Hexokinase II

Author’s Accepted Manuscript Tetrandrine cardioprotection in ischemia– reperfusion (I/R) injury via JAK3/STAT3 /Hexokinase II Tie-jun Zhang, Rui-xia Guo, Xia Li, Yan-wei Wang, Yong-jun Li www.elsevier.com/locate/ejphar

PII: DOI: Reference:

S0014-2999(17)30536-8 http://dx.doi.org/10.1016/j.ejphar.2017.08.019 EJP71360

To appear in: European Journal of Pharmacology Received date: 19 February 2017 Revised date: 9 August 2017 Accepted date: 15 August 2017 Cite this article as: Tie-jun Zhang, Rui-xia Guo, Xia Li, Yan-wei Wang and Yong-jun Li, Tetrandrine cardioprotection in ischemia–reperfusion (I/R) injury via JAK3/STAT3 /Hexokinase II, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2017.08.019 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Tetrandrine cardioprotection in ischemia–reperfusion (I/R) injury via JAK3/STAT3 /Hexokinase II Tie-jun Zhang1, Rui-xia Guo2, Xia Li3, Yan-wei Wang3, Yong-jun Li1,4* 1

2

3

Graduate school, Hebei medical University, Shijiazhuang 050017, China

College of Chemical Engineering, Shijiazhuang College, Shijiazhuang 050035,China

Department of heart medicine, Heibei provincial hospital of traditional Chinese medicine, Shijiazhuang 050000, China

4

Department of cardiology, the second hospital of Hebei medical university, Shijiazhuang 050000, China

Abstract: Tetrandrine(TET), a bisbenzylisoquinoline alkaloid has been used for the treatment of cardiovascular diseases and hypertension. This study was to investigate whether tetrandrine exerts cardioprotection in ischemia–reperfusion (I/R) injury and the mechanisms involved. The cardioprotection effect and mechanisms of tetrandrine was evaluated by I/R injury cardiac cell model. Hexokinase II (HKII) is the critical regulators of mitochondrial dysfunction in cardiac I/R injury and it participate in the regulation of glycolysis and energy metabolism. The effect of tetrandrine on HKII and Janus kinase(JAK), (Protein kinase B)Akt as well as hypoxia inducible factor α (HIF-α) which are HKII’s regulator was also investigated. We found that tetrandrine significantly reduced lactate dehydrogenase, caspase 3 level and apoptosis in I/R injury cardiac cell , meanwhile restored mitochondrial energy metabolism and enhanced glycolysis in model cell. Tetrandrine up-regulated the expression of p-STAT3 and HKII, but has no effect on p-akt and HIF-α. The cardioprotection effect significantly attenuated after tetrandrine combined with JAK3 inhibitor. The expression of p-STAT3 and HK II were also significantly decreased simultaneously. On the contrary, combined with JAK1/2 inhibitor, there was no significant influence. In addition, Corresponding author: Yong-jun Li E-mail address: [email protected]

tetrandrine increased the JAK3 in model cells, but have no impact on the expression of JAK1, JAK2. Taken together, these data revealed that the cardioprotection effect of tetrandrine appears to be involved in the JAK3/STAT3 /HK II. Keywords:

Tetrandrine; Ischemia–reperfusion (I/R) injury; glycolysis ; JAK3/STAT3 /Hexokinase II;

Tetrandrine

Pubmed CID: 73078; Ruxolitinib Pubmed CID: 25126798; Tofacitinib Pubmed CID:

9926791 1. Introduction Ischemia and reperfusion (IR) injury is a primary cause of cardiac failure, morbidity, and mortality after cardiac operations or heart infarctions. In ischemia and reperfusion injury, the short, non-lethal periods of ischemia could activate an endogenous cardiac protection program to against long, lethal, periods of ischemia. This phenomenon was called ischemia preconditioning (Murry et al., 1986). Subsequent research demonstrated that ischemia preconditioning cellular signaling converged on the mitochondrion and was associated with alterations in glycolysis (Murphy et al., 2007). The process of glycolysis starts with glucose uptake by glucose transporters (GLUTs), and phosphorylation by hexokinases II (HKII) (Mueckler., 1994; Printz et al., 1997). HKII mitochondrial translocation increases in response to ischemia, this phenomenon also observed with cardioprotective treatments such as ischemia preconditioning (Abel et al., 1999; Liao et al., 2002; Middleton., 1990). Recently work demonstrated that mitochondrially bound HKII is a major important element of ischaemic preconditioning. HKII which activate by Akt, signal transducer and the activator of the transcription3 (STAT3) and hypoxia inducible factor (HIF)-1α could regulated glycolysis and then affects mitochondrial function, reactive oxygen species (ROS) production and mitochondrial energy metabolism (Hausenloy et al., 2005; Heiss et al., 2016). 2

Tetrandrine [(1b)-6, 6’, 7, 12-tetramethoxy-2, 2’-dimethyl-berbaman], which belongs to the bisbenzylisoquinoline alkaloid family and was isolated from the root of Stephania tetrandra S Moore, possesses multiple pharmacologically relevant classes (Bellik et al., 2013). As a clinical drug in China, tetrandrine has been used for decades to treat patients with cardiovascular diseases and hypertension (Huang et al., 2011; Shen et al., 2010). Tetrandrine plays a significant role in regulating blood pressure, but there are few studies on myocardial ischemia reperfusion. Tetrandrine could inhibit neutrophil adhesion, and reactive oxygen species (ROS) in myocardial ischaemia-reperfusion (I/R) injury (Shen et al., 1999). However, the cardioprotection mechanism of tetrandrine in myocardial ischemia reperfusion

injury still need further investigate. In the present experiment, cultured H9c2 cells were used to investigate the influence of tetrandrine on hypoxia cardiomyocytes, and to explore the mechanism of tetrandrine on mitochondrial and glycolysis, providing the experimental reliance for its clinical application of preventing and treating myocardial ischemia reperfusion injury. 2. Materials and methods 2.1 Reagents The rat heart-derived H9c2 cell line was provided by the Cell Culture Center, Institute of Basic Medical Science, and Chinese Academy of Medical Science. Tetrandrine (Sigma Aldrich, St. Louis, MO, USA).

MTT(3-(4,5-dimethylthia2-yl)-2,5-di-phenyltetrazolium),

Ruxolitinib

(Jak1/2

inhibitor),

Tofacitinib (Jak3 inhibitor) , and the Caspase 3 Activity Assay Kit were from Sigma Chemical Co. Hexokinase II, p-STAT3(phosphor-Y750), p-Akt (phosphor-473),

Akt, HIF-α, JAK1, JAK2, JAK3

GAPDH (Abcam, UK) 2.2 To establish hypoxia–recover (H/R) injury cell model induced by hypoxia and recovered with 3

oxygen in H9c2 cells. H9c2 myocardial cells were inoculated at the concentration of 2×106/ml. Twenty four hours before treatment, To simulate hypoxia, the culture medium was replaced by hypoxia buffer (139mM NaCl, 4.7mM KCl, 0.5mM MgCl2, 1.0mM CaCl2, 5mM Hepes, 20mM sodiumlactate), and then incubated for 12h in a Low oxygen gas (95 % N2, 5 % CO2). Then, the condition was recovered with oxygen, the cells were incubated in serum-free DMEM under normal conditions (20% O2, 5% CO2) at 37℃ for 4h and different drugs were applied simultaneously. 2.3 Methyl thiazolyl tetrazolium (MTT) assay Culture media was refreshed with media containing MTT reagent (5mg/ml) and cells were incubated under standard conditions for an additional 4 h. The culture media was carefully aspirated and 100 μl dimethylsulfoxide was added per well to solubilize the formazan crystals. Following agitation, absorbance was measured spectrophotometrically at a wavelength of 490 nm using a Tecan infinite M200pro Microplate Spectrophotometer (Tecan Laboratories, Inc., Switzerland). Viabilities of the challenged cells were expressed relative to control cells. 2.4 Measurement the LDH levels The H9c2 cells were cultured at 4×105 cells/ml in 6-well plates. The supernatant was collected after the different treatments and the LDH level was measured by the corresponding detection kit in accordance with the manufacturer's instructions (Sun et al., 2012). 2.5 Hoechst 33258 staining Hoechst 33258 nucleic acid staining was used to observed nuclear morphological changes. Briefly, cells were seeded in 24-well microplates and grouped as control, tetrandrine (1, 5, 25mM), TR group (Tetrandrine 5mM+ Ruxolitinib), TT group (Tetrandrine 5mM+Tofacitinib) for 24h. Following treatment, 4

the cells were washed with PBS and fixed with 4% paraformaldehyde for 10min at 37℃, then stained in dark with Hoechst 33258 for 20min at 37℃, washed with PBS for three times and photographed using florescence microscope (Nikon Eclipse Ni; Nikon Corporation, Japan). 2.6 Detection of glucose consumption and lactate production Glucose-uptake experiments were conducted with each group according to the manufacturer’s instructions (Biovision, CA, USA). To measure lactate production, H9c2 cells from different treatment groups were collected and measured for lactate concentration using a lactate ELISA kit (Biovision) according to the manufacturer’s instructions with Infinite M200 Pro type absorbance reader (Tecan Group, Ltd., Switzerland). For detecting the uptake of glucose and the production of lactate, the culture supernatants of H9c2 cells with different treatments were collected and the fresh culture media was used as control. Equal amounts (2–10 μl) of tested samples were added to a 96-well plate and the volumes were then adjusted to 50 μl/well with Glucose or Lactate Assay Buffer respectively. Meantime, the standard curve was prepared with the same protocol. The reactions were incubated in dark for 30 min at 37 °C. Finally, the absorbance (OD 570 nm) or Fluorescence (Ex/Em = 535/590 nm) were measured by microplate reader. The uptake of Glucose was determined by subtracting the concentration of glucose in tested samples from the initial concentration of glucose in fresh culture media. The production of lactate was determined referring standard curve. Considering the cell number of every sample may be different, all the concentration of glucose or productions of lactate were finally normalized to the protein concentration (Jin et al., 2017). 2.7 Measurement of intracellular NAD+/NADH NAD+ and NADH levels were measured using a commercially available fluorimetric assay kit (BioVision, Inc, USA). NAD+ and NADH were specifically recognized and detected using an enzyme 5

cycling reaction and according to the manufacturer’s instructions. In brief, NADH and NAD+ were extracted from H9c2 cells separately and measured at 565 nm after an enzymecatalyzed kinetic reaction, in which the intensity of the product color is proportionate to the NAD+/NADH concentrations in the samples(Jun

et al., 2011).

2.8 ATP assay The level of ATP in H9c2 cell lines was determined using the ATP assay Kit (Abcam, Cambridge, UK) according to the manufacturer’s protocol. Briefly, harvested cultured cells were lysed with a lysis buffer, followed by centrifugation at 10,000 × g for 2 min at 4°C. The level of ATP was determined by mixing 50 μl of the supernatant with 50 μl of luciferase reagent, which catalyzed the light production from ATP and luciferin. The emitted light was linearly related to the ATP concentration and measured using a microplate illuminometer. 2.9 Preparation mitochondrial fractions

Preparation of mitochondrial fractions was achieved using a commercially available mitochondrial fractionation kit in accordance with the manufacturer's protocol (C3601, BiYunTian, Institute of Biotech, Shanghai, China). Briefly, 4 × 107 cells were washed thrice with ice-chilled PBS at 600×g. Cell pellets were resuspended in 2 ml of extraction buffer and incubated at 4°C for 20 min, followed by homogenization. The homogenate was centrifuged at 600×g for 10 min at 4°C. The supernatant was additionally centrifuged at 11,000×g for 10 min (fraction enriched with intact mitochondria). The final pellet was a more purified mitochondrial fraction. 2.10 Western blot The expression quantity of various proteins was determined in H9c2 cells using western blotting 6

according to standard procedures. The antibodies against Hexokinase II, HIF-α, p-Akt, Akt，p-STAT3, STAT3, Jak1, Jak2, Jak3 and GAPDH were purchased from purchased from Abcam. In short, total protein from untreated or treated cells was extracted in RIPA lysis buffer. The same amount of protein (25 μg) from each group were separated with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, USA). Each membrane was incubated with a specific primary antibody (1:1000) at 4°C overnight after blocking with 5% skim milk at room temperature for 1 h. After three washes with washing buffer (20 mmol/L Tris-HCl, 500 mmol/L NaCl, and 0.1% Tween 20), each suitable secondary antibody was incubated in the membrane at room temperature for 2 h. An ECL Advanced Western Blot Detection Kit (Thermo Fisher, Waltham, USA) was used to visualize the specific protein bands. 2.11 Statistical analysis Data are expressed as the mean ± standard deviation (SD) of triplicate samples. Statistical analysis for multiple comparisons was analyzed by a one-way analysis of variance (ANOVA). P values below 0.05 were considered to be statistically significant. 3. Results 3.1 Tetrandrine attenuated H/R-induced H9c2 cell injury In order to investigate the effects of tetrandrine against cell cytotoxicity caused by H/R, the viability of H9c2 cell and LDH leakage were detected. The results showed that the cell viability was distinctly reduced and the LDH leakage was enhanced after H/R exposure, but reversed by pretreatment with tetrandrine at concentrations of 1mM, 5mM, 25mM, in a concentration-dependent manner (Fig. 1a, b). Hoechst 33258 staining was used in evaluation for apoptosis. The result showed that tetrandrine significant attenuated apoptosis in H/R H9c2 cells as shown in Figure 1d. The numbers of apoptotic cells 7

decreased in a dosage-dependent manner. In addition, Caspase-3 activity in H/R H9c2 cells also decreased after pretreatment with tetrandrine (Fig. 1c). These data revealed that tetrandrine could attenuate H/R-induced cell viability loss and cell apoptosis in H9c2 cell.

(a)

(b)

8

(c)

(d) Fig 1 (a) Effect of tetrandrine (TET) on the viability of H9c2 cells, TET at a final concentration of 1, 5 or 25mM to the H/R model; LDH activity was showed in (b) Caspase 3 activity was showed in (c). Hoechst 33258 staining of cell apoptotic (Ⅰ-Ⅴ) in control (Ⅰ), H/R modal (Ⅱ), Tetrandrine(TET) (1, 5, 25mM,ⅢⅤ) were showed in (d) Results shown are mean±SD of three independent experiments performed in duplicate. **p ≤ 0.01 relative to control , *p ≤ 0.05 relative to H/R group

3.2 Tetrandrine positively regulates the glycolysis and energy metabolism in H9c2 cells Glucose consumption and lactate production were the main index to evaluate the cellular glycolysis function. In this experiment, glucose consumption and lactate production were detected. As the result 9

showed in Fig 2a, glucose consumption and lactate production were significantly decreased in H/R H9c2 cells. Pretreatment with tetrandrine significantly increased the consumption of glucose and the production of lactate compared to H/R group. Energy metabolism of H9c2 cells were evaluated by ATP content and NAD+/NADH ratio. As the Fig 2b showed, ATP content decreased in H/R H9c2 cells, but rose when pretreatment with tetrandrine in a concentration-dependent manner. Moreover, the production of key metabolic products- NAD+/NADH significantly increased in H/R group, and decreased when pretreatment with tetrandrine.

(a)

(b) Fig. 2 Tetrandrine (TET) regulates the glycolysis and energy metabolism in H9c2 cells. (a) Glucose consumption 10

and Lactate production in H9c2 cells was evaluated (b), ATP content and NAD+/NADH were also evaluated. Data are presented as means ± SEM, n = 3. **p ≤ 0.01 relative to control , *p ≤ 0.05 relative to H/R group,

3.3 Tetrandrine promote the expression of HKⅡin mitochondrial, and up-regulated the expression of p-STAT3, but has no influence on p-AKt and HIF-α Western blot analysis indicated that the expression of HKⅡin mitochondrial was significantly increased in tetrandrine pretreatment group, these effects were concentration-dependently by tetrandrine (Fig. 3). In addition, the expression of p-STAT3 in H/R H9c2 cell also increased when pretreatment with tetrandrine, but has no impact on the expression of p-AKt and HIF-α. Taken together, these data revealed that tetrandrine could increase the p-STAT3 lever in H/R injury cell and elevated the expression of HKⅡ in mitochondrial.

Fig. 3 The protein levels of p-STAT3,HexokinaseⅡ, p-Akt，HIF-α in H/R H9c2 cells pretreatment with Tetrandrine (TET) were determined by Western blotting.

3.4 Tetrandrine attenuated the H/R-induced H9c2 cell apoptosis, combined with Jak1/2 inhibitor, the protective effect still exist, but attenuated with Jak3 inhibitor. MTT and LDH assays were designed to assess the viability of H9c2 cell which pretreatment with 11

JAK inhibitor and tetrandrine. The results showed that the cell viability was distinctly reduced and the LDH leakage was enhanced after tetrandrine combined with tofacitinib, the myocardial cells protective effect of tetrandrine was disappeared. The difference is that the cardioprotection effect of tetrandrine was unaffected when tetrandrine combined with ruxolitinib. The cell viability and LDH leakage still significantly different between H/R group. (Fig. 4a, b) With Hoechst 33258 staining, cell apoptosis was analyzed (Fig 4d). The apoptotic cell was significantly increased in model cells, but decreased in tetrandrine-pretreatment group. The influence of apoptosis by tetrandrine was not significantly changed when combine with ruxolitinib. But the apoptotic cell was significantly increased when tetrandrine combined with tofacitinib compare with single tetrandrine group. In addition, tetrandrine could decreased the elevated caspase-3 activity in H/R-treated H9c2 cells (Fig. 4c), combined with ruxolitinib, the effect still exist, but attenuated with tofacitinib.

(a)

12

(b)

(c)

13

(d) Fig 4 (a) Effect of tetrandrine (TET) and JAK inhibitor on the viability of H9c2 cells. Experimental grouping: Tetrandrine (TET 5 mM); TT (TET 5 mM+ Tofacitinib); TR(TET 5 mM+ Ruxolitinib); To: Tofacitinib (JAK3 inhibitor); Ru: Ruxolitinib (JAK1/2 inhibitor); LDH activity was showed in (b), Caspase 3 activity were showed in (c). Hoechst 33258 staining of cell apoptotic (Ⅰ-Ⅴ) in control (Ⅰ),

H/R modal (Ⅱ), TET (5mM, Ⅲ),

TT(Ⅳ),TR(Ⅴ) were showed in (d) Results shown are mean ± SD of three independent experiments performed in duplicate. **p ≤ 0.01 relative to control , *p ≤ 0.05 relative to H/R group, #p ≤ 0.05 relative to TET group

3.5 Combined with Jak1/2 inhibitor, the regulation effect of tetrandrine on glycolysis and energy metabolism still exists, but attenuated when combined with Jak3 inhibitor. Tetrandrine increased the lever of glucose consumption and lactate production in H/R-treated H9c2 cells. As the result showed in Fig 5, glucose consumption and lactate production were significantly decreased when tetrandrine combined with tofacitinib, but not with ruxolitinib. Meanwhile, ATP content and NAD+/NADH ratio were also decreased in combined with tofacitinib compared to single tetrandrine group. 14

Western blot analysis indicated that the expression of HKⅡin mitochondrial was significantly increased in tetrandrine pretreatment group, but this effect was attenuated in combined with tofacitinib, not with ruxolitinib. Pretreatment of tetrandrine resulted in an obvious increased in the expression of p-STAT3. However, this effect was attenuated by tofacitinib (Fig. 5C). And also, tetrandrine combined with ruxolitinib have no significantly change on the expression of p-STAT3. In addition, we also detected the expression of JAK. As the result showed in Fig 5, tetrandrine increased JAK3 in H/R-treated H9c2 cells, the lever of JAK3 was significantly decreased when tetrandrine combined with tofacitinib. But pretreatment with tetrandrine have no impact on the expression of JAK1 and JAK2.

(a)

(b) 15

(c)

(d) Fig 5. Representative images and quantitative analysis of protein levels of p-STAT3, Hexokinase Ⅱ, JAK 1,JAK 2 JAK 3 in H9c2 cells were detected by western-bolt. Experimental grouping: Tetrandrine (TET 5 mM);TT (TET 5 mM+ Tofacitinib); TR(TET 5 mM+ Ruxolitinib); To: Tofacitinib (JAK3 inhibitor); Ru: Ruxolitinib (JAK1/2 inhibitor) (a-b); (c) Glucose consumption and Lactate production in H9c2 cells was evaluated; (d) ATP content and NAD+/NADH were also evaluated. Data were presented from three independent experiments; **p ≤ 0.01 relative to control, *p ≤ 0.05 relative toH/R group, #p ≤ 0.05 relative to TET group

4. Discussion Ischemia–reperfusion (IR) injury of organs and/or tissues is main contributor to several acute and chronic pathologies, with injury not only developing during ischemia, but paradoxically also during reperfusion. Glycolysis is the main way to maintain cellular energy supply under hypoxia condition. 16

During acute myocardial ischemia, anaerobic glycolysis and glycogenolysis assume the central role for energy production when oxidative phosphorylation cannot occur because of a lack of oxygen. The shift to anaerobic metabolism entails rapid increases in glucose uptake, glycogenolysis, and glycolytic flux. So promoting glycolysis has been used successfully to protect hearts from I/R injury (Opie LH. 1995). In this study, effect of tetrandrine on cell viability of H/R injury H9c2 cell was measured using the MTT assay firstly. The cell viability of H/R injury H9c2 cell was increased in a concentration and time dependent manner when treated with tetrandrine. Then, we also focus on the glycolysis in H9c2 cells. The glucose consumption and lactate production are key indicators of the glycolysis. In addition, the production of ATP and NAD+/NADH ratio were also detected. In this study we demonstrated that (i) Tetrandrine promoted glucose consumption and lactate production in H/R injury H9c2 cells. (ii) Tetrandrine increased ATP formation and decreased NAD+/NADH ratio, revealing that tetrandrine could promoted energy metabolism in H9c2 cells. HKII is the key speed limiting enzyme in glycolysis. Recent studies show that HKII is also one of the critical regulators of mitochondrial dysfunction in cardiac I/R injury (Aflalo et al., 1998; Azoulay-Zohar et al., 2004; Yeih et al., 2011). HKII contains an N-terminal, 21-amino-acid sequence that forms a hydrophobic α helix, and enables HKII to bind to the outer mitochondrial membrane (Chiara et al., 2008). The binding to mitochondria may also allow preferable access of HKII to primarily directs glucose into glycolysis and mitochondrially-generated ATP and reduce cellular production of reactive oxygen species (Calmettes et al., 2015). In this research, our result showed that tetrandrine could significant increased the protein lever of HKII in H/R injury H9c2 cell. At present, the main protective measures of ischemia reperfusion injury include reperfusion injury Salvage Kinase (RISK) pathway (Murphy et al., 2007; Tamareille et al., 2011) and survivor activating 17

factor enhancement (SAFE) pathway (Lacerda et al., 2009; Lecour., 2009; Xie et al., 1988). Phosphatidylinositol-3-kinase (PI3K)/Akt. and signal transducer and activator of transcription-3 (STAT-3) which were the key points in these pathways. Akt activation is known to regulate cell death, cell cycle progression and cell growth, as well as controlling rates of glucose uptake into the cell via the GLUT1 transporter (Inukai et al., 2015). Previous research had shown that Akt is known to further influence glycolysis by HK II (Robey et al., 2006; John et al., 2011). Many studies have shown that glycolysis is markedly increased during hypoxia, and HIF-1 is the key molecule to regulate glycolysis. HIF-1 activation is able to up-regulate levels of HKII in tumor cells (Calmettes et al., 2015; Lapel et al., 2017). Recently studies also have shown that STAT3 is also involved in the regulation of glucose metabolism and HKII (Liu et al., 2015; Li et al., 2014). So the influence on Akt, HIF-1α, and STAT3 were detected by western-blot to reveal the mechanism of tetrandrine on glycolysis regulation. We found that tetrandrine could significantly up-regulated the expression of p-STAT3, but have no effect on HIF-1α, Akt. Therefore, the mechanism in which tetrandrine up regulated in p-STAT3 still needs to be explored. Previous studies have shown that activation of STAT is associated with HIF-1α in glycolysis, but mainly in tumor cells. Targeting Stat3 with a small-molecule inhibitor blocks HIF-1 expression in vitro and inhibits breast tumor growth and angiogenesis in vivo (Xu et al., 2005). In addition, HIF-1α level is elevated and contributes to STAT3 oncogenic activity by supporting high rates of glycolysis in mouse embryonic fibroblasts (MEFs) (Demaria et al., 2012). However, we found tetrandrine significantly up-regulated the expression of p-STAT3, but have no effect on HIF-1α in our study. Thus, tetrandrine regulation of glycolysis may not be through STAT3/HIF-α in H9c2 cells. Recent evidence indicates that HK2 induction underlies a complex regulation including the control via STAT3 or HIF-1α. Using selective STAT3 or HIF-1α inhibitors as well as siRNA-mediated depletion of STAT3, the result 18

confirms that HK2 expression is dependent on STAT3 or HIF-1α (Heissa et al., 2016). Moreover, transfection of HepG2 and Hep3B cells with STAT3 siRNA significantly decreased glucose consumption and lactate production and HK2 mRNA and protein expression (Li et al., 2017). Thus, tetrandrine regulation of glycolysis should be directed through STAT. JAK/STAT3 is involved in many biological responses and influence cell growth, survival, and metastasis. Many studies have shown that JAK/STAT is also involved in the regulation of glycolysis (Oppermann et al., 2016). The JAK family consists of 4 members: JAK1, JAK2, JAK3, and Tyk2. The phosphorylation of STAT was mainly regulated by JAK1, JAK2 and JAK3. To confirm the involvement of tetrandrine in the regulation of JAK/STAT3 pathway, JAK1/2 inhibitor and JAK3 inhibitor was used. As the result showed that: (i) Cellular proliferation and glycolytic metabolism which increased by tetrandrine were significantly inhibited by JAK3 inhibitor. Although, cellular proliferation and glycolytic metabolism were decreased when combined with JAK1/2 inhibitor in tetrandrine group, there were still significant differences between H/R groups. (ii) Energy metabolism was also examined, tetrandrine combined with JAK3 inhibitor have no effect on ATP content and NAD+/NADH. But combined with JAK1/2 inhibitor, tetrandrine still could enhance the ATP content and decease the ratio of NAD+/NADH. (iii) The expression of HKII was downward in tetrandrine combine with JAK3 inhibitor. But in pretreatment with JAK1/2 inhibitor, tetrandrine still could increase the expression of HKII in H/R H9c2 cells. (iv) Tetrandrine increased the JAK3 in H/R-treated H9c2 cells, have no impact on the expression of JAK1 and JAK2. In conclusion, we demonstrated in our study that tetrandrine enhanced glycolysis by targeting JAK3/STAT3/HKII in H9c2 cells. Additionally, tetrandrine may increase the scope of potential therapeutics for ischemia and reperfusion injury in heart. These mechanisms may be involved in 19

mediating cardiac protective actions of tetrandrine in hypoxia and ischemic conditions.

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