Pien Tze Huang Gan Bao attenuates carbon tetrachloride‑induced hepatocyte apoptosis in rats, associated with suppression of p53 activation and oxidative stress

Zhao,Jinyan; Zhang,Yuchen; Wan,Yun; Hu,Haixia; Hong,Zhenfeng

doi:10.3892/mmr.2017.6936

Pien Tze Huang Gan Bao attenuates carbon tetrachloride‑induced hepatocyte apoptosis in rats, associated with suppression of p53 activation and oxidative stress

Authors:
- Jinyan Zhao
- Yuchen Zhang
- Yun Wan
- Haixia Hu
- Zhenfeng Hong
View Affiliations

Affiliations: Biomedical Research Center, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, P.R. China
Published online on: July 6, 2017 https://doi.org/10.3892/mmr.2017.6936
Pages: 2611-2619
Copyright: © Zhao et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Pien Tze Huang Gan Bao (PZH‑GB), a traditional Chinese medicine, has been used for thousands of years as a protective remedy effective against liver injury induced by excessive alcohol and smoking. The present study aimed to evaluate the protective effects and potential mechanisms of PZH‑GB against carbon tetrachloride (CCl4)‑induced hepatic injury. Rats were pre‑treated with silymarin (50 mg/kg) or different doses of PZH‑GB (150, 300 or 600 mg/kg) orally administered for 7 days. At the end of treatment, the rats were intraperitoneally injected with CCl4, or control rats received a corn oil injection. The lactate dehydrogenase (LDH) levels in serum were evaluated. Apoptosis was assessed via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. p53, B‑cell lymphoma 2 (Bcl‑2), B cell‑lymphoma 2‑associated X protein (Bax), cyclooxygenase‑2 (COX‑2), inducible nitric oxide synthase (iNOS) and cytochrome P450 family 2 subfamily E member 1 (CYP2E1) were measured by reverse transcription‑quantitative polymerase chain reaction and western blotting. The activity of caspase‑9 and caspase‑3 were measured by a colorimetric assay. The results indicated that silymarin and PZH‑GB prevented CCl4‑induced serum LDH elevations, and CCl4 induced high levels of LDH. Compared with the CCl4 group, silymarin and PZH‑GB treatment significantly decreased LDH levels. Histopathological results revealed that silymarin and PZH‑GB ameliorated the CCl4‑induced liver histological alterations. The TUNEL results showed that compared with the control group, CCl4 induced liver cell apoptosis, while silymarin and PZH‑GB treatment inhibited apoptosis and the TUNEL‑positive cells. The elevated expression of Bax, p53, iNOS, COX‑2 and CYP2E1 were reduced by silymarin or PZH‑GB pretreatment, whereas reduced Bcl‑2 expression levels were increased. CCl4 increased the activity of caspase‑9 and ‑3 by 6.86‑ and 7.42‑fold, respectively; however, silymarin and PZH‑GB ameliorated this effect. In conclusion, silymarin and PZH‑GB treatment prevented the deleterious effects on liver functions by attenuation of oxidative stress, inflammation and mitochondrial apoptosis via the p53 signaling pathway.

View Figures

View References

1	Kumar CH, Ramesh A, Kumar JN Suresh and Ishaq BM: A review on hepatoprotective activity of medicinal plants. Int J Pharmaceut Sci Res. 23:501–515. 2011.
2	Ahsan R, Islam KM, Bulbul IJ, Musaddik A and Haque E: Hepatoprotective activity of methanol extract of some medicinal plants against carbon tetrachloride-induced hepatotoxicity in rats. Eur J Sci Res. 37:302–310. 2009.
3	Chattopadhyay RR: Possible mechanism of hepatoprotective activity of Azadirachta indica leaf extract: part II. J Ethnopharmacol. 89:217–219. 2003. View Article : Google Scholar : PubMed/NCBI
4	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
5	Recknagel RO, Glende EA Jr, Dolak JA and Waller RL: Mechanisms of carbon tetrachloride toxicity. Pharmacol Ther. 43:139–154. 1989. View Article : Google Scholar : PubMed/NCBI
6	Williams AT and Burk RF: Carbon tetrachloride hepatotoxicity: An example of free radical-mediated injury. Semin Liver Dis. 10:279–284. 1990. View Article : Google Scholar : PubMed/NCBI
7	Slater TF: Free radicals as reactive intermediates in tissue injury. Adv Exp Med Biol. 136:575–589. 1981.PubMed/NCBI
8	Brattin WJ, Glende EA Jr and Recknagel RO: Pathological mechanisms in carbon tetrachloride hepatotoxicity. J Free Radic Biol Med. 1:27–38. 1985. View Article : Google Scholar : PubMed/NCBI
9	Cederbaum AI: Role of CYP2E1 in ethanol-induced oxidant stress, fatty liver and hepatotoxicity. Dig Dis. 28:802–811. 2010. View Article : Google Scholar : PubMed/NCBI
10	Bartosz G: Reactive oxygen species: Destroyers or messengers? Biochem Pharmacol. 77:1303–1315. 2009. View Article : Google Scholar : PubMed/NCBI
11	Cadenas E and Davies KJ: Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med. 29:222–230. 2000. View Article : Google Scholar : PubMed/NCBI
12	Circu ML and Aw TY: Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic Biol Med. 48:749–762. 2010. View Article : Google Scholar : PubMed/NCBI
13	Shi J, Aisaki K, Ikawa Y and Wake K: Evidence of hepatocyte apoptosis in rat liver after the administration of carbon tetrachloride. Am J Pathol. 153:515–525. 1998. View Article : Google Scholar : PubMed/NCBI
14	Hoek JB, Cahill A and Pastorino JG: Alcohol and mitochondria: A dysfunctional relationship. Gastroenterology. 122:2049–2063. 2002. View Article : Google Scholar : PubMed/NCBI
15	Volkmann N, Marassi FM, Newmeyer DD and Hanein D: The rheostat in the membrane: BCL-2 family proteins and apoptosis. Cell Death Differ. 21:206–215. 2014. View Article : Google Scholar : PubMed/NCBI
16	Takebe H, Sato I, Tajima S, Ikeda Y, Ito K and Nose T: Effects of cianidanol (KB-53) on liver cirrhosis induced by CCl₄ in rats: A pathological investigation. Nihon Yakurigaku Zasshi. 81:585–591. 1983.(In Japanese). View Article : Google Scholar : PubMed/NCBI
17	Merino N, González R, González A and Remirez D: Histopathological evaluation on the effect of red propolis on liver damage induced by CCl₄ in rats. Arch Med Res. 27:285–289. 1996.PubMed/NCBI
18	Yadav SS, Sindram D, Perry DK and Clavien PA: Ischemic preconditioning protects the mouse liver by inhibition of apoptosis through a caspase-dependent pathway. Hepatology. 30:1223–1231. 1999. View Article : Google Scholar : PubMed/NCBI
19	Perry DK, Smyth MJ, Stennicke HR, Salvesen GS, Duriez P, Poirier GG and Hannun YA: Zinc is a potent inhibitor of the apoptotic protease, caspase-3. A novel target for zinc in the inhibition of apoptosis. J Biol Chem. 272:18530–18533. 1997. View Article : Google Scholar : PubMed/NCBI
20	Yao Y, Huang C, Li ZF, Wang AY, Liu LY, Zhao XG, Luo Y, Ni L, Zhang WG and Song TS: Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/Bax pathway. World J Gastroenterol. 15:1751–1758. 2009. View Article : Google Scholar : PubMed/NCBI
21	Jin S and Dai CL: Attenuation of reperfusion-induced hepatocyte apoptosis is associated with reversed bcl-2/bax ratio in hemi-hepatic artery-preserved portal occlusion. J Surg Res. 174:298–304. 2012. View Article : Google Scholar : PubMed/NCBI
22	Liu B, Shi Y, Peng W, Zhang Q, Liu J, Chen N and Zhu R: Diosmetin induces apoptosis by upregulating p53 via the TGF-β signal pathway in HepG2 hepatoma cells. Mol Med Rep. 14:159–164. 2016.PubMed/NCBI
23	Ortega JF, de Conti A, Tryndyak V, Furtado KS, Heidor R, Horst MA, Fernandes LH, Tavares PE, Pogribna M, Shpyleva S, et al: Suppressing activity of tributyrin on hepatocarcinogenesis is associated with inhibiting the p53-CRM1 interaction and changing the cellular compartmentalization of p53 protein. Oncotarget. 7:24339–24347. 2016. View Article : Google Scholar : PubMed/NCBI
24	Allam A, Gabr S, Ajarem J and Abdel-Maksoud M: Bcl-2 and p53 expression in hepatic tissues of Egyptian patients with Chronic Hepatitis C. J Pak Med Assoc. 65:1186–1192. 2015.PubMed/NCBI
25	Li X, Yu J, Brock MV, Tao Q, Herman JG, Liang P and Guo M: Epigenetic silencing of BCL6B inactivates p53 signaling and causes human hepatocellular carcinoma cell resist to 5-FU. Oncotarget. 6:11547–11560. 2015. View Article : Google Scholar : PubMed/NCBI
26	Guo XL, Liang B, Wang XW, Fan FG, Jin J, Lan R, Yang JH, Wang XC, Jin L and Cao Q: Glycyrrhizic acid attenuates CCl₄-induced hepatocyte apoptosis in rats via a p53-mediated pathway. World J Gastroenterol. 19:3781–3791. 2013. View Article : Google Scholar : PubMed/NCBI
27	Rechnagel RO and Glende EA Jr: Carbon tetrachloride hepatotoxicity: An example of lethal cleavage. CRC Crit Rev Toxicol. 2:263–297. 1973. View Article : Google Scholar : PubMed/NCBI
28	Al-Shabanah OA, Alam K, Nagi MN, Al-Rikabi AC and Al-Bekairi AM: Protective effect of aminoguanidine, a nitric oxide synthase inhibitor, against carbon tetrachloride induced hepatotoxicity in mice. Life Sci. 66:265–270. 2000. View Article : Google Scholar : PubMed/NCBI
29	Badger DA, Sauer JM, Hoglen NC, Jolley CS and Sipes IG: The role of inflammatory cells and cytochrome P450 in the potentiation of CCl₄-induced liver injury by a single dose of retinol. Toxicol Appl Pharmacol. 141:507–519. 1996. View Article : Google Scholar : PubMed/NCBI
30	Lowenstein CJ and Snyder SH: Nitric oxide, a novel biologic messenger. Cell. 70:705–707. 1992. View Article : Google Scholar : PubMed/NCBI
31	Inoue T, Kwon AH, Oda M, Kaibori M, Kamiyama Y, Nishizawa M, Ito S and Okumura T: Hypoxia and heat inhibit inducible nitric oxide synthase gene expression by different mechanisms in rat hepatocytes. Hepatology. 32:1037–1044. 2000. View Article : Google Scholar : PubMed/NCBI
32	Nadler EP, Dickinson EC, Beer-Stolz D, Alber SM, Watkins SC, Pratt DW and Ford HR: Scavenging nitric oxide reduces hepatocellular injury after endotoxin challenge. Am J Physiol Gastrointest Liver Physiol. 281:G173–G181. 2001.PubMed/NCBI
33	Chun KS and Surh YJ: Signal transduction pathways regulating cyclooxygenase-2 expression: Potential molecular targets for chemoprevention. Biochem Pharmacol. 68:1089–1100. 2004. View Article : Google Scholar : PubMed/NCBI
34	Chang YC, Li PC, Chen BC, Chang MS, Wang JL, Chiu WT and Lin CH: Lipoteichoic acid-induced nitric oxide synthase expression in RAW 264.7 macrophages is mediated by cyclooxygenase-2, prostaglandin E2, protein kinase A, p38 MAPK, and nuclear factor-κB pathways. Cell Signal. 18:1235–1243. 2006. View Article : Google Scholar : PubMed/NCBI