Study on the synergistic protective effect of Lycium barbarum L. polysaccharides and zinc sulfate on chronic alcoholic liver injury in rats

Abstract Both Lycium barbarum L. polysaccharides (LBP) and zinc have protective effects on liver injuries. In this paper, LBP and ZnSO4 were combined to study the effects on the prevention of alcoholic liver injury. The rats were divided into six groups, the normal group, alcohol group, zinc sulfate group, LBP group, low‐dose group of ZnSO4, and high‐dose group of ZnSO4 and LBP, used to explore the impact of LBP and ZnSO4 complex on liver lipid metabolism of alcohol, alcohol‐metabolizing enzymes, oxidative damage, and inflammation of the liver. The experimental model was established by gavage treatment, observation, and determination of indexes of rats. The results showed that the combination of LBP and ZnSO4 could significantly decrease the levels of triglyceride (TG), total cholesterol (TC), tumor necrosis factor‐α(TNF‐ɑ), malondialdehyde (MDA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and the activity of enzyme subtype 2E1 (CYP2E1). It also significantly increased the activities of total superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH‐Px), glutathione peptide (GSH), and alcohol dehydrogenase, effectively improved the liver tissue lesion. What is more, the combination of LBP and ZnSO4 had a synergistic effect on the remission of alcoholic fatty liver, and alleviated chronic alcoholic liver injury by promoting lipid metabolism, inhibiting oxidative stress, controlling inflammatory responses, and regulating the expression and activity of alcohol‐metabolizing enzymes in rats.

Moreover, alcohol consumption increases a small intestinal bacterial overgrowth and intestinal permeability of endotoxins. The endotoxin-mediated inflammatory signaling plays a major role in alcoholic liver fibrosis (Altamirano & Bataller, 2011). However, no treatment has been approved for patients with ALD yet, and the only recognized management strategies were alcohol cessation (Orman et al., 2013); therefore, development of novel pathophysiological-targeted adjuvant therapies are urgently needed (Ghorbani, Hajizadeh, & Hekmatdoost, 2016).
Lycium barbarum L. is a traditional Chinese geoherbalism medicine (Bartosz & Anna, 2016), which can nourish liver, improve eyesight, and exhibit protective effects for liver function as recorded by the Compendium of Materia Medica. Modern medicine shows that Lycium barbarum L. is rich in polysaccharides, which are natural antioxidant and a hepatoprotective derivative (Masci et al., 2018). Gan et al. (2018) showed that an alleviating effect of LBPs on CCl 4 -induced liver fibrosis in Wistar rats may be through inhibiting the TLRs/ NF-κB signaling pathway expression. Cheng Daye and Kong Hong showed LBP administration protected liver cells from the damage induced by ethanol (Cheng & Kong, 2011).
Zinc plays an important role in maintaining the stability of antioxidant enzymes and scavenging oxygen-free radicals. It also plays a protective role in alcoholic liver injury (McClain, Vatsalya, & Cave, 2017).
Approximately 30%-50% of individuals with alcohol dependency have a low zinc status because alcohol consumption decreases intestinal absorption of zinc and increases urinary excretion of zinc (Skalny, Skalnaya, Grabeklis, Skalnaya, & Tinkov, 2017). Zinc deficiency may also give rise to oxidative stress. Increased oxidative stress and oxidative stress-induced damage have been observed in humans with a suboptimal zinc intake (Rajapakse, Curtis, & Chen, &Xu, 2017). A significant increase in the MDA levels and decrease in the GSH content and SOD activity were observed in the liver of rats fed on a zinc-deficient diet; however, zinc supplementation resulted in a decrease in the MDA levels and increase in GSH content and SOD activity (Tupe, Tupe, & Agte, 2011;Tupe, Tupe, Tarwadi, & Agte, 2010). Additionally, zinc deficiency is linked to alcohol-induced intestinal barrier dysfunction, as well as alveolar epithelial cell and macrophage dysfunction (Lenz et al., 2013;Zhong, Zhao, McClain, Kang, & Zhou, 2010).
As both LBP and Zn are potent antioxidants and could potentially help to protect the alcohol liver injury, we aimed to explore whether LBP-ZnSO 4 has a synergistic effect in alleviating the detrimental alterations induced by ethanol in rats, such as the imbalance between oxidation and antioxidants, liver injury, and abnormal hemorheology.

| Materials
SD rats were purchased from the Experimental Animal Center of Xi'an Jiaotong University, license number SCXK (Shanxi) 2013-001. LBP was purchased from Zhejiang Genk Pharmaceutical Co., Ltd.. Food grade zinc sulfate was purchased from Shanxi Parnir Biotechnology Co., Ltd..

| Animal treatment and modeling
Thirty-six female SD rats of clean grade, weighing about 150 g were put into cages, maintained in a specific pathogen-free environment (25 ± 4°C, 60% ~ 70% relative humidity, 12 hr light alternated), with 1 week of adaptive feeding and then divided into six groups ran- normal saline (0.5 ml 100 g/day) during the experiment, meanwhile, the medicine treatment groups (group C, D, E, and F) were given gavage (0.5 ml/100 g B.W./day) with ZnSO 4 or LBP or different dosage of ZnSO 4 and LBP compound solution to rats once a day, as shown in Table 1. After 1 hr, all the groups except the normal group were treated with 56% alcohol (V/V), and the first week with the dose 2 g kg −1 day −1 , then increased to 8 g kg −1 day −1 for 8 weeks.

| Rats specimens collection
All rats were weighed daily and killed at the end of 9 weeks. Blood samples were centrifuged and collected at 2,000 r/min at 4°C for 15 min to obtain serum. Livers were totally excised from the rats and stored at −80°C for the subsequent experiments.

| Body weight and liver coefficient
SD rats were weighed by everyday during experiment period. The liver coefficient was calculated as follows: where W 1 and W 0 are wet liver weight and body weight, respectively.

| Liver histopathology studies
Rat livers from all groups were removed and fixed immediately in 4% neutral buffered formalin, dehydrated in gradual ethanol (30%-100%), cleaned in xylene, and embedded in paraffin. Sections were prepared and stained with hematoxylin and eosin (H&E) for photomicroscopic observation.

| Measurement of lipid levels in serum
TG and TC were measured by a phosphoglycerate oxidase-PAP (GPO-PAP) enzyme kit and cholesterol oxidase-PAP (CHOD-PAP) enzymatic kit, respectively.

| Oxidative stress parameters and inflammatory factors in rat livers
The levels of Serum transaminase (ALT and AST), MDA, SOD, CAT, GSH-Px, GSH, and tumor necrosis factor-α(TNF-ɑ) were allied using the kits of Nanjing Jiancheng Bioengineering Institute.

| Determination of the activity and the contents of ADH and CYP2E1 in rat liver
The activity of ADH and CYP2E1 were analyzed by the double antibody sandwich method using commercially available kits (Shanghai Xin Le Biotechnology Co., Ltd.).

| Analysis of the interaction between LBP and zinc sulfate
The synergistic effect of the drug combination was analyzed using the drug interaction coefficient (CDI), which was calculated as follows (Liu et al., 2015): N AB is the ratio of the corresponding parameters in ZnSO 4 and LBP complex group to alcohol group; N A or N B is the ratio of these parameters in the single group to the alcohol group. If CDI < 1, it means that there is a synergic effect between the two drugs; CDI = 1 means that there is an additive effect between the two drugs; CDI > 1 means that there is an antagonistic effect between the two drugs.

| Statistical analysis
All statistical analysis was carried out using SPSS 21.0 software (SPSS). All data were expressed as mean ± SD, Duncan's multiple comparison (DMRT) was used to analyze the significant difference.
A value of p < .05 was considered to be statistically significant.

| Effect of LBP and ZnSO 4 on body weight (BW) and liver coefficient in experimental rats
The body weight in experimental rats was shown in Table 2. At the start of the experiment there was no significant difference in body weight between groups (p > .05), and then increased in varying de- (1) Compared the normal model group, the liver coefficient was increased significantly (p < .05) in alcohol group as shown in Figure 1; compared the alcohol group, the liver coefficient of group C, E, F are decreased significantly (p < .05) except group D, it indicated that compound of LBP and Zn can make the liver coefficient return to a normal level.

| Effect of LBP and ZnSO 4 on serum lipid metabolism in experimental rats
As shown in Figure 2, the levels of TG and TC in rat serum in the alcohol group (B) were significantly increased (p < .05) after liver injury induced by alcohol gavage, which were 1.2 times and 2 times the normal model group (A), indicating that alcohol gavage led to the liver lipid metabolism disorder. Compared with the group B, the decrease of TG in the LBP group (D) was not significant, and the decrease of TC in the ZnSO 4 group (C) was not significant (p > .05).
However, the reduction of TG and TC in group E and F had a significant difference (p < .05) correspondingly, and decreased by 25% and 60%, respectively.

| Effect of LBP and ZnSO 4 on serum transaminase levels in liver of rats
Oxidative stress is one of the important mechanisms leading to liver damage (Ana, Alma, Vázquez, Natalia, & Javier, 2016;Gao et al., 2017). Many investigations strongly suggest that liver damage produced by alcohol is mediated through oxidative stress (Albano, 2008;Ambade & Mandrekar, 2012).

| Effect of LBP and ZnSO 4 on antioxidant levels in liver of rats
As shown in Table 3, compared with group A, the SOD, CAT, GSH-Px, GSH levels were significantly decreased (p < .05) in group B, but compared with group B, these antioxidant indicators in the group C, E and F were increased significantly (p < .05)inversely. The results show that the effect of LBP and Zn on oxidative damage of alcoholic liver were different, the synergistic effect of LBP and Zn is not a simple dose-relationship.
F I G U R E 1 Effect of Complex solution on liver index in alcoholinduced chronic hepatic injury. Note: The data are presented as the mean ± SD (n ≥ 3); # means compared to normal model group, p < .05; * means compared to alcohol group, p < .05 F I G U R E 2 Effects of complexes on serum total triacylglycerol and total cholesterol levels. The data are presented as the mean ± SD (n ≥ 3), # means compared to control group (A), p < .05; * means compared to the alcohol group (B), p < .05 F I G U R E 3 Effects of complexes on serum total triacylglycerol and total cholesterol levels. The data are presented as the mean ± SD (n ≥ 3), # means compared to control group (A), p < .05; * means compared to the alcohol group (B), p < .05

| Effect of LBP and ZnSO 4 on serum inflammatory factor and lipid peroxidation in liver of rats
As shown in Figure 4, compared with the group A, the TNF-α and MDA level were increased significantly (p < .05) in group B. However, compared with the group B, the TNF-α levels in the groups C and D showed no significant difference (p > .05), the groups E and F could significantly reduce the level of TNF-α in serum (p < .05), while MDA level was decreased significantly (p < .05) in group C, D, E and F. The results showed that the combination of ZnSO 4 and LBP had a synergistic effect on relieving lipid peroxide and chronic inflammation in rats.

| Effect of ZnSO 4 /LBP on alcohol metabolism enzymes in liver of rats
As shown in Figure 5a, compared with the group A, the activity of ADH decreased significantly (p < .05) in group B, compared with the group B, groups were increased significantly (p < .05) in group C, D and E, while the content of ADH could be improved only in the group E (p < .05) compared with group B.
However, for the CYP2E1, as shown in Figure 5b, compared with the group A, both expression content and activity were significantly improved in group B (p < .05), which reduced in intervention groups (C, D, E, F) compared with the group B. Genetic polymorphism of enzymes involved in alcohol metabolism plays a relevant role in etiopathogenesis of alcohol disease and alcohol liver cirrhosis (Caro & Cederbaum, 2007;Cichoz, Partycka, Nesina, Celiński, & Saomka, 2006). The results showed that the complex of ZnSO 4 and LBP could improve ADH activity to inhibit the production of acetaldehyde, on the contrary, reduce CYP2E1 activity, which could decrease alcoholderived reactive oxygen species (ROS).

| Interaction between ZnSO 4 and LBP on alcohol liver injury of rats
The pathophysiological process of alcohol-induced liver injury is a complex process involving multiple factors. As shown in Table 4, in the group E, the CDI values of every index were ≤1, except CAT and GSH. While in the group F, the CDI values of ALT, AST, CAT, GSH were ≥1. These results showed that LBP and ZnSO 4 have a synergistic effect on alcohol liver injury of rats, but was not a simple dose-relationship.

| Histopathological analysis
Liver biopsies are useful to evaluate the stage and severity of ALD.
As shown in Figure 6, in the normal model group (A), the liver tissue structure of rats was complete and clear, the hepatic lobule structure is normal, the arrangement of the liver cells was a regular cord-like shape, distributed radially around the central vein. Central venous endothelial was integrity, the liver cells were arranged in neat rows and closely, the cells structure was integrity, and the nuclei were

| D ISCUSS I ON
Alcohol-induced liver injury has an impact on the body's nutritional status (Campillo, Bories, Pornin, & Devanlay, 1997), and different alcohols have different impacts on body weight (Wang, Chen, Hu, Nan, & Chen, 2014). LBP exhibited the most significant treatment in reducing the body weight loss in rats, the body weight of LBP group was almost close to the normal model group, which could be related to the immune enhancement of LBP to the body (Zhang et al., 2014).
However, there is no similar result in Daye Cheng's study (Cheng & Kong, 2011), the reason may be that animals of different genders and genotypes have different responses to the alcohol model (Gao et al., 2017).
It was showed that ZnSO 4 and LBP can be more synergistic to repair a damaged lipid metabolism or reverse the lipid dysfunction caused by alcohol administration in rats and prevent alcohol-induced fatty liver deteriorate in this study, which similar to their search that both LBP and Zn could reduce high fat-induced liver damage and significantly reduce lipid accumulation (Cheng & Kong, 2011;Gan et al., 2018;Masci et al., 2018;McClain et al., 2017). Previous studies have shown that the mechanisms of ethanol impair oxidative balance within hepatic cells is complicated, autoimmune reactions associated with oxidative stress might contribute to fueling hepatic inflammation in ALD (Vidali, Stewart, & Albano, 2008); signaling intermediates regulated by oxidative stress that provokes proinflammatory responses in alcoholic liver disease (Ana et al., 2016);chronic ethanol-associated alterations of mitochondria influenced the production of reactive oxygen and nitrogen species, which disrupted hepatic energy conservation in the chronic alcohol abuser (Bailey, 2003). The results of Table 2 showed that the effect of LBP and Zn on oxidative damage of alcoholic liver were different, the synergistic effect of LBP and Zn is not a simple dose-relationship. Thus, the mechanisms of LBP and Zn on preventing oxidative damage of alcoholic liver should be research in-depth in the future.
Genetic polymorphism of enzymes involved in alcohol metabolism plays a relevant role in etiopathogenesis of alcohol disease and alcohol liver cirrhosis (Caro & Cederbaum, 2007;Cichoz et al., 2006).
It was shown that the complex of ZnSO 4 and LBP could improve the activity and of ADH, reduces the activity and expression of CYP2E1, which indicated that a synergistic effects of ZnSO 4 and LBP on the regulation of alcohol metabolism enzymes, although the details of the synergistic mechanisms were still unclear.

| CON CLUS IONS
In conclusion, compared with the alcohol group, the complex of LBP and ZnSO 4 could significantly decrease the levels of TG, TC, TNF-α, MDA, ALT, AST and the activity of CYP2E1 in rats, which suffered chronic alcoholic liver injury, on the contrary, increased the activity of SOD, CAT, GSH-PX, GSH and activity of ADH, effectively alleviate the liver tissue lesion. It was the combination of LBP and ZnSO 4 that had a synergistic effect on the remission of alcoholic fatty liver in rats, but they are not a simple dose-relationship. Ingestion of LBP and ZnSO 4 could alleviate chronic alcoholic liver injury in rats by promoting lipid metabolism, inhibiting oxidative stress, controlling inflammatory responses, and regulating expression and activity of alcohol-metabolizing enzymes. These results implied that LBP and zinc complexes may be applied for the treatment of ALD. However, clinical trials are needed to validate the beneficiary role of these supplements in patients with ALD, and further study is warranted.

ACK N OWLED G M ENTS
This research was funded by National Natural Science Foundation

CO N FLI C T O F I NTE R E S T
This study has not any potential sources of conflict of interest.

E TH I C A L A PPROVA L
All animals were housed and cared for in accordance with the Chinese Pharmacological Society Guidelines for Animal Use.