1. Introduction

The liver is a vital organ in the human body which functions to detoxify exogenous xenobiotics, drugs, viral infections, and alcohol from the body. Liver disease is a primary causes of mortality and morbidity worldwide. Specifically, drug-induced liver toxicity has been shown to be strongly associated with the onset of hepatic dysfunction ^[1]. Liver damage is a widespread pathology that is often characterized by oxidative stress and a progressive evolution from steatosis to chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma ^[2]. In recent years, a great deal of attention has focused on the transformation of chemicals into highly reactive metabolites associated with cellular toxicity. Carbon tetrachloride- (CCl₄-) induced hepatotoxicity has been widely used in animal models to investigate the hepatoprotective effects of natural compounds ^{[3, 4]}.

Oxidative stress is considered to be a contributing mechanism towards the initiation and progression of hepatic damage in a range of different liver disorders. Excess reactive species derived from oxygen and nitrogen or an antioxidant deficiency are known to be causative agents of oxidative stress, ultimately leading to cell death ^[5]. Thus, antioxidants isolated from plant sources represent a promising therapeutic strategy for the treatment of liver disease. Both flavonoids and polysaccharides possess unique antioxidant properties as well as other pharmacological activities that could play a role in the protection of the liver from CCl₄-induced hepatotoxicity ^{[6, 7]}. It is thought that flavonoids and polysaccharides function to prevent the production of oxidants through the inhibition of xanthine oxidase and the chelation of transition metals. By inhibiting the production of oxidants, cellular targets would be protected from oxidative damage, and the propagation of oxidative reactions would be prevented. In addition, flavonoids and polysaccharides could function to reinforce cellular antioxidant capacity by both minimizing the effects of oxidants on antioxidants and inducing the expression of endogenous antioxidants ^{[8, 9]}. Flavonoids and polysaccharides have also been described to possess anti-inflammatory properties. These actions are carried out through their ability to inhibit enzymes and signaling pathways associated with decreased oxidant production ^{[10, 11]}. These multifaceted activities of flavonoids and polysaccharides support their use as potential therapeutic mechanism for CCl₄-induced hepatotoxicity injury.

Dendrobium officinale Kimura et Migo is a traditional Chinese medicinal plant species and has been used as an herbal medicine component in numerous Asian countries for hundreds of years. Since the Qing dynasty, Dendrobium officinale Kimura et Migo stems have been used as a traditional Chinese tonic medicine known as “Tiepi Fengdou”. According to records found in both “Ben Cao Gang Mu” and “Pharmacopoeia of the People’s Republic of China”, this plant has been described to provide numerous benefits for human health. The Huoshan Dendrobium officinale Kimura et Migo is scarcity among all of them, and large quantities of polysaccharides and flavonoids were isolated from this plant ^[12]. In this study, we investigate the antioxidant properties of water extracts from Huoshan Dendrobium officinale Kimura et Migo and the hepatoprotective effects these extracts impart on CCl₄-induced hepatotoxicity.

2. Materials and Methods

Huoshan Dendrobium officinale Kimura et Migo was purchased from Sannor Biotechnology in Luan, China. The authenticity of the material was verified by an author of this paper, and further confirmed by a botanist.

Water extract was prepared by adding 20 g of Huoshan Dendrobium officinale Kimura et Migo to 1 L of boiling water. The mixture was then stored at room temperature for 4 hours to enable the infusion process to occur. Following the infusion step, the mixture was filtered through Whatman No.1 filter paper and the extract was dried by Bain-marie. The extract was then stored in a sterile bottle at 4C. The water extract was concentrated under vacuum (-40C) and lyophilized. The extract yield was determined to be 52% (10.4 g of dried extract from an initial 20 g of Huoshan Dendrobium officinale Kimura et Migo.).

Kunming mice weighing 20–30 g were obtained from Xinjiang Medicine University Medical Laboratory Animal Center [License Number: SCXK(xin)2011-0003]. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Institute Pharmaceutical Education and Research.

Mice were randomly divided into five groups (n = 12 mice /group): (1) control group, mice that received distilled water for 5 days, (2) the CCl₄ group with mice that received distilled water for 5 days and were intraperitoneally (i.p.) administered 10 mL/kg body weight CCl₄ diluted with 1:500 corn oil once on day 6 ^[13], (3) the three HDW + CCl₄ groups with mice who were treated with HDW once daily for 5 days (20, 100 or 500 mg/kg/day) followed by a single i.p. dose of CCl₄ (10 ml/kg body weight) on day 6.

Twenty-four hours post-CCl₄ administration, animals were anesthetized ether. A total of 1 ml of blood was collected from each mouse via cardiac puncture. The blood was allowed to clot and was then centrifuged at 4000 g for 10 min. Following centrifugation, the serum was isolated and used to assay for ALT and AST using standard enzyme assay kits (Nanjing Jiancheng Biological Product, Nanjing, China).

SOD activity, MDA levels, GSH levels, and GSSG levels in serum were measured using spectrophotometry according to the ELISA assay instructions (Tsz Biosciences, Greater Boston, USA).

Tumor necrosis factor-α (TNF-α), C-reactive protein (CRP), and interleukin-6 (IL-6) levels in serum were measured using spectrophotometry by an ELISA assay (Tsz Biosciences, Greater Boston, USA) according to the manufacturer’s instructions.

Mice were sacrificed by cervical dislocation. The livers were then excised, washed in phosphate buffer, and dried using tissue paper. Each mouse liver was fixed in 10% formaldehyde and preserved at normal temperature (20°C-25°C). A small piece of hepatic tissue (2 mm × 1 mm × 1 mm) was obtained and fixed in 0.1 mM phosphate buffer (pH 7.2) containing 3% glutaraldehyde and 1.5% paraformaldehyde at 4°C. This piece of hepatic tissue was then further cut into smaller 1 mm³ sized pieces, and subsequently fixed in the abovementioned solution for 4 hours. The piece was then rinsed with phosphate buffer and fixed in 1% osmic acid at 4°C for 1.5 hours. Finally, the tissue was dehydrated using alcohol followed by treatment with dimethylbenzene. The sample was then embedded in epoxy resin 618. The tissue was first located by semi-thin sectioning, and then sliced into ultrathin sections (60 nm). Slides were examined under a microscope (Olympus, Japan) at 100 × magnification in order to observe any histopathological changes.

Data are presented as the mean ± SD from at least three independent experiments. Statistical significance was evaluated using ANOVA followed by Student’s t-test. Statistical significance was considered at p < 0.05. Statistical analysis was performed using SPSS (IBM SPASS, International Business Machines Corporation, Armonk, NY, USA).

3. Results

As shown in Figure 2, CCl₄–induced hepatotoxicity in rats resulted in a marked increase in levels of liver function serum markers. Specifically, ALT (251.61±17.18 U/L) (Figure 1A) and AST (166.21±8.15 U/L) levels were observed to be increased compared to the control group (p < 0.01, Figure 1B). We show that the increased levels of liver function markers observed with CCl₄-induced hepatotoxicity returned to control levels with the ameliorative effect of HDW treatment. Significant hepatoprotective effects were observed in animals treated with HDW at 100 mg/kg (p < 0.05) and 500 mg/kg (p < 0.01), with a dose-dependent effect of HDW observed.

As shown in Table 1, oxidative stress markers in liver homogenates from mice revealed that CCl₄-induced hepatotoxicity resulted in a significant decrease in activity levels of oxidative stress marker enzymes in serum, such as SOD (24.07 ± 2.56 U/mL) and GSH (412.13 ± 20.70 ng/L) in comparison to the toxic control group (SOD, 51.19±4.53 U/mL and GSH, 789.84±18.64 ng/L). In addition, MDA (10.00 ± 0.35 nmol/mL) and GSSG (9.67 ± 0.22 nmol/mL) levels was observed to be significantly increased in CCl₄-intoxicated mice compared to control animals.

Download as

• PPT
  PowerPoint Slide
• PNG
  Larger image(png format)

Veiw figureFigures index

•  NEW
  View larger figure in new window

View next figure

Figure 1. Prophylactic effect of HDW on the restoration of liver function markers in CCl₄-intoxicated mice. Values are expressed as mean ± SD (n = 8/group). ^#p < 0.05 and ^##p < 0.01 compared to the normal control group, *p < 0.05 and **p < 0.01 compared to the CCl₄ treatment group

Download as

• PPT
  PowerPoint Slide
• PNG
  Larger image(png format)

Veiw figureFigures index

•  NEW
  View larger figure in new window
• PREV
  View previous figure

View next figure

Figure 2. Effect of HDW on the levels of IL-6, TNF-α and CRP in mice subjected to CCl4-induced hepatotoxicity (values are presented as mean ± SD, n = 8). ^#p < 0.05 and ^##p < 0.01 compared to the normal control group, *p < 0.05 and **p < 0.01 compared to the CCl₄ treatment group

Table 1. The effect of HDW pretreatment on oxidative stress parameters of mice in CCl₄-induced hepatotoxicity, Values are expressed as mean ± SD (n = 8/group)

Download as

PowerPoint Slide

Larger image(png format)

• Tables index

Veiw figure

View current table in a new window

Compared with CCl₄ treatment group, the group pretreated with HDW (100 or 500 mg/kg) showed a significant ameliorative effect with a recovery observed in both SOD (32.78 ± 3.34 U/mL and 43.49 ± 2.78 U/mL) and GSH (535.91 ± 79.25 ng/L and 659.10 ± 25.21 ng/L) levels (p < 0.01). In addition, increased levels of MDA and GSSG in the CCl4-treatment group were observed to be lowered closer to control levels in the case of animals which received a 500 mg/kg HDW pretreatment (7.8 ± 0.46 nmol/mL MDA and 7.73 ± 0.61 nmol/mL GSSG). Finally, in animals that received a 100 or 500 mg/kg HDW pretreatment, levels of oxidative stress marker enzymes were observed to be restored to levels close to that of control animals.

Download as

• PPT
  PowerPoint Slide
• PNG
  Larger image(png format)

Veiw figureFigures index

•  NEW
  View larger figure in new window
• PREV
  View previous figure

Figure 3. Histopathology of liver tissue. (A) Liver section of a normal control mouse showing normal architecture; (B) Liver section of a CCl₄-treated mouse showing a massive presence of inflammatory cells and cellular necrosis; (C) Liver section of a mouse treated with CCl₄ and 20 mg/kg HDW showing a massive presence of inflammatory cells and cellular necrosis; (D) Liver section of a mouse treated with CCl₄ and 100 mg/kg HDW showing mild necrosis and mild presence of inflammatory cells; (E) Liver section of a mouse treated with CCl₄ and 500 mg/kg HDW showing an absence of inflammatory cells and an absence of necrosis

Inflammation is a key mechanism which underlies hepatotoxic injury. We measured levels of inflammatory cytokines in serum (IL-6, CRP and TNF-α) associated with hepatotoxic injury in order to identify potential mechanisms underlying the hepatoprotective activity of HDW. We observed significantly lower levels of IL-6 in animals pretreated with HDW at 100 mg/kg (98.55 ± 1.08 pg/mL) (p < 0.05) and 500 mg/kg (87.60 ± 1.26 pg/mL) (p < 0.01) compared to CCl₄-treated animals (115.95 ± 8.21 pg/mL) (Figure 2A). CRP levels were also observed to be significantly decreased in animals pretreated with 100 mg/kg HDW (1983.15 ± 128.33 μg/L) (p < 0.05) and 500 mg/kg HDW (1073.15 ± 91.46 μg/L) (p < 0.01) compared to that of the I/R group (2284.95 ± 186.55 µg/L) (Fig. 2B). TNF-α activity was also observed to be significantly decreased in animals pretreated with HDW at 100 mg/kg (396.63 ± 26.50 ng/L) and 500 mg/kg (321.63 ± 18.03 ng/L) compared to that of the CCl₄ group (496.83 ± 24.93 ng/L) (p < 0.05, p < 0.01) (Figure 2C).

The histological profile of liver sections from control animals showed normal hepatic architecture consisting of a well-preserved cytoplasm, a prominent nucleus, a central vein, and a compact arrangement of hepatocytes with no fatty lobulation (Figure 3A). In contrast, we observed hydropic changes in centrilobular hepatocytes and cell necrosis surrounded by neutrophils in liver sections from CCl₄-treated animals. In addition, congestion of the central vein and sinusoids was observed along with infiltration of inflammatory cells within sinusoids mainly in the central zone (Figure 3B). Liver sections of mice that were administered 20 mg/kg HDW appeared similar to sections from CCl₄-treated animals, with hydropic changes in centrilobular hepatocytes, cell necrosis surrounded by neutrophils, and inflammatory cells infiltrating sinusoids mainly in the central zone (Figure 3C). However, in the case of liver sections from mice that were administered 100 mg/kg HDW, we observed only mild necrosis and a mild presence of inflammatory cells (Figure 3D). Animals that were administered with 500 mg/kg HDW were observed to exhibit significant liver protection against CCl₄-induced liver damage. This was evident by the presence of hepatic cords and the absence of both inflammatory cells and necrosis (Figure 3E).

4. Discussion

In this study, the hepatoprotective effects of HDW were examined in a mouse model of CCl₄-induced liver toxicity. We show that HDW treatment results in a suppression of the CCl₄-induced increase in MDA, IL-6, CRP, GSSG and TNF-α levels, and a concomitant decrease in SOD activity and GSH levels attributed with the CCl4-induced hepatotoxicity model. Thus, the hepatoprotective effects of HDW could be attributed to its antioxidant and anti-inflammatory properties.

CCl₄ is a well established hepatotoxin known to induce toxic liver injury. This system is used widely to study the cellular mechanisms that underlie oxidative damage in animal systems ^[14]. Carbon tetrachloride is metabolized by cytochrome P4502E1 (CYP2E1) to form trichloromethyl (CCl₃∙) and trichloromethyl peroxy (CCl₃OO∙) radicals that are largely associated with CCl₄-induced hepatic damage ^[15]. These radicals bind covalently to macromolecules within the cell, with a known preference towards polyunsaturated fatty acids (PUFA) within the cell membrane. This leads to the generation of fatty acid free radicals, known to initiate autocatalytic lipid peroxidation which ultimately causes a loss in membrane integrity and the leakage of microsomal enzymes. This process is characterized by elevated levels of serum marker enzymes, such as AST and ALT, following administration of CCl₄ in animals ^{[16, 17]}. Here, we show that levels of serum marker enzymes (AST and ALT) are significantly elevated in animals treated with CCl₄. The administration of HDW was observed to reduce the toxic effect of CCl₄ by restoring the serum marker enzyme levels to that of control levels.

ROS generation has been described to be one of the major factors involved in liver toxicity ^[18]. The antioxidant-like molecules, SOD and GSH, play important roles as defense mechanisms against the damaging effects of reactive oxygen species (ROS) and free radicals in biological systems ^[19]. We observed increased levels of MDA and GSSG in liver tissue homogenate of CCl₄-treated mice treated in our study. These increased levels of MDA and GSSG are thought to reflect lipid peroxidation and plasma membrane damage caused by oxidative stress ^{[19, 20]}. Interestingly, we show that HDW treatment in these systems caused SOD and GSH levels to increase back to levels similar to that of control animals, while MDA and GSSG levels were observed to decrease back to levels similar to control animals.

A histopathological profile of a CCl₄-treated mouse liver demonstrated necrosis and an infiltration of inflammatory mediators. Animals treated with HDW showed a significant improvement of CCl₄-induced liver injury, as demonstrated by the presence of normal hepatic cords and an absence of necrosis. In the present study, we found that CCl₄-induced liver injury resulted in an increase in CRP, IL-6, and TNF-α levels in serum. However, HDW treatment was found to reduce the levels of these cytokines. Therefore, we suggest that the suppressed infiltration of inflammatory cytokines with HDW treatment could contribute to its hepatoprotective properties.

5. Conclusion

This study provides evidence that HDW possesses hepatoprotective properties that act to protect against hepatic damage induced by carbontetrachloride. The hepatoprotective effects of HDW may be attributed to its antioxidant and anti-inflammatory properties.

Acknowledgments

This study was supported by the fund of Binzhou Medical University (BY2014KYQD01), the Xinjiang Production and Construction Corps Funds for innovation team in key areas (2015BD005) to Q.S. Zheng. This study was supported by the Dominant Disciplines' Talent Team Development Scheme of Higher Education of Shandong Province.

Conflict of Interests

The authors declare that they have no financial conflict of interests.

References

[1]	Rabinowich L, Shibolet O, Drug Induced Steatohepatitis: An Uncommon Culprit of a Common Disease. Biomed Res Int, 2015.
	In article
[2]	Suzek H, Celik I, Dogan A, Yildirim S, Protective effect and antioxidant role of sweetgum (Liquidambar orientalis) oil against carbon tetrachloride-induced hepatotoxicity and oxidative stress in rats. Pharm Biol, 54(3). 451-7. 2016.
	In article
[3]	Liang D, Zhou Q, Gong W, Wang Y, Nie Z, He H, Li J, Wu J, Wu C, and Zhang J, Studies on the antioxidant and hepatoprotective activities of polysaccharides from Talinum triangulare. J Ethnopharmacol, 136(2). 316-21. 2011.
	In article
[4]	Abdou RH, Saleh SY, and Khalil WF.-, Toxicological and biochemical studies on Schinus terebinthifolius concerning its curative and hepatoprotective effects against carbon tetrachloride-induced liver injury. Pharmacogn Mag, 11(Suppl 1), S93-S101. 2015.
	In article
[5]	Abenavoli L, Peta V, and Milic N, Lifestyle changes associated with a new antioxidant formulation in non-alcoholic fatty liver disease: a case series. nn Hepatol, 14(1), 121-6. 2015.
	In article
[6]	Pisonero-Vaquero S, Gonzalez-Gallego J, Sanchez-Campos S, and Garcia-Mediavilla MV, Flavonoids and Related Compounds in Non-Alcoholic Fatty Liver Disease Therapy. Curr Med Chem, 22(25), 2991-3012. 2015.
	In article
[7]	Zhao X, Qian Y, Li GJ, and Tan J, Preventive effects of the polysaccharide of Larimichthys crocea swim bladder on carbon tetrachloride (CCl4)-induced hepatic damage. Chin J Nat Med, 13(7), 521-528. 2015.
	In article
[8]	Akhlaghi M, and Bandy B, Mechanisms of flavonoid protection against myocardial ischemia-reperfusion injury. J Mol Cell Cardiol, 46(3), 309-17. 2009.
	In article
[9]	Khaskheli SG, Zheng W, Sheikh SA, Khaskheli AA, Liu Y, Soomro AH, Feng X, Sauer MB, Wang YF, and Huang W, Characterization of Auricularia auricula polysaccharides and its antioxidant properties in fresh and pickled product. Int J Biol Macromol, 81, 387-395. 2015.
	In article
[10]	Yang J, Yang G, Hou G, Liu Q, Hu W, Zhao PU, and He YI, Scutellaria barbata D. Don polysaccharides inhibit the growth of Calu-3 xenograft tumors via suppression of the HER2 pathway and angiogenesis. Oncol Lett, 9(6), 2721-2725. 2015.
	In article
[11]	Huang Y, Zhou LS, Yan L, Ren J, Zhou DX, and Li SS, Alpinetin inhibits lipopolysaccharide-induced acute kidney injury in mice. Int Immunopharmacol, 28(2), 1003-1008. 2015.
	In article
[12]	Li X, Ding X, Chu B, Zhou Q, Ding G, and Gu S, Genetic diversity analysis and conservation of the endangered Chinese endemic herb Dendrobium officinale Kimura et Migo (Orchidaceae) based on AFLP. Genetica, 133(2), 159-166. 2008.
	In article
[13]	Niu L, Cui X, Qi Y, Xie D, Wu Q, Chen X, Ge J, and Liu Z, Involvement of TGF-β1/Smad3 Signaling in Carbon Tetrachloride-Induced Acute Liver Injury in Mice. PLoS One, 11(5), e0156090. 2016.
	In article
[14]	Girish C, and Pradhan SC, Hepatoprotective activities of picroliv, curcumin, and ellagic acid compared to silymarin on carbon-tetrachloride-induced liver toxicity in mice. J Pharmacol Pharmacother, 3(2), 149-155. 2012.
	In article
[15]	Augustin W, Wiswedel I, Noack H, Reinheckel T, and Reichelt O, Role of endogenous and exogenous antioxidants in the defence against functional damage and lipid peroxidation in rat liver mitochondria. Mol Cell Biochem, 174(1-2), 199-205. 1997.
	In article
[16]	Krishnappa P, Venkatarangaiah K, Venkatesh, Shivamogga Rajanna SK, and Kashi Prakash Gupta R, Antioxidant and prophylactic effects of Delonix elata L., stem bark extracts, and flavonoid isolated quercetin against carbon tetrachloride-induced hepatotoxicity in rats. Biomed Res Int, 2014.
	In article
[17]	Yu Z, Sun W, Peng W, Yu R, Li G, and Jiang T, Pharmacokinetics in Vitro and in Vivo of Two Novel Prodrugs of Oleanolic Acid in Rats and Its Hepatoprotective Effects against Liver Injury Induced by CCl4. Mol Pharm, 13(5), 1699-1710. 2016.
	In article
[18]	Faedmaleki F, Shirazi FH, Ejtemaeimehr S, Anjarani S, Salarian AA, Ahmadi Ashtiani H, and Rastegar H, Study of Silymarin and Vitamin E Protective Effects on Silver Nanoparticle Toxicity on Mice Liver Primary Cell Culture. Acta Med Iran, 54(2), 85-95. 2016.
	In article
[19]	Zhang JQ, Shi L, Xu XN, Huang SC, Lu B, Ji LL, and Wang ZT, Therapeutic detoxification of quercetin against carbon tetrachloride-induced acute liver injury in mice and its mechanism. J Zhejiang Univ Sci B, 15(12), 1039-1047. 2014.
	In article
[20]	Ray S, Murmu N, Adhikari J, Bhattacharyya S, Adhikari S, and Banerjee S, Inhibition of Hep G2 hepatic cancer cell growth and CCl4 induced liver cytotoxicity in Swiss albino mice by Mahua extract. J Environ Pathol Toxicol Oncol, 33(4), 295-314. 2014.
	In article