The protective effect of propolis against aluminum chloride-induced hepatorenal toxicity in albino rats

Background: The use of natural products is an essential way to new pharmaceutical leads for the discovery and development of new drugs to treat diseases. Propolis (Pro) is a natural resinous product produced by honey bees. It has a strong cytoprotective effect against various exogenous toxic agents. The current study was designed to evaluate the possible protective effect of propolis against the toxicity of aluminum chloride (AlCl3) on hepatorenal structure and function in male white albino rats. Methods: Thirty mature males of albino rat, Rattus rattus, weighing about 80-90g were divided into five groups contained 6 rats each. The first group acts as a control (received only saline solution); the second group (Al) had given orally 40 mg/kg b.w. of AlC13, the third group (Pro) had administrated orally 150 mg/kg b.w. of propolis and the fourth group (Al+Pro) had given 40 mg/kg b.w. of AlCl3 in the morning and 150 mg/kg b.w. of propolis in the evening. These four groups had given the treatments for two months. The fifth group (Al-Pro) had given 40 mg/kg b.w. of AlC13 chloride for one month then had given 40 mg/kg b.w. of AlCl3 combined with 150 mg/kg b.w. of propolis for another month. Results: The AlCl3-treated group showed a significant increase in the activities of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), acid phosphatase (AP), and lactate dehydrogenase (LDH) in the plasma. Also, glucose, total protein, albumin, creatinine, uric acid, urea, cholesterol, and triglycerides in the plasma were significantly increased. The histological examination for the liver and kidney sections revealed marked histopathological alternations. The treatment with propolis combined with AlCl3 improved the previous mentioned biochemical and histological alterations induced by AlCl3. Conclusion: It can be concluded that the combination of propolis with AlCl3 alleviated the toxic effects of AlCl3. The propolis has protective influences on the hepatorenal structure and function and could be able to resist AlCl3 intoxication.

The use of natural products is an important way to new pharmaceutical leads for the discovery and development of new drugs for treating diseases (Watanabe, Amarante, Conti, & Sforcin, 2011). Propolis, bee glue, is a natural resinous product produced by honey bees from substances gathered from exudates and buds of plants. It has a strong cytoprotective effect against various exogenous harmful and toxic agents (Rizk, Zaki, & Mina, 2014). The chemical compositions of propolis are beneficial to bees and also have an important pharmacological value as a natural mixture (Oršolić, 2010). Currently, propolis is used in medical and dental sciences based on its chemical composition and its therapeutic properties (Elmenoufy, 2012). Essentially, it is widely used as a component in cosmetic and pharmaceutical products such as facial and body creams, ointments and lotions, antiacne creams, and several formulations for oral hygiene (Castaldo & Capasso, 2002).

Experimental animals
Males of albino rats, Rattus rattus, were obtained from the animal house of Assiut University, Egypt. For this study, animals were reserved in cages at room temperature for a period of 2 weeks to reach their optimal conditions of weight and maturity. All animal experimental protocols were approved by the Committee of Scientific Ethics at Sohag University and were carried out in accordance with its guidelines for animal use.

Drugs
Aluminum chloride (AlCl 3 ) (Merk), propolis was obtained as crude from Faculty of Agriculture, Sohag University, Egypt. A water-soluble derivative of propolis (WSDP) was prepared according to a method described by Orsolic, Sver, Terzic, and Basic (2005). Briefly, it was extracted with 96% ethanol, and then, extract was filtered and left to dry. The resultant resinous product was added to a stirred solution of 8% L-lysine (sigma) and freeze-dried to yield WSDP (a yellow-brownish powder). WSDP was stored at -20°C until use. The WSDP was dissolved in distilled water and was given to rat orally at a dose of 150 mg/kg b.w. (Orsolic et al., 2005).

Animal grouping
Thirty mature males of albino rat, Rattus rattus, weighing about 80-90 g were divided into five groups contained 6 rats each. The first group acts as a control (received only saline solution); the second group (Al) had given orally 40 mg/kg b.w. of AlC1 3 It has been reported that the LD 50 of AlCl 3 was 400 mg/kg b.w. (Krasovskiĭ, Vasukovich, & Chariev, 1979;Yousef, 2004). The third group (Pro) had administrated orally 150 mg/kg b.w. of propolis, and the fourth group (Al + Pro) had given 40 mg/kg b.w. of AlCl 3 in the morning and 150 mg/kg b.w. of propolis in the evening. These four groups had given the treatments for 2 months. The fifth group (Al − Pro) had given 40 mg/kg b.w. of AlC1 3 chloride for 1 month then had given 40 mg/ kg b.w. of AlCl 3 combined with 150 mg/kg b.w. of propolis for another month.

Biochemical analysis
After 2 months of treatments, the animals of each group were scarified, and the collected blood was putted in a tube containing anticoagulant. It was centrifuged at 3000 rpm for 20 min to obtain plasma. The obtained plasma was stored frozen at -20°C until use.
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined as described by Reitman & Frankel, 1957. Alkaline phosphatase (ALP) activity was measured by the method of Bablok, Passing, Bender, and Schneider (1988). Acid phosphatase (AP) activity was measured spectrophotometry at 405 nm by Moss, 1984. Lactate dehydrogenase (LDH) was performed as described by Cabaud, Wróblewski, ok, and Ruggiero (1958). Plasma glucose level was measured as described by Hyvarinen and Nikkila (1962). The estimation of total protein was carried out by the method of Henry (1964). The method of Doumas and Biggs (1976) was used for the estimation of albumin. Plasma urea, uric acid, and creatinine concentrations were measured by the methods of Patton and Crouch (1977), Barham and Trinder (1972), and Weissman, Pileggi, Henry, Cannon, and Winkelman (1974), respectively. The estimation of cholesterol in plasma was carried out according to the method of Watson (1960). The estimation of triglycerides was carried out according to the method of Bablok et al. (1988).

Histological examination
After 2 months of treatments, the liver and kidney of different animal groups were prepared for histological examination through the routine technique according to the method of Bancroft and Gamble (2008) and stained with couple stain haematoxalin (H) and eosin (E). The slides were examined and photographed as required.

Statistical analysis
Results were presented as means ± SE. The student's ttest was used for comparison of different experimental animal groups and control ones, and the results were considered significant at P < 0.05.

Biochemical results
The influences of AlC1 3 (40 mg/kg b.w.), propolis (150 mg/ kg b.w.), propolis combined with AlC1 3 , and the administration of AlC1 3 for 1 month followed by administration of AlC1 3 combined with propolis for another month on the plasma level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), acid phosphatase (AP), and lactate dehydrogenase (LDH) in the albino rats were evaluated. The AlCl 3 treatment led to a highly significant increase (P < 0.001) in plasma level of ALT, AST, ALP, AP, and LDH relative to that of control. Propolis and AlCl 3 combined with propolis groups had nonsignificant effect (P > 0.05) on the plasma level of the previous mention parameters relative to that of the control. The administration of AlCl 3 for month followed by administration of AlCl 3 combined with >propolis for another month led to non-significant effect (P > 0.05) on the plasma level of AST and LDH, and a significant increase (P > 0.05) on the plasma level of ALT, ALP, and AP relative to that of control. In comparison with the effect of AlCl 3 , the treatments with propolis, AlCl 3 combined with propolis, and the administration of AlCl 3 for month and combined with propolis for another month led to highly significant decrease (P < 0.001) in the levels of ALT, AST, ALP, AP, and LDH in the plasma (Table 1, Fig. 1). Table 1 The effect of AlC1 3 chloride (40 mg/kg b.w.), propolis (150 mg/kg b.w.), AlC1 3 combined with propolis and AlC1 3 choloride for one month followed by administration of AlCl 3 combined with propolis for another month on the biochemical parameters in plasma Also, the treatment with AlCl 3 led to a highly significant increase (P < 0.001) on the plasma level of glucose and albumin while it led to a significant increase (P < 0.05) on the plasma level of total protein relative to that of the control. Propolis, AlCl 3 combined with propolis, and the administration of AlCl 3 for 1 month followed by administration of AlCl 3 combined with propolis for another month had non-significant effect (P > 0.05) on the plasma level of glucose, total proteins, and albumin as compared to control. In comparison with the effect of AlCl 3 , propolis, AlCl 3 combined with propolis, and administration of AlCl 3 for 1 month followed by administration of AlCl 3 combined with propolis for another month led to a highly significant decrease (P < 0.001) in the plasma level of glucose and a significant decrease (P < 0.05) in the level of plasma total protein and plasma albumin (Table 1, Fig. 2). Table 1 showed that AlCl 3 treatment led to a highly significant increase (P < 0.001) in the plasma level of creatinine, urea, and uric acid relative to that of the control, whereas propolis, AlCl 3 combined with propolis and the administration of AlCl 3 for one month followed by administration of AlCl 3 combined with propolis for another month had non-significant effect (P > 0.05) on the plasma level of creatinine, urea, and uric acid as compared to control. In comparison with the effect of AlC1 3 on the plasma level of creatinine, urea and uric acid, the propolis, AlC1 3 combined with propolis and administration of AlCl 3 for one month followed by administration of AlCl 3 combined with propolis for another month led to a highly significant decrease (P < 0.001) in the plasma level of creatinine, urea and uric acid (Table 1, Fig. 3). The AlCl 3 administration led to a highly significant increase (P < 0.001) in the plasma level of cholesterol and triglycerides, relative to that of control. Propolis and AlCl 3 combined with propolis led to a non-significant effect (P > 0.05) on the plasma level of cholesterol and triglycerides as compared with that of control, while the administration of AlCl 3 for 1 month followed by administration of AlCl 3 combined with propolis for another month had a highly significant increase (P < 0.001) on the plasma level of cholesterol and triglycerides as compared with that of control (Table 1, Fig. 3). In comparison with the effect of AlCl 3 on the level of plasma cholesterol and triglycerides, propolis, AlCl 3 combined with propolis, and administration of AlCl 3 for 1 month followed by administration of AlCl 3 combined with propolis for another month led to a highly significant decrease (P < 0.001) in the plasma level of cholesterol and triglycerides as compared to control (Table 1, Fig.  3).

Histological results Liver
Histological examination of the liver sections of different animal groups obtained after 2 months of treatments is given in Fig. 4. The control liver tissue is shown in Fig. 4a. It consists of liver cells arranged in cords that interspersed with sinusoids. In the AlCl 3treated group, the liver tissue shows severe vacuolation with increased inflammatory infiltrated cells among the sinusoids and necrosis of cells with cell nuclei pyknosis (Fig. 4b), as compared to that of the control tissue. No such vacuolation or massive inflammatory cells were detected in either the propolis-treated group (Fig. 4c) or those received AlCl 3 combined with propolis (Fig. 4d). The role of propolis in AlCl 3 detoxicating efficiency is obvious on comparing the tissue of the liver in the AlCl 3 -treated group for 1 month followed by AlCl 3 combined with propolis for another month with that of AlCl 3 combined with propolis (Fig. 4e). However, in both groups nuclear pyknosis is obvious regardless the recovered cell degeneration and inflammatory infiltration observed in AlCl 3 -treated group only.

Kidney
Histological examination of the kidney sections of different animal groups obtained after 2 months of treatments is given in Fig. 5. The control kidney tissue is shown in Fig. 5a. It consists of well-developed glomeruli and tubules. Examination of the kidney sections from rats treated with AlCl 3 revealed a severe histological damage (Fig. 5b), as compared to that of the control. These changes were mainly confined to shrinkage in glomeruli and infiltration with intensive inflammatory cells. No such shrinkage of glomeruli or intensive inflammatory cells was detected in either the propolis-treated animals (Fig. 5c) or those received aluminum chloride combined with propolis (Fig. 5d). The group of the animals which was treated with AlCl 3 for 1 month followed by administration of AlCl 3 combined with propolis for another month indicates the protective role of propolis; there are no pathological changes were detected (Fig. 5e), as compared to that of AlCl 3 -treated group.

Discussion
The present study was carried out to evaluate the protective role of propolis against aluminum toxicity induced biochemical and histological alterations in the liver and kidney of rats. However, it is well known that the plasma enzymes are hepatic health markers, and the alteration in their levels is an indicator of the disturbances in the histological structure of hepatocytes. In the present study, the AlCl 3 -treated group showed a highly significant increase in the plasma level of Roshanaei, 2019; Geyikoglu, Türkez, Bakir, & Cicek, 2013;Imam, Khalifa, Hussein, & Ali, 2016). However, the elevated liver enzymes in the plasma after administration of AlC1 3 might be due to cellular degeneration and changes in permeability of hepatic cell membranes (Abdel-Wahab, 2012;Hassoun & Stohs, 1995;Yeh, Lee, Hsieh, & Hwang, 2009). It had been reported that the accumulation of AlC1 3 in the liver tissue is associated with necrosis and degeneration of hepatic tissue to escape of liver enzymes from the injured cells to the plasma (Imam et al., 2016;Yousef, 2004). In addition, histological changes in the liver and the increase of plasma enzyme activities may be due to free radicals production and oxidative stress after AlC1 3 administration in the liver tissue (Abdel-Wahab, 2012; Cheraghi & Roshanaei, 2019). On the other hand, the administration of propolis, AlCl 3 combined with propolis and administration of AlCl 3 for 1 month, and administration of AlCl 3 combined with propolis for another month showed non- significant change on the plasma level of enzymes, ALT, AST, ALP, AP, and LDH with normal liver histological structure, indicating that propolis tended to prevent damage and blocked the enzymes leakage through cellular membranes. These results are in agreements with findings of many studies (Yeh et al., 2009;Wen, Zhao, Nirala, & Bhadauria, 2012;Al-Qayim & Saadoon, 2013) who concluded that propolis mostly recovered the action of AlCl 3 on the function and structure of the liver. Moreover, the hepatoprotective effect of propolis may be attributed directly to stabilization of redox state in the cells (El-Guendouz et al., 2017;Ibrahim et al., 2019). The present study revealed that AlCl 3 treatment induced a significant increase in the plasma glucose which may indicate disruption in carbohydrate metabolism (Geyikoglu et al., 2013). The hyperglycemia observed in the present study is in accordance with the results of El-Demerdash (2004), Shati and Alamri (2010), and Wen et al. (2012). It was suggested that increase in the plasma glucose level observed in AlC1 3 -treated group may be due to the inhibition of pancreatic B-cell activity and insufficient insulin secretion (Ibrahim et al., 2019;Yousef, 2004). In contrary, the administration of propolis, AlCl 3 combined with propolis, and administration of AlCl 3 for 1 month and the administration of AlCl 3 combined with propolis for another month showed non-significant change on the level of plasma glucose. These results are in agreements with findings of Wen et al. (2012). In addition, Fuliang et al. (2004) suggested that propolis can control the metabolism of glucose. It has been reported that the potential positive effects of propolis on glucose metabolism in experimental models of diabetic rats (El Araby et al., 2017).
The present study demonstrated that AlC1 3 administration induced a significant increase in the plasma level of total protein and albumin. This finding is not in agreement with El-Demerdash (2004) who reported that the decrease of plasma total protein and albumin in AlCl 3treated animals might be related to alterations in synthesis and/or metabolism of protein (Chinoy & Memon, 2001). On the other hand, the present findings revealed that the propolis and the combined treated groups were similar to that of control in the plasma level of total protein indicating that propolis contains a great amount of flavonoids and proteins, and the propolis interaction with plasma proteins caused conformational changes in the protein (Olinescu, Gidoiu, Safta, & Popescu, 1982).
The present results showed that the administration of AlCl 3 led to a significant increase in the level of plasma creatinine, urea, and uric acid, relative to that of the control group. These biochemical changes were in confirmations with extensive histolopathogica changes including shrinkage in glomeruli and intensive inflammatory cells infiltration in the kidney. Our results are in agreement with Abdel-Wahab (2012) and Imam et al. (2016). They reported that the elevated plasma urea and creatinine levels in AlCl 3 -treated rats are considered as a substantial marker of renal dysfunction. In addition, Szilagyi et al. (1994) and Geyikoglu et al. (2013) reported that changes in serum urea level may be related to metabolic disturbances. Also, the increase in urea levels in plasma of AlCl 3 -treated animals may be due to the liver dysfunction as proved by the increase in serum AST, ALT, and ALP activities (Yousef, 2004).
Regarding to the results of cholesterol and triglycerides, the increase in plasma cholesterol and triglycerides is due to loss of membranes integrity (Abdel-Wahab, 2012;Sarin, Gupta, & Gill, 1997). Also, the increase in plasma triglycerides may be due to hypoactivity of lipoprotein lipase in the blood vessels which break up the triglycerides (Wen et al., 2012). Similarly, Whihelm, Jaeger, Schüll-Cablitz, Hafner, and Idel (1996) reported that AlCl 3 exposure can cause Al accumulation in the liver leading to disturbance of lipid metabolism and an elevation of serum cholesterol.
On the other hand, the effect of propolis or AlCl 3 treated with propolis did not show any significant increase in plasma creatinine, urea, and uric acid with normal kidney histological structure in propolis-treated group, while the AlCl 3 combined with propolis induced an improvement of the previous histological alternations caused by AlCl 3 treatment. With regard to lipid metabolism, plasma concentrations of triglycerides and cholesterol showed no alteration after propolis treatment. Our results are in agreement with Jasprica et al. (2007) who reported normal levels of cholesterol, triglycerides, and uric acid after the propolis supplementation and also are in agreement with Wen et al. (2012) who observed normal levels of urea, uric acid, cholesterol, and triglycerides in combined treatment of HEDTA + propolis-treated animals (Mani, Damasceno, Novelli, Martins, & Sforcin, 2006).

Conclusion
The present results demonstrated that the exposure of animals to aluminum is able to induce marked detectable alterations in histological and biochemical characteristics and enzymatic activities. Also, our study demonstrated that the propolis minimized the toxic effects of AlCl 3 by reducing the degenerative changes in the liver and kidney tissues and alleviated of biochemical parameters. Consequently, it can be recommended that the exposure to aluminum in our daily life should be reduced, and the intake of diets rich with propolis might be a beneficial method to avoid the aluminum toxicity. efforts in coordination, planning, and reporting of this study. We are also greatful to Sohag University for supporting this study.