhibition of PKM2 by Shikonin. Study has demonstrated that Shikonin, a naphthoquinone isolated from the traditional Chinese medicine Lithospermum, inhibits the function of PKM2 by inhibiting the pyruvate kinase activity and nuclear translocation of PKM2, and decreasing both dimerization and tetramerization of PKM2 in macrophages [ 29 ] . Angiogenesis is achieved through the proliferation, migration, and tube formation of ECs. Our results indicated that LIQ pretreatment significantly enhanced the transfer and tubular ability of endothelial cells after H 2 O 2 treatment, and inhibition of PKM2 significantly reversed this phenomenon. VEGF and FGF2 are the main regulators that stimulate the migration and proliferation of ECs to form new blood vessels, and are key molecular targets that promote angiogenesis in ischemic diseases such as myocardial infarction and myocardial ischemia [ 30 ] . In this study, we found that VEGF and FGF2 significantly decreased after H 2 O 2 treatment, while LIQ pretreatment reversed this result. The beneficial effect of LIQ is obviously reduced after inhibiting PKM2. These data suggest that LIQ may promote angiogenesis after I/R by up regulating PKM2, promoting PKM2 phosphorylation and increasing the ratio of p-PKM2 to PKM2. Oxidative stress can mediate the process of I/R injury, causing endothelial dysfunction, leading to endothelial cell apoptosis, and subsequently altering the function and structure of blood vessels [ 1 , 31 ] . Low levels of ROS can maintain normal physiological metabolism, however, excessive ROS can lead to oxidative stress and oxidative damage, which damages cellular macromolecules including DNA, lipids and proteins [ 32 ] . In this study, LIQ pretreatment demonstrate clearance of H 2 O 2 . Transcription factor Nrf2 plays a key role in clearing ROS, and the antioxidant gene HO-1 regulated by Nrf2 helps cells resist oxidative stress [ 33 ] . This study found that after H 2 O 2 treatment the expression of Nrf2 and HO-1 was significantly increased, while LIQ pretreatment further increased the levels of Nrf2 and HO-1. These effects were significantly reversed after inhibition of PKM2, which indicated that LIQ protected HUVECs from oxidative stress in a PKM2 dependent manner. The apoptosis of ECs is an important factor causing endothelial dysfunction, therefore the apoptosis status of ECs in each group was evaluated. Research has shown that anti-apoptotic protein Bcl-2 and proapoptotic protein Bax play important roles in cell apoptosis [ 32 ] . Consequently, we detected the above indicators through Western blot, and the results showed that H 2 O 2 treatment significantly reduced the Bcl-2/Bax ratio, decreased the expression of anti-apoptotic Bcl-2, and increased proapoptotic Bax. Although LIQ pretreatment significantly improved the ratio of Bcl-2/Bax, this increase evidently weakened evidently after PKM2 inhibition which further confirmed that LIQ plays an anti-apoptotic role partly in a PKM2-dependent manner. Studies have confirmed that inflammation is regarded as an indicator of endothelial status, and after endothelial dysfunction, the pro-inflammatory cytokines increase, thereby promoting the inflammatory response of ECs [ 34 ] . The inflammatory cytokines TNF-α and IL-1β play a major role in endothelial dysfunction, and the increased level of them is associated with more severe clinical conditions and worse outcomes [ 34 , 35 ] . Therefore, we detected the TNF- α and IL-1 β protein expression levels through Western blot, and our results showed that H 2 O 2 treatment significantly induced the accumulation of IL-1β and TNF-α, while LIQ remarkably reduced the levels of these inflammatory cytokines and exerts an anti-inflammatory effect. The STAT3 signaling pathway plays an important role in enhancing cell proliferation and other aspects [ 36 ] . In ischemic brain injury, the activation of STAT3 signaling could promote tissue repair and functional recovery by enhancing cell migration, promoting angiogenesis and regulating cell inflammation [ 14 , 15 ] . In the present study, H 2 O 2 treatment increased the levels and phosphorylation of STAT3, which indicates that the STAT3 signaling pathway plays an important role in H 2 O 2 -induced cell damage. Moreover, LIQ pretreatment significantly enhanced this result, and significantly increasesd the ratio of p-STAT3 to STAT3. while inhibiting PKM2 weakened the expression and phosphorylation of STAT3, and the ratio of p-STAT3 to STAT3. After inhibiting STAT3, the expression of p-PKM2 did not exhibit a decrease, but the protective effect of LIQ on I/R-induced endothelial injury and the promoting effect of angiogenesis were partially eliminated. Therefore, it is evident that STAT3 is the key downstream mediator of PKM2 and LIQ plays a protective role in I/R induced endothelial injury by upregulating PKM2, promoting PKM2 phosphorylation, increasing the ratio of p-PKM2 to PKM2, and activating the STAT3 signaling pathway. In conclusion, LIQ can alleviate endothelial dysfunction induced by I/R by promoting angiogenesis, inhibiting oxidative stress, apoptosis, and inflammation. The beneficial effects of LIQ are mainly mediated by upregulating PKM2, promoting PKM2 phosphorylation, increasing the ratio of p-PKM2 to PKM2, and activing of the STAT3 signaling pathway. LIQ preconditioning will be a potential strategy in clinical as the repair of injured heart. Declarations Ethics approval and consent to participate The animal protocols in this experiment were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. License NO. SCXK(鄂)2018 − 0265. Consent for publication All the authors confirm that the work described has not been published before, and all the authors agree to publication in the Journal. Competing interests The authors declare that there is no competing interest. Author contributing Bin Zeng contributed to the conception, revised this manuscript and provided the funding. Hanyang Sun, Xiaoting Liao and Yan Zhang carried out the experiments, performed data analysis and wrote the draft of the manuscript. Lei Liu and Xiang Zhang performed statistical analyses. All authors reviewed the results and approved the final version. Funding This work was supported by the Chinese National Nature Science Foundation [30900609, 81270271, 81570333 and 81670304] and the Fundamental research funds for the Central Universities [2042020kf1014]. Author Contribution Bin Zeng contributed to the conception, revised this manuscript and provided the funding. Hanyang Sun, Xiaoting Liao and Yan Zhang carried out the experiments, performed data analysis and wrote the draft of the manuscript.Xiang Zhang performed statistical analyses. All authors reviewed the results and approved the final version. Data Availability Data is provided within the manuscript or supplementary information files References Daiber, A. & Chlopicki, S. Revisiting pharmacology of oxidative stress and endothelial dysfunction in cardiovascular disease: Evidence for redox-based therapies[J]. Free Radic. Biol. 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(2022). 10.1073/pnas.2202631119 Martins, T. F. et al. Cocoa Flavanols Protect Human Endothelial Cells from Oxidative Stress[J]. Plant. Foods Hum. Nutr. 75 (2), 161–168. 10.1007/s11130-020-00807-1 (2020). Zeng, B. et al. Thyroid hormone protects cardiomyocytes from H2O2-induced oxidative stress via the PI3K-AKT signaling pathway[J]. Exp. Cell Res. 380 (2), 205–215. 10.1016/j.yexcr.2019.05.003 (2019). Choi, E. S. et al. Ligustilide attenuates vascular inflammation and activates Nrf2/HO-1 induction and, NO synthesis in HUVECs[J]. Phytomedicine 38 , 12–23. 10.1016/j.phymed.2017.09.022 (2018). Park, J. H. et al. Notch1-mediated inflammation is associated with endothelial dysfunction in human brain microvascular endothelial cells upon particulate matter exposure[J]. Arch. Toxicol. 95 (2), 529–540. 10.1007/s00204-020-02942-9 (2021). Zeng, B. et al. Thyroid hormone mediates cardioprotection against postinfarction remodeling and dysfunction through the IGF-1/PI3K/AKT signaling pathway[J]. Life Sci. 267 , 118977. 10.1016/j.lfs.2020.118977 (2021). Wang, B. et al. PKM2 is involved in neuropathic pain by regulating ERK and STAT3 activation in rat spinal cord[J]. J. Headache Pain . 19 (1), 7. 10.1186/s10194-018-0836-4 (2018). Additional Declarations No competing interests reported. Supplementary Files OriginalData9.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7283996","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":512350708,"identity":"08d4d200-79ca-48e9-8536-77ed4f5c9bb5","order_by":0,"name":"Bin Zeng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYJACZgYDBiBiPnDgww/StLAlHpzZQ7QWBpAWHuPDHGxEKJdv7z38uqDgTuJ2iZwPhxl4GOT5xQ7g18LYcy7NeobBs8SdM3I3HC6wYDCcOTuBgKMkcsyMeQwO5264AdQyg4chweA2AS1s8m9gWnIeHOZhI0ILjwSP8WOoFgbitEjw5JgxzzA4XL/hzDMDYCBLEPaLfPsZ488Ffw4bGxxPfvzhww8beX5pAlpA3pFAtpWgchBg/kCUslEwCkbBKBi5AACL/Ed/4CtduwAAAABJRU5ErkJggg==","orcid":"","institution":"Renmin Hospital of Wuhan University, Wuhan University","correspondingAuthor":true,"prefix":"","firstName":"Bin","middleName":"","lastName":"Zeng","suffix":""},{"id":512350710,"identity":"c5382037-481a-4ecc-89fb-ce96f21dc1cb","order_by":1,"name":"Hanyang Sun","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University, Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Hanyang","middleName":"","lastName":"Sun","suffix":""},{"id":512350711,"identity":"767e5aa8-aeae-4b20-929c-6b235a6f1600","order_by":2,"name":"Yan Zhang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University, Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhang","suffix":""},{"id":512350712,"identity":"0390fe18-0fc0-4126-9b3c-3f7ccf67a387","order_by":3,"name":"Xiaoting Liao","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University, Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoting","middleName":"","lastName":"Liao","suffix":""}],"badges":[],"createdAt":"2025-08-03 14:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7283996/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7283996/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91025409,"identity":"f4fcf2df-bdb4-4df3-9ba1-3b47ceb408fe","added_by":"auto","created_at":"2025-09-10 20:02:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":956406,"visible":true,"origin":"","legend":"\u003cp\u003eLIQ improved cardiac function and enhanced angiogenesis after I/R. (A) Left ventricular mode echocardiograms: Left ventricular ejection fraction (%LVEF) and Fractional shortening (%FS). (B-C) The total area of CD31 positive endothelial cells was considered as the angiogenesis area. Values are expressed as the mean ± SD from 5 experiments (*P\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001, ns meaningless).\u003c/p\u003e","description":"","filename":"LIQFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-7283996/v1/346fa3b02834c640b11e3f52.png"},{"id":91025806,"identity":"1b8979ce-ecde-4b97-ae46-de4345a4ae25","added_by":"auto","created_at":"2025-09-10 20:10:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5830694,"visible":true,"origin":"","legend":"\u003cp\u003eLIQ promotes the migration and the tube formation of HUVECs induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (A) The wound healing in each group at 0, 24, 36 hours. Scale bars represented 100 μm and the images are brightness/contrast-adjusted. (B) Relative mobility rates of each group at 0, 24, and 36 hours. (C) Images representing the formation of HUVECs tubes in each group (Scale bar:100μm). (D) Quantification of the tube formation is presented as the tube length. Values are expressed as the mean ± SD from 3 experiments (*P\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001, ns meaningless).\u003c/p\u003e","description":"","filename":"LIQFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-7283996/v1/5d9743ce8fa52e4b722c702d.png"},{"id":91025217,"identity":"51413cfd-b57e-4e5c-b984-6835dbb1e4d8","added_by":"auto","created_at":"2025-09-10 19:54:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":257748,"visible":true,"origin":"","legend":"\u003cp\u003eLIQ enhances the production of VEGF and FGF2 in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HUVECs. VEGF and FGF2 protein expression levels were analyzed by western blot in each group. (A) Grayscale value of the VEGF. (B) Grayscale value of the FGF2. The above data represent three independent replicates and are expressed as the mean ± SD (*P\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001, ns meaningless).\u003c/p\u003e","description":"","filename":"LIQFigures3.png","url":"https://assets-eu.researchsquare.com/files/rs-7283996/v1/09ca34f1850ac9f69be0fc9f.png"},{"id":91025410,"identity":"4643476c-f26d-4a1f-8840-a8a1a54c5b0b","added_by":"auto","created_at":"2025-09-10 20:02:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":792422,"visible":true,"origin":"","legend":"\u003cp\u003eLIQ reduced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced ROS production and oxidative stress in HUVECs. (A) ROS production of each group was observed using a fluorescence microscope (Scale bar:50μm). (B) Bar chart showing quantitative data of cell fluorescence intensity.\u003c/p\u003e\n\u003cp\u003eThe Nrf2 and HO-1 protein expression levels in each group were analyzed by western blot. (C) Grayscale value of the HO-1. (D) Grayscale value of the Nrf2. The above data represent three independent replicates and are expressed as the mean ± SD (*P\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001, ns meaningless).\u003c/p\u003e","description":"","filename":"LIQFigures4.png","url":"https://assets-eu.researchsquare.com/files/rs-7283996/v1/c1fab522a4166df547f920bf.png"},{"id":91025219,"identity":"5fb406e4-7447-45dd-b40b-d567ea546cd3","added_by":"auto","created_at":"2025-09-10 19:54:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":338662,"visible":true,"origin":"","legend":"\u003cp\u003eLIQ prevents HUVECs apoptosis caused by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. The Bcl-2 and Bax protein expression levels in each group were analyzed by western blot. (A) Grayscale value of the Bcl-2 and Bax. (B) The ratio of Bcl-2/Bax protein expression. The above data represent three independent replicates and are expressed as the mean ± SD (*P\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001, ns meaningless).\u003c/p\u003e","description":"","filename":"LIQFigures5.png","url":"https://assets-eu.researchsquare.com/files/rs-7283996/v1/1da15aceb9377a96b5f6a61e.png"},{"id":91025218,"identity":"e653fa57-19ca-45ac-a8db-bef50acfe21b","added_by":"auto","created_at":"2025-09-10 19:54:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":367627,"visible":true,"origin":"","legend":"\u003cp\u003eLIQ reduces H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced inflammation in HUVECs. The IL-1β and TNF-α protein expression levels in each group were analyzed by western blot. (A) Grayscale value of the IL-1β. (B) Grayscale value of the TNF-α. The above data represent three independent replicates and are expressed as the mean ± SD (*P\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001, ns meaningless).\u003c/p\u003e","description":"","filename":"LIQFigures6.png","url":"https://assets-eu.researchsquare.com/files/rs-7283996/v1/79bf576e8877ba534f485d09.png"},{"id":91025220,"identity":"5bc4b2f3-224e-45ab-b08f-328bc3af2836","added_by":"auto","created_at":"2025-09-10 19:54:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":182838,"visible":true,"origin":"","legend":"\u003cp\u003ePKM2 changes in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treated HUVECs.\u0026nbsp; (A-B) Grayscale value of the PKM2 and p-PKM2 in each group. Every experiment was performed more than three times and are presented in the form of mean ± SD (*P\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001, ns meaningless).\u003c/p\u003e","description":"","filename":"LIQFigures7.png","url":"https://assets-eu.researchsquare.com/files/rs-7283996/v1/c3a975fb99a067f61fc0ac73.png"},{"id":91025228,"identity":"4630dbff-1884-4673-8713-cf86c356333b","added_by":"auto","created_at":"2025-09-10 19:54:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":118369,"visible":true,"origin":"","legend":"\u003cp\u003eLIQ promotes activation of STAT3 signaling pathway in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HUVECs. The expression of STAT3 and p-STAT3 was analyzed by Western blot and representative bands quantified in the corresponding bar graph. β-actin protein expression was used for normalization. (A-B) Grayscale value of the STAT3 and p-STAT3 in each group. The above data represent three independent replicates and are expressed as the mean ± SD (*P\u0026lt;0.05, **\u0026lt;0.01, ***\u0026lt;0.001, ns meaningless).\u003c/p\u003e","description":"","filename":"LIQFigures8.png","url":"https://assets-eu.researchsquare.com/files/rs-7283996/v1/93b2d7a1daf80a166ba73e16.png"},{"id":97142512,"identity":"8efe24e7-b380-4747-9816-4caf3f08278d","added_by":"auto","created_at":"2025-12-01 10:07:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10014900,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7283996/v1/d572a6e6-616a-4155-844d-06ef11803221.pdf"},{"id":91025214,"identity":"7a6476e6-185a-47f2-b8fb-11fa381da2fa","added_by":"auto","created_at":"2025-09-10 19:54:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2020261,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalData9.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7283996/v1/e4bf803be0333faea59cc5a5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"PKM2 regulated by liquiritin improves ischemia and reperfusion induced endothelial injury and promotes angiogenesis through STAT3 pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eRevascularization is a common method to save most patients with myocardial infarction, but it can lead to ischemia/reperfusion (I/R) injury, cause irreversible myocardial necrosis, and lead to pathological cardiac remodeling and heart failure, and eventually death. The excessive production of reactive oxygen species (ROS) during reperfusion disrupts the body's antioxidant defense system, leading to oxidative stress, dysfunction of endothelial cells (ECs), and damage to vascular wall cells\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Vascular endothelial dysfunction is the initial stage of vascular injury and also the first critical step in the progression of myocardial I/R injury\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e, which can cause an increase in infarct size, thereby increasing the perioperative mortality rate of patients\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Therefore, improving vascular function and preventing endothelial injury may be an effective means of preventing and treating myocardial I/R injury.\u003c/p\u003e\u003cp\u003ePyruvate Kinase M2 (PKM2), a critical isozyme of pyruvate kinase (PK), maintains glycolytic functions under normoxic conditions while exhibiting hypoxia-enhanced catalytic activity. Under oxygen deprivation, PKM2 dynamically modulates glucose flux through both glycolytic and alternative hexose metabolic pathways, coordinating ATP production with biosynthetic precursor synthesis. Research has shown that PKM2 expression is low in adult cardiomyocytes, but the expression of PKM subtypes in ischemic myocardium shows significant changes, with a significant increase in PKM2 expression after myocardial ischemic injury\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. PKM2 has been shown to play an effective role in cardiovascular function and disease, which can regulate the postnatal cardiomyocyte cycle and protect cell survival from ischemic damage\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Cardiomyocytes lacking PKM2 are manifested as reduced cardiac energy, stress response, angiogenesis and survival pathways\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. In addition, ECs mainly express PKM2, which is essential for the growth of ECs and the maintenance of vascular integrity\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. PKM2 knockout mice showed that the proliferation, migration and tubular ability of ECs were severely inhibited, and the innate immune signal of ECs was triggered\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSignal transducers and activators of transcription3 (STAT3), is expressed in cardiomyocytes, ECs and other cardiac cell types, and can be activated by ischemia, hypoxia and ROS, which plays a key role in cardiovascular disease\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. In addition, STAT3 produced by ECs can improve cardiac function after I/R injury by maintaining vascular integrity and inhibiting cardiomyocytes apoptosis\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Research has shown that PKM2 is related to the expression of STAT3, PKM2 overexpress can activate STAT3, while PKM2 knockout inhibits STAT3 expression\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. As the upstream kinase of STAT3, PKM2 can promote angiogenesis and nerve regeneration through phosphorylation of STAT3, thereby restoring brain function in ischemic stroke mice, inhibiting apoptosis and regulating post-ischemic inflammation of peripheral neutrophils\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eLicorice also known as \"Glycyrrhizae Radix et Rhizoma\" is the dried root or rhizome of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., and Glycyrrhiza glabra L. according to the Pharmacopoeia of the People's Republic of China, which is widely used as a traditional Chinese medicine\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Liquiritin (LIQ) is a flavonoid compound (C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e, molecular weight\u0026thinsp;=\u0026thinsp;418.39, as shown in the figure). It is an important monomeric active component in licorice, which has a variety of pharmacological effects such as antidepressant, anti-tumor, protection of the nervous and heart system, antioxidant and anti-inflammatory\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that LIQ exerts a neuroprotective effect on cerebral I/R-induced injury in mice through antioxidant and anti-apoptotic mechanisms\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, and can reduce the expression of inflammatory factors in lipopolysaccharide (LPS) -induced mice cardiomyocytes, inhibits cardiac oxidative stress and apoptosis\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. LIQ can also reduce Adriamycin-induced cardiotoxicity\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that PKM2 can be regulated by various traditional Chinese medicines\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. Given that PKM2 not only regulates energy metabolism but also directly modulates oxidative stress and inflammatory pathways, we hypothesize that LIQ may protect against I/R injury by targeting PKM2 to activate the STAT3 signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn this study, we investigated the role of LIQ with a focus on the function of PKM2. We found that the expression of PKM2 and PKM2 phosphorylation increased, and the ratio of p-PKM2 to PKM2 increased after LIQ pretreatment. LIQ pretreatment prevented endothelial cell injury, promoted angiogenesis, and mitigated I/R injury in a PKM2-dependent manner, and the activation of the STAT3 signaling pathway played a crucial role in this process.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003eLIQ (C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e9\u003c/sub\u003e, PubChem CID: 503737, purity\u0026thinsp;\u0026gt;\u0026thinsp;98%) was purchased from Shanghai Yuanye Bio Technology. Shikonin (C\u003csub\u003e16\u003c/sub\u003eH\u003csub\u003e16\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e, PubChem CID: 479503) as an effective PKM2 activation inhibitor\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e was purchased from Shanghai Yuanye Bio Technology, STAT3 activation inhibitor Stattic and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were both purchased from Sigma.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Animal I/R model and study protocols\u003c/h2\u003e\u003cp\u003eC57BL/6 mice aged 8\u0026ndash;10 weeks were purchased from Skobes Biotechnology Co., Ltd. (Henan, China). The animal protocol used in this experiment was approved by the Animal Care and Use Committee of Wuhan University People's Hospital and complies with the Guidelines for the Care and Use of Experimental Animals issued by the National Institutes of Health in the United States and ARRIVE guidelines of National Centre for the Replacement, Refinement \u0026amp; Reduction of Animals in Research Randomly divide the mice into 5 groups: (1) Sham group, silk was placed underneath the LAD without ligated; (2) I/R group, LAD was ligated for 30 min and received 2 hours of reperfusion; (3) I/R\u0026thinsp;+\u0026thinsp;LIQ group, LIQ(20mg/kg);༈4༉ I/R\u0026thinsp;+\u0026thinsp;LIQ\u0026thinsp;+\u0026thinsp;Shikonin group, LIQ༈20mg/kg༉and Shikonin༈40mg/kg༉;(5) I/R\u0026thinsp;+\u0026thinsp;LIQ\u0026thinsp;+\u0026thinsp;Stattic group, LIQ༈20mg/kg༉and Stattic༈15mg/kg༉. The drug was administered intraperitoneally daily for 3 days before surgery, and each mouse was given the same total dose daily by adjusting the dose of saline.\u003c/p\u003e\u003cp\u003eMice were anesthetized with 80mg/kg 3% pentobarbital sodium, fixed in supine position, and breathing was controlled by a small ventilator. Perform left thoracotomy between the 3rd and 4th ribs of the left chest, the anterior descending coronary artery (LAD) was ligation for 30 min, and then the ligation line was cut and blood perfusion was restored for 2 h. In the blank group, the left anterior descending coronary artery was separated and only threaded without ligature, and the ligature was pulled out 30 minutes later.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Echocardiogram\u003c/h2\u003e\u003cp\u003eMice were lightly anesthetized with 2% isoflurane and transthoracic echocardiography was performed with MyLab30CV ultrasound (Biosound Esaote Inc Indianapolis) to assess cardiac function on day 7 post-reperfusion in mice.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Immunohistochemical detection of myocardial neovascularization density\u003c/h2\u003e\u003cp\u003eAfter function measurements were recorded, mice were euthanized with 3% sodium pentobarbital and the hearts were removed immediately, the excised hearts were placed in 10% KCl solution to induce cardiac arrest during diastole.To obtain cardiac pathological samples, the atriums above the ligation of the heart were removed, the remaining tissues were fixed immediately after dissection in 4% paraformaldehyde for 30\u0026ndash;60 min. Tissues were embedded in OCT compound (Sigma), then quickly frozen in liquid nitrogen and stored at -80\u0026deg;C. Cryostat sections were cut into 5 \u0026micro;m thin sections. Perform endothelial cell specific marker CD31 staining on each group of specimens, and staining the cytoplasm of endothelial cells with a brown color indicates positive expression. Five visual fields were randomly selected to calculate the number of CD31 positive microvessels, and Image J was used to detect mean optical density (MOD) of positive cells, which was used as a quantitative index to evaluate neovascularization density.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cell Culture\u003c/h2\u003e\u003cp\u003eHUVECs (from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were cultured by DMEM, which contained 10% FBS and 1% penicillin/streptomycin. According to pre experiments, 400\u0026micro;mol/L H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was used as the concentration of subsequent experiments. HUVECs were separated into five groups: (a) Control group: Cells cultured in normal medium without any treatment; (b) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group: Cells induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (400\u0026micro;mol/L) for 12h; (c) LIQ group (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;LIQ): Cells were cultured by LIQ (1\u0026micro;mol/L) for 24h before H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment; (d) Shikonin group (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;LIQ\u0026thinsp;+\u0026thinsp;Shikonin): Cells were co-cultured by LIQ and Shikonin (1\u0026micro;mol/L) for 24h before H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment; (e) Stattic group (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;+\u0026thinsp;LIQ\u0026thinsp;+\u0026thinsp;Stattic): Cells were co-cultured by LIQ and Stattic (2\u0026micro;mol/L) for 24h before H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Cell wound healing assay\u003c/h2\u003e\u003cp\u003eA wound-healing assay was performed as described previously\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. HUVECs were seeded on 6-well plates (5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well), after overnight, cells were treated in groups. A straight line was drawn along the bottom of the plate with a 200 \u0026micro;L pipette and the detached cells were washed with PBS, then replaced with serum-free basic medium, and observe the healing of scratches and take photos (at 0, 12, 24, 36 and 48 h). Measure the scratch area using Image J, and the scratch healing rate is (scratch area of 0h\u0026thinsp;~\u0026thinsp;scratch area of 24h)/0h scratch area \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Tube formation in HUVEC\u003c/h2\u003e\u003cp\u003eTube formation assay was performed as described before\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Chilled liquid Matrigel (356234, Corning) was dispensed into 96-well plates (50 \u0026micro;l/well) and incubated at 37℃ for 1 h for solidification. Then inoculate each group of HUVECs onto a Matrigel bed (2\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) and incubate for 6 hours. Use ImageJ software to image the formation of the tube and quantify the length of the tube.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Measurement of ROS in HUVEC\u003c/h2\u003e\u003cp\u003eThe intracellular ROS production was measured by the ROS Assay Kit (Beyotime Biotechnology, China). In brief, cells were collected and resuspended in DMEM medium after treatment, and then measured based on the fluorescence intensity of 2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescin-diacetate (DCFH-DA). The accumulation of ROS was visualized using a fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.9 Western blot analysis\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe cells were harvested and lysed in RIPA buffer after being treated in groups, and the concentration was determined with the BCA kit (Beyotime, Beijing, China). After protein samples were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes, the membranes were blocked into 5% of skimmed milk for 30min and then incubated overnight at 4℃ with antibodies against PKM2(1:2000 dilution), p-PKM2 (1:2000 dilution), STAT3 (1:2000 dilution), p-STAT3 (1:10000 dilution), VEGF (1:2000 dilution), FGF2 (1:2000 dilution), IL-1β(1:2000 dilution), Bax (1:20000 dilution), Bcl-2 (1:4000 dilution), Nrf2 (1:1000 dilution), and HO-1 (1:20000 dilution), TNF-α (1:10000 dilution), β-actin (1:20000 dilution). In addition, rabbit anti-PKM2(4053), STAT3 (9139), and p-STAT3 (Tyr705, 9145) were procured from Cell Signaling Technology (CST, Danvers, MA, USA) and rabbit anti-p- PKM2 (Ser37) (ab205678) was obtained from Abcam (Cambridge, UK). After the membranes washing, the secondary antibodies were incubated at room temperature for 60min. The Image J software was used to quantitatively analyze the intensity of each protein band.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e2.10 Statistical analysis\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eGraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA) is used for statistical analysis. All the results were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Unpaired t-test was used to compare two independent samples, and one-way analysis of variance (ANOVA) was used to compare multiple groups. Values of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 LIQ improved cardiac function and enhanced angiogenesis after I/R in a PKM2-dependent manner\u003c/h2\u003e\u003cp\u003eTo evaluate cardiac function, we performed echocardiography 7 days after surgery. The results showed that left ventricular function was impaired in mice after I/R injury, and LVEF and FS were significantly reduced (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while LIQ pretreatment significantly reversed this result and improved cardiac function (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). CD31 is an important marker of angiogenesis that is highly expressed on the surface of ECs. The vascular density in the surrounding area of left ventricular infarction was analyzed by CD31 staining to determine endothelial cell proliferation or angiogenesis. Our results showed an increase in CD31 expression in the I/R group, while LIQ pretreatment further increased the expression of CD31(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), which indicated some improvement in angiogenesis and microcirculation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-C). It is worth noting that after inhibiting PKM2, the beneficial effect of LIQ on the heart was significantly weakened (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 LIQ promoted H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HUVECs migration and tube formation in a PKM2-dependent manner\u003c/h2\u003e\u003cp\u003eTo evaluate the effect of LIQ on the migration ability of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HUVECs injury and the role of PKM2, wound healing assay was examined for healing rate. Compared with the control group, the migration capacity of HUVECs reduced after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, while LIQ could significantly reverse this phenomenon, but this phenomenon was significantly weakened after using Shikonin to inhibit PKM2. In addition, compared to the LIQ group, the migration area of HUVECs significantly decreased after the treatment of Stattic (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMoreover, to assess the effect of LIQ on tube formation of the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced injury on HUVECs, Matrigel assay was used to simulate the angiogenesis experiment. In the control group robust and well-formed capillary-like tubes could be detected. However, the tube formation was significantly inhibited after treatment of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while compared with the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group, LIQ treatment obviously encouraged tube formation and the tube length was increased, and this protective effect was obviously abolished by cotreatment with Shikonin (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Additionally, the use of Stattic significantly inhibited tube formation compared to the LIQ group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 LIQ enhanced the production of pro-angiogenic molecules in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HUVECs in a PKM2-dependent manner\u003c/h2\u003e\u003cp\u003eWestern blot was used to detect the protein expression of pro-angiogenic molecules VEGF and FGF2 in each group to investigate the effect of LIQ on angiogenesis after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HUVECs injury. The results showed that the decreased VEGF and FGF2 in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group could be reversed through LIQ treatment, however, this beneficial effect was significantly eliminated by PKM2 inhibitor Shikonin (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Stattic prominently inhibited the increase of VEGF and FGF2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These results suggest that PKM2 is required by LIQ to promote angiogenesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 LIQ inhibited H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced oxidative stress in HUVECs by up-regulating PKM2\u003c/h2\u003e\u003cp\u003eDCFH-DA was used to evaluate the intracellular ROS levels in each group, and ROS production was quantified by measuring cell fluorescence intensity to detect the levels of oxidative stress in each group. Compared with the control group, the red fluorescence of HUVECs induced by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was significantly enhanced, which indicated that the ROS generation increased after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In addition, LIQ pretreatment significantly reduced fluorescence intensity, but inhibition of PKM2 prominently reversed this change (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, we observed the expression levels of antioxidant proteins Nrf2 and HO-1 in each group. After H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment the Nrf2 and HO-1 protein expression levels had significantly increased compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, LIQ pretreatment remarkably enhanced the Nrf2 and HO-1 expression levels compared to those in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while Shikonin can inhibit this effect. In addition, Stattic significantly inhibited the anti-oxidative stress effect of LIQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-E).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5 LIQ prevented H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced apoptosis of HUVECs by up-regulating PKM2\u003c/h2\u003e\u003cp\u003eTo evaluate the apoptosis of HUVECs, Western blot was used to analyze the protein expression of BCL-2 and Bax. The results showed that the decline in the Bcl-2/Bax ratio in the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e group could be reversed by LIQ treatment, and this beneficial effect was significantly mitigated after PKM2 inhibition (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Moreover, Stattic prominently inhibited the increase of the Bcl-2/Bax ratio (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6 LIQ reduced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced inflammation of HUVECs induced by up-regulating PKM2\u003c/h2\u003e\u003cp\u003eIn order to detect cellular inflammation, Western blot was used to detect the levels of pro-inflammatory cytokines TNF-α and IL-1β. IL-1β and TNF-α expression was significantly upregulated in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced HUVECs compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). While LIQ obviously mitigated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced increases in TNF-α and IL-1β (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), however, these effects were significantly reversed after treatment with Shikonin or Stattic (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.7 LIQ up-regulated the ratio of p-PKM2 to PKM2 and activated STAT3 signaling pathway\u003c/h2\u003e\u003cp\u003eWestern blot analysis of the expression of PKM2 protein in each group showed that LIQ pretreatment significantly increased the expression level of PKM2 protein, the level of phosphorylated PKM2, and the ratio of p-PKM2 to PKM2 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). PKM2 inhibitor Shikonin significantly reversed this result (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)(Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSince PKM2 is known as a STAT3 transcriptional coactivator, we also evaluated the role of STAT3 in LIQ-induced PKM2 protein accumulation in HUVECs. The results showed that LIQ pretreatment significantly increased STAT3 phosphorylation and the ratio of p-STAT3 to STAT3, but inhibition of PKM2 by shikonin significantly reduced LIQ-induced p-STAT3 activation and the ratio of p-STAT3 to STAT3 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). In addition, there was no significant difference in the expression of p-PKM2 compared with the LIQ group after Stattic inhibited STAT3, but the beneficial effect of LIQ remarkably decreased. These data indicate that STAT3 is the key downstream mediator of PKM2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eECs are the most abundant cell types in the cardiac\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e, the blood vessels constituted by them cover the cardiac and lymphatic, which participate in the pathophysiological process of the cardiac. I/R can cause endothelial damage through various mechanisms, so reducing endothelial damage and promoting angiogenesis may be an effective treatment strategy for myocardial I/R injury\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. PK isoenzyme PKM2 is highly expressed in ECs and plays an important role in endothelial growth and vascular integrity. Studies have shown that PKM2 loss alters endothelial metabolism and inhibits endothelial growth and angiogenesis\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. LIQ, as an important monomer active component in licorice, has anti-inflammatory, anti-oxidant, neuroprotective and other pharmacological effects. This study first confirmed the regulatory effects of LIQ on PKM2 by establishing in vivo I/R injury models and in vitro H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e induced HUVECs injury models. LIQ can upregulate PKM2 expression, promote PKM2 phosphorylation, increase the ratio of p-PKM2 to PKM2 and activate the STAT3 signaling pathway to rescue I/R-induced ECs injury.\u003c/p\u003e\u003cp\u003eThis study used echocardiography to evaluate whether LIQ pretreatment can protect cardiac function in vivo after I/R injury. The results showed that the LVEF and FS values of LIQ pretreated mice were significantly increased, indicating that LIQ pre-treatment significantly improved left ventricular function in mice after I/R injury. After inhibiting PKM2, this protective effect of LIQ is partially eliminated. Angiogenesis is a crucial step in tissue repair and regeneration after ischemia. The reconstruction of blood flow in the infarcted area can save the infarcted myocardium by inhibiting apoptosis, removing scar tissue and recruiting progenitor cells\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. CD31 is a specific marker of endothelial cells, which can reflect angiogenesis. Our results indicate that LIQ pretreatment significantly increased the CD31 in the infarct boundary zone of mice after I/R, indicating that LIQ can significantly promote angiogenesis in the ischemic zone to improve impaired cardiac function. However, this beneficial effect was significantly weakened after inhibition of PKM2 by Shikonin. Study has demonstrated that Shikonin, a naphthoquinone isolated from the traditional Chinese medicine Lithospermum, inhibits the function of PKM2 by inhibiting the pyruvate kinase activity and nuclear translocation of PKM2, and decreasing both dimerization and tetramerization of PKM2 in macrophages\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Angiogenesis is achieved through the proliferation, migration, and tube formation of ECs. Our results indicated that LIQ pretreatment significantly enhanced the transfer and tubular ability of endothelial cells after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, and inhibition of PKM2 significantly reversed this phenomenon. VEGF and FGF2 are the main regulators that stimulate the migration and proliferation of ECs to form new blood vessels, and are key molecular targets that promote angiogenesis in ischemic diseases such as myocardial infarction and myocardial ischemia\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. In this study, we found that VEGF and FGF2 significantly decreased after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, while LIQ pretreatment reversed this result. The beneficial effect of LIQ is obviously reduced after inhibiting PKM2. These data suggest that LIQ may promote angiogenesis after I/R by up regulating PKM2, promoting PKM2 phosphorylation and increasing the ratio of p-PKM2 to PKM2.\u003c/p\u003e\u003cp\u003eOxidative stress can mediate the process of I/R injury, causing endothelial dysfunction, leading to endothelial cell apoptosis, and subsequently altering the function and structure of blood vessels\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Low levels of ROS can maintain normal physiological metabolism, however, excessive ROS can lead to oxidative stress and oxidative damage, which damages cellular macromolecules including DNA, lipids and proteins\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. In this study, LIQ pretreatment demonstrate clearance of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Transcription factor Nrf2 plays a key role in clearing ROS, and the antioxidant gene HO-1 regulated by Nrf2 helps cells resist oxidative stress\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. This study found that after H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment the expression of Nrf2 and HO-1 was significantly increased, while LIQ pretreatment further increased the levels of Nrf2 and HO-1. These effects were significantly reversed after inhibition of PKM2, which indicated that LIQ protected HUVECs from oxidative stress in a PKM2 dependent manner.\u003c/p\u003e\u003cp\u003eThe apoptosis of ECs is an important factor causing endothelial dysfunction, therefore the apoptosis status of ECs in each group was evaluated. Research has shown that anti-apoptotic protein Bcl-2 and proapoptotic protein Bax play important roles in cell apoptosis\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Consequently, we detected the above indicators through Western blot, and the results showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment significantly reduced the Bcl-2/Bax ratio, decreased the expression of anti-apoptotic Bcl-2, and increased proapoptotic Bax. Although LIQ pretreatment significantly improved the ratio of Bcl-2/Bax, this increase evidently weakened evidently after PKM2 inhibition which further confirmed that LIQ plays an anti-apoptotic role partly in a PKM2-dependent manner.\u003c/p\u003e\u003cp\u003eStudies have confirmed that inflammation is regarded as an indicator of endothelial status, and after endothelial dysfunction, the pro-inflammatory cytokines increase, thereby promoting the inflammatory response of ECs\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. The inflammatory cytokines TNF-α and IL-1β play a major role in endothelial dysfunction, and the increased level of them is associated with more severe clinical conditions and worse outcomes\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Therefore, we detected the TNF- α and IL-1 β protein expression levels through Western blot, and our results showed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment significantly induced the accumulation of IL-1β and TNF-α, while LIQ remarkably reduced the levels of these inflammatory cytokines and exerts an anti-inflammatory effect.\u003c/p\u003e\u003cp\u003eThe STAT3 signaling pathway plays an important role in enhancing cell proliferation and other aspects\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. In ischemic brain injury, the activation of STAT3 signaling could promote tissue repair and functional recovery by enhancing cell migration, promoting angiogenesis and regulating cell inflammation\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. In the present study, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment increased the levels and phosphorylation of STAT3, which indicates that the STAT3 signaling pathway plays an important role in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced cell damage. Moreover, LIQ pretreatment significantly enhanced this result, and significantly increasesd the ratio of p-STAT3 to STAT3. while inhibiting PKM2 weakened the expression and phosphorylation of STAT3, and the ratio of p-STAT3 to STAT3. After inhibiting STAT3, the expression of p-PKM2 did not exhibit a decrease, but the protective effect of LIQ on I/R-induced endothelial injury and the promoting effect of angiogenesis were partially eliminated. Therefore, it is evident that STAT3 is the key downstream mediator of PKM2 and LIQ plays a protective role in I/R induced endothelial injury by upregulating PKM2, promoting PKM2 phosphorylation, increasing the ratio of p-PKM2 to PKM2, and activating the STAT3 signaling pathway.\u003c/p\u003e\u003cp\u003eIn conclusion, LIQ can alleviate endothelial dysfunction induced by I/R by promoting angiogenesis, inhibiting oxidative stress, apoptosis, and inflammation. The beneficial effects of LIQ are mainly mediated by upregulating PKM2, promoting PKM2 phosphorylation, increasing the ratio of p-PKM2 to PKM2, and activing of the STAT3 signaling pathway. LIQ preconditioning will be a potential strategy in clinical as the repair of injured heart.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003e The animal protocols in this experiment were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University. License NO. SCXK(鄂)2018\u0026thinsp;\u0026minus;\u0026thinsp;0265.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eAll the authors confirm that the work described has not been published before, and all the authors agree to publication in the Journal.\u003c/p\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare that there is no competing interest.\u003c/p\u003e\u003ch2\u003eAuthor contributing\u003c/h2\u003e\u003cp\u003eBin Zeng contributed to the conception, revised this manuscript and provided the funding. Hanyang Sun, Xiaoting Liao and Yan Zhang carried out the experiments, performed data analysis and wrote the draft of the manuscript. Lei Liu and Xiang Zhang performed statistical analyses. All authors reviewed the results and approved the final version.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the Chinese National Nature Science Foundation [30900609, 81270271, 81570333 and 81670304] and the Fundamental research funds for the Central Universities [2042020kf1014].\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBin Zeng contributed to the conception, revised this manuscript and provided the funding. Hanyang Sun, Xiaoting Liao and Yan Zhang carried out the experiments, performed data analysis and wrote the draft of the manuscript.Xiang Zhang performed statistical analyses. 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Headache Pain\u003c/em\u003e. \u003cb\u003e19\u003c/b\u003e (1), 7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s10194-018-0836-4\u003c/span\u003e\u003cspan address=\"10.1186/s10194-018-0836-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Liquiritin, PKM2, STAT3 pathway, endothelial dysfunction, angiogenesis","lastPublishedDoi":"10.21203/rs.3.rs-7283996/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7283996/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlleviating ischemia/reperfusion (I/R) induced cardiac vascular endothelial dysfunction may effectively prevent myocardial I/R injury. Studies showed that PKM2 are associated with a reduction in cardiomyocyte apoptosis and ischemic insult. Liquiritin (LIQ) has attracted attention for its cardioprotective effects. However, whether LIQ can reduce endothelial injury induced by I/R has not been fully explored and the mechanism of PKM2 in this process remains to be elucidated. In our study, we identified that LIQ preconditioning significantly improved cardiac dysfunction and increased angiogenesis after I/R injury, but inhibition of PKM2 markedly deteriorated cardiac function and angiogenesis in vivo. In vitro, experiments have revealed that LIQ significantly promoted angiogenesis, inhibited oxidative stress, apoptosis and inflammation, and then effectively prevented H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced umbilical vein endothelial cell (HUVECs) damage. LIQ up-regulated PKM2 expression, promoted PKM2 phosphorylation, increased the ratio of p-PKM2 to PKM2, and significantly increased the expression of p-STAT3. However, suppression of PKM2 by shikonin significantly abolished these protective effects. Additionally, it is worth noting that a significant decline in the beneficial effect of LIQ was observed in models that inhibit STAT3. In conclusion, LIQ upregulates PKM2, promotes PKM2 phosphorylation, increases the ratio of p-PKM2 to PKM2, and activates STAT3 pathway to effectively alleviate ECs dysfunction and stimulate angiogenesis, which may be a potential treatment for myocardial I/R injury.\u003c/p\u003e","manuscriptTitle":"PKM2 regulated by liquiritin improves ischemia and reperfusion induced endothelial injury and promotes angiogenesis through STAT3 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-10 19:54:03","doi":"10.21203/rs.3.rs-7283996/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8210dfad-0222-4cd5-b99f-1fb5e7f290d1","owner":[],"postedDate":"September 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54417207,"name":"Health sciences/Cardiology"},{"id":54417208,"name":"Biological sciences/Cell biology"},{"id":54417209,"name":"Health sciences/Diseases"},{"id":54417210,"name":"Biological sciences/Molecular biology"},{"id":54417211,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2025-12-01T08:39:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-10 19:54:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7283996","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7283996","identity":"rs-7283996","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}