Ca²⁺-ATPase and cAMP-mediated Anti-Apoptotic Effects of Acanthopanax senticosus Extracts on Ischemia/Reperfusion Liver Damages

Introduction

Clinical or experimental hepatic vascular exclusion, clamping of the portal pedicle along with the inferior vena cava below was useful methods on the clinical and/or liver-related disease model. These methods were conventional protocols for resection of tumorous liver by close the hepatic veins or the vena cava (Serracino-Inglott et al., 2001; Huguet et al., 1992, Garden., 1994). A period of ischemia is required for a many surgical procedures on the liver such as transplantation, dealing with extensive hepatic trauma, and resecting large intrahepatic lesions (Huguet et al., 1992;

Delva et al., 1989). Restoration of blood after liver ischemia, the liver subjected to a further insult, accelerating the cellular

injury by ischemia. This is termed ischemia-reperfusion (IR) injury, and in the field of hepatic transplantation, it covers a various problems with the common clinical phenomenon of a poorly functioning graft (Henderson, 1999; Serracino-Inglott et al., 2001).

Acanthopanax senticosus (or Eleutherococcus senticosus, Araliaceae; also called Siberian Ginseng) has been used as a traditional medicine to management of various internal medicines (Nishibe et al., 1990; Fujikawa et al., 2005). The leaves, stems, and roots of A. senticosus have also been used clinically for anti-stress, treatment of anaphylaxis, rheumatoid, chronic bronchitis, hypertension, ischemic heart disease, gastric ulcer (Yi et al., 2001), diabetes (Kim et al., 2010) and liver ischemic diseases (Xie et al., 2012). Recently, effective contents from A. senticosus were reported and have been shown to be responsible for the adaptogenic properties

Ca ²⁺ -ATPase and cAMP-mediated Anti-Apoptotic Effects of Acanthopanax senticosus Extracts on Ischemia/Reperfusion

Liver Damages

Guang-Hua Xie ¹ , Jae-Hun Jeong ² , Sun Eun Choi ³ , Seung Il Jeong ⁴ and Kwang-Hyun Park ⁵ *

1

Department of General Surgery, YanBian University Hospital, JiLin 133-000, China

2

Department of Food Science & Bio-Technology, Jeonnam State University, Damyang 57337, Korea

3

Department of Cosmetology Science, Nambu University, Gwangju 62271, Korea

4

Jeonju AgroBio-Materials Institute, Jeonju 54810, Korea

5

Department of Oriental Pharmaceutical Development, Nambu University, Gwangju 62271, Korea

Abstract - Hepatic ischemia-reperfusion injury (HIRI) is linked with high mortality rate. Several agents have been developed so far to reduce the risk of HIRI. In this study, we investigated the effects of Acanthopanax senticosus extract (AS) on hepatic ischemia-reperfusion. To explore the protective effects of A. senticosus extract injection (ASI) on hepatic ischemia-reperfusion injury rats animal model were used. After the development of HIRI by using clamping method rats were then randomly divided into five groups. Different doses of AS were administered in HIRI rat model. The level of ALT, AST, and MDA content in serum were detected in sham and HIRI groups. The activity of SOD, MPO and Ca

-ATPase, content of MDA, and cAMP in hepatic tissue were also measured. Expression of Bcl-2 and Bax protein were detected by immunohistochemical staining method. Compared with sham group, ASI has the protective effect on the HIRI model in rats.

Blood levels of ALT, AST, SOD, MPO, and MDA were significantly lower in ASI group compared with HIRI. Indeed SOD and Ca

-ATPase activities, MDA content, and cAMP level were improved in ASI group. Furthermore, Bcl-2 and Bax protein were improved in ASI group compared with only HIRI group. These results suggest that AS may provide potential ameliorative therapy by inhibiting the damage signaling mechanism in hepatic ischemia/reperfusion injury model.

Key words - Acanthopanax senticosus, Apoptosis, Ca

-ATPase, cAMP, Hepatic ischemia-reperfusion injury

*Corresponding author. E-mail : [email protected] Tel. +82-62-970-0220

ⓒ 2017 by The Plant Resources Society of Korea

Original Research Article

(Hikino et al., 1986). Especially, syringin and its alycone (eleutherosides B) are widely regarded as two important substances of the eleutheroside group (Niu et al., 2008) and eleutherosides etc.

In this study, we investigated the protective effects of both ASI in HIRI model. Specifically, we examined the abilities of each to regulate hepatic-related enzymes activities in blood and inflammatory molecules expression, using a rat model of ischemia/reperfusion injury induced by vascular clamping.

Materials and Methods

Materials

The AS was obtained from Wusuli River Pharmaceutical Co. Ltd. (Cat# Z23023215; Heilongjiang, China). Each vial of 20 ㎖ was contained 300 ㎎ as total flavonoids in manuf- acturer instruction.

Animal experiments

The 50 male Wistar rats (290-320 g) were purchased from Charles River (Shanghai, China). The experiments were performed in accordance with the principles and with the approval of the Ethics Committee of the YanBian University, China (Approval No. Z23021162). All animals maintained in a temperature/humidity-controlled room with a 12 hr/12 hr light/dark cycle, and acclimatized to the laboratory environment while housed in individual cages for 1 week before the experiments. Animals were randomly divided into five groups: sham operation group (normal group): Only open without occlusion of blood vessels; ischemia-reperfusion group (control group); treatment group (experimental group).

Sham group and ischemia-reperfusion group calculated according to the weight given saline.

Preparation of hepatic ischemia-reperfusion model Male Wistar rats weighing 290-320g of fasting before surgery 12h, After weighing accuracy with 10% chloral hydrate 4 ㎖/㎏ intraperitoneal injection to anesthetized rats.

After induction of anesthesia with intramuscular ketamine hydrochloride (100 ㎎/㎏), The AS injected intravenously with indicated dose (3 ㎖) to experimental groups during 15 min. Normal and control group were also injected equal

volume of saline by same procedures. After 30 minute of injection with AS or saline, an indwelling intravenous line was placed via the right external jugular vein for serial blood sampling and administration of intravenous fluids and medications. By positioning the catheter tip in the suprahepatic vena cava just above the dome of the liver, immediately posthepatic blood samples were obtained; correct catheter position was confirmed at laparotomy. After intravenous heparinization with 100 U/100 g of body weight, midline laparotomy was performed. Hepatic ischemia was initiated by application of a Heifitz clip, ischemia was maintained for 60 min, and the Heifitz clip was then removed at a second laparotomy. Intravenous lactated Ringer's solution in a dose of 0.75 ㎖ was administered to replace operative fluid and blood losses. Sham-operated control animals were treated in an identical fashion with the omission of vascular occlusion (Colletti et al., 1990).

Specimen collection and testing

Animals after reperfusion 2h, inferior vena cava blood 2 ㎖ and centrifuged at 3000 rpm for 10 min for separation of serum. Detectable levels ALT and AST's, SOD activity and MDA were measured. The left lobe of liver tissue were obtained and moved to 0.85% ice-cold saline. The serially diluted liver homogenate were used for detecting protein content, SOD activity, MDA, GSH content. Another portion of liver lobe were fixed with 10% neutral formalin for 48h at 4℃, embedded in paraffin for immunohistochemistry and measured Bcl-2 and Bax protein.

Measurement of biochemical parameters

To assess hepatic function and injury after liver ischemia,

we measured serum ALT and AST levels were measured

using a clinical chemistry system (Bayer Co. Tarrytown,

NY). SOD activity was measured through the inhibition of

nitroblue tetrazolium (NBT) reduction by O

generated by

the xanthine/xanthine oxidase system. One SOD activity unit

was defined as the enzyme amount causing 50% inhibition in

1 ㎖ reaction solution per milligram tissue protein and the

result expressed as U/ ㎎protein. MPO activities of serum can

catalyze the redox reaction of H

O

and 3,3,5,5-tetrame-

thylbenzidine and produce yellow- colored compounds,

through whose absorbance at 460 ㎚MPO activity was calculated and expressed as U/L of serum. One unit of MPO activity was defined as the quantity of enzyme that degraded 1 nmol H

O

at 37℃ per ㎖ serum. MDA levels of serum and liver tissues were assayed by the measurement of thiobarbituric acid-reactive substances (TBARS) levels at 532 ㎚. Results were expressed as μmol/L serum and nmol/

㎎ protein, respectively.

Ca

-ATPase activities assay

The enzymatic activity was measured in the presence of 100 nM thapsigargin and 5mM sodium azide to inhibit the contamination of the endoplasmic reticulum Ca

-ATPase (SERCA) and other ATPases. Calmodulin (0.32 ㎍/㎖) was added when indicated. The reaction was started by the addition of 1 mM ATP. The Ca

-ATPase activity of these fractions was obtained after subtraction of the Mg

-ATPase activity, measured in the presence of 6 mM EGTA, and was given in U/L (Sepúlveda et al., 2004)

Immunohistochemistry

For immunohistochemistry, skin tissues were fixed in 10%

formaldehyde, embedded in paraffin and sectioned at 4 μm thickness. The sections were deparaffinized and antigen- retrieval was performed in sodium-citrate buffer (10 mM, pH 6.0) for 10 min at 90 ℃. Sections were blocked in 1% BSA in PBS for 30 minute and incubated 2 hours with a polyclonal mouse antibody against indicated antibodies. After 4 times washes for 5 minutes each with PBS, sections were incubated with horseradish peroxidase-conjugated secondary antibodies.

Stained sections were mounted and visualized with a Leica microscope with digital camera (DC200, Germany) (Park et al., 2015).

cAMP measurement

The liver tissue was placed in 6% TCA to stop the reaction, after grinding tissue by centrifugation for 5 minutes after the supernatant ether layer was removed. Quantitative determination of dissolved with buffer detected by radioimmunoassay.

cAMP measurements to quantify the amount of radioactivity (Yuan et al., 2008).

Statistical analysis

All data were expressed as mean ± SEM, and differences between groups were analyzed using one-way ANOVA and Duncan’s multiple-range test. All analyses were performed using SPSS 10.0 (SPSS Inc. USA). Each value was the mean of at least 3 separate experiments in each group, and data with different superscript letters are significantly different when p value is less than 0.05.

Results

Effects of ASI injection on ALT and AST level in serum To investigate the therapeutic potential of ASI on HIRI- induced liver damages, we first measured only showing serum ALT and AST levels compared with normal and HIRI processed rats. ASI has been shown to reduce serum ALT and AST levels increases on HIRI rats (Fig. 1). HIRI rat shown increase of ALT and AST levels in serum however ASI reduced levels in a dose dependent manner. Thus these results suggest that ASI reduced HIRI mediated liver cells damages.

Fig. 1. Effect of the pretreatment with ASI on the serum levels of ALT and AST in HIRI model rats. (Mean ± SEM). ASI-AS injection; HIRI-hepatic ischemia/reperfusion injury. *P < 0.05 vs negative control group and **P < 0.01 vs HIRI group. #P <

0.01 vs negative control group and ##P < 0.05 vs HIRI group.

Effects of ASI on activities of serum SOD, MDA, and MPO in HIRI rat

To examine the effects of ASI on HIRI-mediated liver damage, we measured serum SOD, MDA, and MPO levels.

In Fig. 2, significant reduces of SOD levels were detected in control group compared with the normal group. However, those were reduced by ASI in a dose dependent manner. The MDA and MPO levels were significantly increased by HIRI processes. MDA and MPO levels in HIRI processed rats changed by 6.1 and 1.4 fold compared with normal rats, respectively. In contrast, ASI processes were reduced serum MDA and MPO levels compared with HIRI processed rats in a dose dependent manner. Although ASI with low concen-

tration did not shown significant results but 50 and 100 ㎎/㎏

groups were recovered the HIRI-mediated increase in MDA and MPO activity, These results suggest that ASI reduced HIRI-mediated liver damages via regulation of oxidative stress.

Effects of ASI on SOD activity, MDA content, and Ca

-ATPase activity in tissue of HIRI rats

Similar to serum SOD activity, SOD activity in HIRI- processed liver tissue was also reduced. In Fig. 3, SOD activity in liver tissue was reduced to 45% compared with normal group but these activities were significantly recovered by ASI process in a dose dependent manner. Indeed, HIRI-

Fig. 2. Effect of the pretreatment with ASI on the serum levels of SOD, MDA and MPO ALT and AST levels in HIRI model rats. (Mean ± SEM). ASI-AS injection; HIRI-hepatic ischemia/

reperfusion injury. *P < 0.01 vs negative control group and

**P < 0.01 vs HIRI group. #P < 0.005 vs negative control group and ##P < 0.05 vs HIRI group. ¶P < 0.05 vs negative control group and ¶¶P < 0.05 vs HIRI group.

Fig. 3. Effect of the pretreatment with ASI on the tissue levels of SOD, MDA, and activity of Ca

-ATPase in HIRI model rats. (Mean ± SEM). ASI-AS injection; HIRI-hepatic ischemia/

reperfusion injury. *P < 0.005 vs negative control group and

**P < 0.05 vs HIRI group. #P < 0.01 vs negative control group

and ##P < 0.05 vs HIRI group. ¶P < 0.001 vs negative control

group and ¶¶P < 0.05 vs HIRI group.

mediated increases of MDA contents in liver tissue were significantly reduced in all dosage groups but not shown significant changes in a dose dependent manner. Moreover, we measured the activity of Ca

-ATPase, major component for calcium homeostasis, in liver tissues. HIRI process were significantly decreased Ca

-ATPase activity. In contrast, ASI was recovered Ca

-ATPase activity in liver tissues in a dose dependent manner but not reached to normal ranges (approximately ~50%). Thus these results suggest that ASI was effective to HIRI-mediated liver damage via anti-oxidative mechanisms and parallel cellular signaling.

Effects of ASI on cAMP level in liver tissue of HIRI rats We determined whether IR itself triggers cAMP level elevation in rats livers subjected to 60 minutes of ischemia.

There was a notable increases in tissue cAMP levels in the control group. In contrast, cAMP levels decreased in the ASI groups in a dose dependent manner. cAMP levels differed significantly from the control during the observation period (Fig. 4).

Effects of ASI on expression of Bcl-2 and Bax in tissue of HIRI rats

ASI on HIRI in rat liver tissue Bcl-2, Bax expression: The experimental results show (Fig. 5 and Fig. 6). ASI of small, medium high-dose group could significantly increase hepatic ischemia-reperfusion injury in rat liver tissue expression of Bcl-2 protein, compared with model group were significantly

Fig. 4. Effect of the pretreatment with ASI on the tissue level of cAMP in HIRI model rats. (Mean ± SEM). ASI-AS injection;

HIRI-hepatic ischemia/reperfusion injury. *P < 0.05 vs negative control group and **P < 0.01 vs HIRI group.

Fig. 5. Immunohistochemical observations showing the effects of ASI on changes of BAX protein expression in HIRI model rats. (A) Sham, (B) HIRI control, (C) 25 ㎎/㎏ ASI + HIRI, (D) 50 ㎎/㎏ ASI + HIRI, and (E) 100 ㎎/㎏ ASI + HIRI.

Magnification : ×200. Scale bar = 50 ㎛. ASI-AS injection;

HIRI-hepatic ischemia/reperfusion injury.

Fig. 6. Immunohistochemical observations showing the effects of ASI on changes of Bcl-2 protein expression in HIRI model rats. (A) Sham, (B) HIRI control, (C) 25 ㎎/㎏ ASI + HIRI, (D) 50 ㎎/㎏ ASI + HIRI, and (E) 100 ㎎/㎏ ASI + HIRI.

Magnification : ×200. Scale bar = 50 ㎛. ASI-AS injection;

HIRI-hepatic ischemia/reperfusion injury.

different. ASI of low-dose group could reduce the expression of Bax protein, compared with model group was statistically significant; ASI, the high-dose group can significantly reduce the expression of bax protein, with the model group there was a significant difference (P < 0.01).

Discussion

In present study, we observed the protective effects of ASI in HIRI model. The A. senticosus was widely adopted as a folk medicine in China, Soviet, however little adopted in western countries and investigated by alternative medicine in worldwide (Huang et al., 2011). This plant is contained various biological effective molecules for oxidative stress, inflammation, etc. Indeed, most available AS extracts can purchased on the market as edible foods such as tea, capsule, or powder but not reported as intravenous injects in western countries.

Several second messengers such as cyclic adenosine monophosphate (cAMP), inositol 1,4,5-triphosphate (IP3), cyclic adenosine diphosphate ribose (cADPR), nicotinic acid adenine dinucleotide (NAADP), translates a variety of extracellular stimuli into intracellular biological responses in heart (Xie et al., 2003), pancreas (Park et al., 2013), liver, skin (Park et al., 2015), kidney, and sperm (Park et al., 2011).

Especially, cAMP was serves initial intracellular signaling by various stimulation cascades such as electric shock (Park et al,. 2015), β-adrenergic receptor (βAR) (Xie et al., 2005).

cAMP binds to protein kinase A (PKA) via a complex of catalytic or mutated regulatory subunits, and the activation of cAMP-dependent PKA leads to an array of regulatory cell functions, including proliferation, differentiation, apoptosis, migration, and immune responses (Ji et al., 2012). Hepatocytes are very much negatively affected when HIRI condition, especially occurred anoxic status in mitochondria (Peralta et al., 2013). Thus, increase the intracellular NADH/NAD+

ratio leads to cellular ATP depletion, and increases of cAMP level by hepatic ischemia. .In other reports, cAMP-PKA activation can ameliorate the apoptosis of parenchymal cells, including hepatocytes (Wang et al., 2006; Kullhanek-Heinze et al., 2004). In pathophysiologically, we observed that ASI rescued anti-apoptotic molecule (Bcl-2) expression and

reduced the apoptotic signaling molecule (BAX) in liver tissue on HIRI model mice (Fig 5 and 6), However, more quantitative investigation required for identification of action mechanism in further study. Indeed, although ASI has been shown to mediate ameliorative effects in rat livers ischemia/

reperfusion (Lim et al., 2013), it remains unknown whether and how cAMP-PKA activation can affect liver HIRI in vivo.

Therefore, regulation of cAMP/PKA pathway has crucial roles on treatment and care of hepatic ischemia/reperfusion condition in pathophysiologic and experimental model.

These results suggest that AS exert potent ameliorative effects and inhibit the damage signaling on hepatic ischemia/

reperfusion injury.

These results suggest that A. senticosus exert potent ameliorative effects and inhibit the damage signaling on hepatic ischemia/reperfusion injury via Ca

-ATPase and cAMP signaling cascade.

Acknowledgement

These works supported by Science and Technology Research Projects of The Education Department of Jilin province (12-5, G-H-X).

Conflict of Interest

The authors declares that there is no conflict of interest regarding the publication of this paper.

References

Colletti, L.M., D.G. Remick, G.D. Burtch, S.L. Kunkel, R.M.

Strieter and D.A. Campbell Jr. 1990. Role of tumor necrosis factor-alpha in the pathophysiologic alterations after hepatic ischemia/reperfusion injury in the rat. J. Clin. Invest. 85:

1936-1943.

Delva, E., Y. Camus, B. Nordlinger, L. Hannoun, R. Parc, H.

Deriaz, A. Lienhart and C. Huguet. 1989. Vascular occlusion for liver resections. Operative management and tolerance to ischemia: 142 cases. Ann Surg 209:211-218.

Fujikawa, T., S. Miguchi, N. Kanada, N. Nakai, M. Ogata, I.

Suzuki and K. Nakashima. 2005. Acanthopanax senticosus

Harms as a prophylactic for MPTP-induced Parkinson’s disease in rats. J. Ethnopharmacol 97:375-381.

Garden, O.J. 1994. Technique of vascular isolation for liver resection. HPB Surg 7:332-334.

Henderson, J.M. 1999. Liver transplantation and rejection: an overview. Hepato-Gastroenterol 46 (suppl 2):1482-1484.

Hikino, H., M. Takahashi, K. Otake and C. Konno. 1986.

Isolation and hypoglycemic activity of eleutherans A, B, C, D, E, F, and G: glycans of Eleutherococcus senticosus roots.

J. Nat. Prod. 49:293-297.

Huang, L., H. Zhao, B. Huang, C. Zheng, W. Peng and L. Qin.

2011. Acanthopanax senticosus: review of botany, chemistry and pharmacology. Pharmazie 66:83-97.

Huguet, C., P. Addario-Chieco, A. Gavelli, E. Arrigo, J. Harb and R.R. Clement. 1992. Technique of hepatic vascular exclusion for extensive liver resection. Am. J. Surg 163:602-605.

Ji, H., X.D. Shen, Y. Zhang, F. Gao, C.Y. Huang, W.W. Chang, C. Lee, B. Ke, R.W. Busuttil and J.W. Kupiec-Weglinski.

2012. Activation of cyclic adenosine monophosphate- dependent protein kinase a signaling prevents liver ischemia/

reperfusion injury in mice. Liver Transpl. 18:659-670.

Kim, D.J., J.M. Kim, T.H. Kim, J.M. Baek, H.S. Kim and M.

Choe. 2010. Anti-diabetic effects of mixed extracts from Lycium chinense, Cordyceps militaris, and Acanthopanax senticosus. Korean J. Plant Res. 23(5):423-429

Lim, E.J., G.M. Do, J.H. Shin and O. Kwon. 2013. Protective effects of Acanthopanax divaricatus vat. albeofructus and its active compound on ischemia-reperfusion injury of rat liver.

Biochem. Biophys. Res Commun. 432:599-605.

Nishibe, S., H. Kinoshita, H. Takeda and G. Okano. 1990.

Phenolic compounds from stem bark of Acanthopanax senticosus and their pharmacological effect in chronic swimming stressed rats. Chem. Pharm. Bull. (Tokyo) 38:

1763-1765.

Niu, H.S., F.L. Hsu and I.M. Liu. 2008. Role of sympathetic tone in the loss of syringin-induced plasma glucose lowering action in conscious Wistar rats. Neurosci. Lett. 445:113-116.

Peralta, C., M.B. Jiménez-Castro and J. Gracia-Sancho. 2013.

Hepatic ischemia and reperfusion injury: effects on the liver sinusoidal milieu. J. Hepatol. 59:1094-1106.

Park, K.H., B.J. Kim, J. Kang, T.S. Nam, J.M Lim, H.T Kim, J.K. Park, Y.G. Kim, S.W. Chae and U.H. Kim. 2011. Ca

signaling tools acquired from prostasomes are required for

progesterone-induced sperm motility. Sci. Signal. 173(4):ra31.

Park, K.H., B.J. Kim, A.I. Shawl, M.K. Han, H.C. Lee and U.H.

Kim. 2013. Autocrine/paracrine function of nicotinic acid adenine dinucleotide phosphate (NAADP) for glucose homeostasis in pancreatic β-cells and adipocytes. J. Biol.

Chem. 288:35548-35558.

Park, K.H., K.N. Kim, D.R. Park, K.Y. Jang and U.H. Kim.

2015. Role of nicotinic acid adenine dinucleotide phosphate (NAADP) in keratinocyte differentiation. J. Invest Dermatol.

135:1692-1694.

Park, D.R., K.H. Park, B.J. Kim, C.S. Yoon and U.H. Kim.

2015. Exercise ameliorates insulin resistance via Ca

signals distinct from those of insulin for GLUT4 trans- location in skeletal muscles. Diabetes 64:1224-1234.

Sepúlveda, M.R., M. Hidalgo-Sánchez and A.M. Mata. 2004.

Localization of endoplasmic reticulum and plasma membrane Ca

-ATPases in subcellular fractions and sections of pig cerebellum. Eur. J. Neurosci. 19:542-551.

Serracino-Inglott, F., N.A. Habiba and R.T. Mathie. 2001.

Hepatic ischemia-reperfusion injury. Am. J. Surg. 181:160-166.

Wang, Y., P.K. Kim, X. Peng, P. Loughran, Y. Vodovotz, B.

Zhang and T.R. Billiar. 2006. Cyclic AMP and cyclic GMP suppress TNFa-induced hepatocyte apoptosis by inhibiting FADD up-regulation via a protein kinase A-dependent pathway. Apoptosis 11:441-451.

Xie, G.H., S.Y. Rah, K.S. Yi, M.K. Han, S.W. Chae, M.J. Im and U.H. Kim. 2003. Increase of intracellular Ca

during ischemia/reperfusion injury of heart is mediated by cyclic ADP-ribose. Biochem. Biophys. Res. Commun. 307:713-718.

Xie, G.H, J.H. Jeong and K.H. Park. 2012. The protective effects of Acanthopanax senticosus extracts on hepatic ischemia-reperfusion injury in rats. Korean J. Plant Res.

symposium 127.

Xie, G.H., S.Y. Rah, S.J. Kim, T.S. Nam, K.C. Ha, S.W. Chae, M.J. Im and U.H. Kim. 2005. ADP-ribosyl cyclase couples to cyclic AMP signaling in the cardiomyocytes. Biochem.

Biophys. Res. Commun. 330:1290-1298.

Yi, J.M., M.S. Kim, S.W. Seo, K.N. Lee, C.S. Yook and H.M Kim. 2001. Acanthopanax senticosus root inhibits mast cell-dependent anaphylaxis. Clin. Chim. Acta. 312:163-168.

Yuan, K., G.Y. Bai, W.H. Park, S.Z, Kim and S.H. Kim. 2008.

Stimulation of ANP secretion by 2-Cl-IB-MECA through A(3) receptor and CaMKII. Peptides 29:2216-2224.

(Received 3 August 2017 ; Revised 16 November 2017 ; Accepted 23 November 2017)