Our study provided an advanced model and more information concerning using treatment mixtures and important measurements for the provided model of diabetes [32]. Oxidative stress plays an important role in the pathogenesis and etiology of diabetes [33]. The formation of ROS plays a role in the pathophysiology of diabetic retinopathy, possibly as a causal agent [34]. Many biochemical mechanisms strictly related to hyperglycemia, including glucose autoxidation and protein glycation, are enhanced under oxidative stress, which supports this theory. An increase in serum glucose was observed in the STZ-treated group; and an appreciable reduction in the groups treated with CUR and when combined with ZnCl2. Endothelial cells exposed to high glucose levels produce more superoxide anions, which may release nitric oxide, a potent endothelium-derived vasodilator that promotes the vasculature's overall homeostasis. The discovery that several negative effects of excessive hyperglycemia on endothelial functions are overcome by antioxidants adds to the harmful role of oxidative stress [35]. A study showed that the oral digestion of CUR at the rate of 100 mg/kg/day for eight weeks could appreciably remove all STZ induced abnormalities and hyperglycemia, including blood glucose elevation [36]. Administration of CUR could prevent STZ-induced diabetes, which was supported by regular performance of glucose clearance and continued normoglycemia [37]. The capacity of CUR for improving the pancreatic islets was also reported [38]. Besides, it was shown that the death of isolated β-cells and dysfunction could be prevented by CUR in diabetic STZ-induced animals [39]. Balbaa et al. [40] showed a considerable boost in the plasma glucose level in animals after STZ vaccination. It has been found that zinc deficiency and altered zinc metabolism are linked to reduced glucose tolerance [41]. Oral administration of CUR and its combination with ZnCl2 may reveal a significant impact on the levels of blood glucose.

Table 2 summarized the influence of CUR+ZnCl2 on the serum insulin contents of diabetic rats. CUR of 100 and 200 mg/kg BW and combined with ZnCl2 could enhance the level of serum insulin in STZ-induced diabetes in rats. Primarily, the hyperglycemia in the b-cells and the deficiency of insulin were cropped up because of the direct effects from cytotoxic assisted by STZ. Furthermore, it has been postulated that the worsening in glucose tolerance in free radical initiating systems is due to reduced insulin activity [42]. It was supposed that inducing lipid peroxidation in the lipid moiety of the cell membrane by free radicals would change the functional and structural integrity of the cell membrane or the interior cellular components. This would impede insulin's ability to commence and spread its regular sequence of actions, which could partially explain STZ-induced hyperglycemia [43]. However, the inclusion of STZ that disrupted the Langerhans islet cells in the pancreas was slowly restored when treated with CUR, thus saving b cells from further damages. This indicated the defensive activity of CUR against pancreas cell damages caused by STZ induction in the rats [37]. Research on diabetic rats revealed a considerable reduction in the insulin level than control (p<0.001), wherein an insignificant alteration was observed after treating the rats with curcumin [44]. Diabetes has been related to zinc deficiency and altered zinc metabolism, which in turn might be attributed to impaired insulin sensitivity [41]. Similarly, Zn exerted insulin suppression activities by slowing down the insulin hypersecretion related to the pre-diabetic pancreas, thereby allowing the preservation of the remaining bcells' structure and functionality [45].

Table 3 exhibited that the serum LDL-C, TG, and TC were appreciably amplified in PC as well as other treated groups. In contrast, the HDL-C concentration was considerably reduced in diabetic rats. The observed significant decrease in the serum TC, TG, and LDL-C contents in all treated groups of diabetic rats indicated their efficacy against diabetes. Nevertheless, the serum HDL-C concentration was higher compared to PC. Moreover, the group treated with CUR 200 mg/kg b.w + ZnCl2 revealed the optimum outcome. Levels of serum TG and TC in diabetic rats were raised compared to the normal ones. However, such abnormalities were considerably attenuated when treated with CUR [39]. A similar study on diabetic rats disclosed a noteworthy amplification in the serum TC and TG levels compared to healthy (without diabetes) groups. A considerable reduction in the serum HDL-C level of diabetic rats was evidenced. Interestingly, after treating the rats with Zn, the serum HDL-C level was significantly augmented [42]. In short, CUR treatment in diabetic rats could appreciably lower the serum TC and TG concentrations [36, 40].

Table 4 described the influence of CUR+ZnCl2 on the serum AST and ALT contents in STZ induced diabetic rats. Results showed that serum ALT and AST levels notably increased in PC as well as other groups treated with CUR and CUR+ZnCl2. These treated groups disclosed a lower level of AST and ALT compared to PC, especially at 200 mg/kg BW of CUR+ZnCl2. Furthermore, hyperglycemia in hepatocytes could reflect the boost of blood serum aminotransferases as major effects of the diabetic problem. This enhancement in the serum markers can be attributed to the leaking of liver cytosol enzymes into the bloodstream because of hepatomegaly (fatty liver) [42]. Stimulation of diabetes by STZ in rats could produce hepatotoxicity and thus amplified the activity of serum marker enzymes, including ALP, ALT [46, 47]. Zinc can minimize the perturbations in serum AST, ALT, ALP in STZ-diabetic rats [48]. Necrosis or semipermeable membrane injury causes the discharge of those enzymes into the blood. However, the serum level of those enzymes is expounded with liver action. This is in agreement with the liver microscopic anatomy results of this study.

In Table 4, the exposure of rats to STZ could considerably increase the serum SOD, TBA, and CAT levels compared to NC, while it decreased the GSH levels. CUR and CUR+ZnCl2 could attenuate those levels. The serum GSH and SOD levels in 200 mg/kg BW CUR+ZnCl2 treated diabetic rats were almost close to NC. Peroxyl radicals and molecular oxidants are the main kinds of ROS rat cells. Upon reacting with the injected CUR, these free radicals led to the formation of H atom from the phenolic-OH that existed in CUR, which in turn released H. The released H further reacted to form phenoxy, which eventually became stable via resonance around the keto-enol structures. Such oxidants were less reactive compared to the peroxyl radicals that defend cells from ROS-induced oxidative stress [46]. The incorporation of STZ in the rats resulted in a decrease in the liver serum SOD contents. Therefore, weakening in the activities of SOD was ascribed to the excessive production of ROS. In this mechanism, the superoxides were neutralized by ROS due to their inability to cross the lipid membranes by generating H2O2 which can traverse biological membranes. The produced H2O2 being the catalase detoxifiers, played a vital role in damaging the tissues. Thus, such an effective decrease in the SOD level can be majorly ascribed to the disruption of frontline enzymatic defense against H2O2 and superoxide anion [8, 47]. It was asserted that due to the exposure of STZ in the rats, the GSH was depleted and oxidized, which in turn created oxidative stress-mediated hyperglycemia when compared with normal (non-diabetic) rats [37]. It was arguing that hyperglycemic condition led to the formation of reactive species of oxygen [47], which in sequence inhibited antioxidant enzymes' activities, including SOD, CAT, and GSH-Px via the glycosylation mechanism in erythrocytes [49]. Gosh, et al. [46] revealed that exposure of animals to STZ could considerably enhance the hepatic lipid peroxidation than CUR treated ones. Furthermore, it was shown that CUR exposure to diabetic animals could appreciably inhibit hepatic lipid peroxidation. The exposure to STZ could cause fast depletion in the hepatic GSH, thus lowering the GSH to GSSG ratio in STZ induced animals than the normal ones. In brief, the levels of GSH, CUR treated animal groups remained unaltered (like normal animals) [47]. The exposure of STZ lowered the activities of antioxidant enzymes in the hepatic tissues, whereas the administration of CUR helped to restore these activities to make them normal. Thus, the protection of rat liver cells by CUR against the diabetic pathophysiology induced by the STZ exposure was primarily attributed to the antioxidant action of CUR [39, 44]. The presence of CUR in diabetic rats could normalize the activities of erythrocyte and hepatic antioxidant enzymes such as SOD, CAT, GSH-Px, and so forth, causing a considerable decrease in lipid peroxidation [42]. A recent study revealed that the presence of Zn is responsible for the efficient protection of free radicals from occasional injuries [45]. Such protective traits of Zn against enhanced lipid peroxidation and disintegration rates can be majorly ascribed to its capacity for effectively binding and stabilizing the cell membranes [45]. On top of that, the existence of Zn in the biological system is accountable for protecting the antioxidants against diabetic problems. Firstly, such protective action of Zn can be attributed to its excellent capacity to control hypoglycemic conditions, thereby inhibiting the occurrences of the injurious outcomes of hyperglycemia. Secondly, Zn can halt the development of the intrinsic alterations in the diabetic tissues, which gradually cause the disintegrations of cells [49]. The catalase activities revealed a considerable reduction in diabetic rats than the control group. Overall, the presence of Zn and CUR caused a considerable improvement in the specific activities in the diabetic rat's serum and hepatic catalase than those induced by STZ [41]. The observed noteworthy augmentation in the catalase by Zn in the diabetic cells can be ascribed to the competitive action of Zn to both Fe and Cu responsible for binding the cell membranes in diverse biological systems. Consequently, the formation of OH groups was hindered, and the generation of transition metal-free radicals was prevented [46-49]. The presence of Zn in the diabetic rats played an efficient and defensive role against diabetic syndrome that could produce injurious cell radicals to weaken the immune systems [44].

The current study showed that the valuable results of the combined treatment of CUR with ZnCl2 on diabetes could be reached via 2 strategies: (i) the best results of the isolated treatments were kept, including the decrease in glucose and (AST, ALT) levels in (CUR and ZnCl2 effects), (ii) additive effects were achieved, mainly by a further reduction in blood serum TG, cholesterol, and TBA, in which levels were lower compared with those of isolated treatments. The highest benefits reached by combined CUR with ZnCl2 were the antioxidant potential of this treatment and the development of dyslipidemia.

As seen in (Figure 2), in the negative control group, the liver tissue section had a hepatocytes arrangement. The blood sinusoids perforated the strands of hepatocytes and were bordered by endothelial cells. Likewise, Kupffer cells were observed. Histological investigation of the liver tissue exposed to STZ (60 mg/kg b.w) showed a high degree of hydropic degeneration accompanied by coagulative necrosis (Figure 2). Treatment with CUR (100+200 mg/kg b.w) (Figure 2) showed distinct marked improvement of the hepatocytes and nuclei, while treatment with CUR (100+ 200 mg/kg b.w) + ZnCl2 15mg/kg b.w (Figure 2) showed more improvement appeared in hepatocytes and normal nuclei were markedly noticed. The results also indicated that treatment with (CUR 200 + ZnCl2 15 mg/kg BW) (Figure 2) showed the best results. Nuclei and blood sinusoids were clearly with normal architecture.

The present data reveal that CUR combined with ZnCl2 treatment to STZ-diabetic rats (especially at a high dose) produce a remarkable amelioration better than CUR treated groups. The combination of CUR with ZnCl2 exhibited a synergistic and powerful control over hyperglycemia; it was able to scavenge free radicals, normalize liver function as well as exerting a principal role in treating and ameliorating liver damage at the cellular level. Thus, the most promising therapeutic dose currently used in this study can be effective in the treatment and strengthening of liver tissue from damage caused by diabetes and may be used as a candidate anti-diabetes drug. According to Holemans et al. [41], beta cells are destroyed by necrosis as a result of streptozotocin activity. STZ causes degenerative changes and necrosis in pancreatic β-cells; in turn, this leads to insulin insufficiency and glucose oxidation impairment. Mitra et al. [49] found that after injecting STZ, diabetic livers became degenerated and congested. Hyperglycemia is characterized by a drop in blood insulin levels, followed by hypoglycemia that is caused by a decline in insulin levels.  Experimental research indicated that nitric oxide (NO) could be the cause of the increased liver injury (42). Nitic oxide toxicity is increased when it reacts with superoxide radicals to produce powerful secondary toxic oxidizing species called peroxynitrite (ONOO). Such compound (ONOO) can oxidize cellular structure and cause lipid peroxidation [33], a process that causes membrane damage and is considered to be the fundamental cause of cell death.

This study proposes that CUR combined with ZnCl2 treatment acts in the liver as a potent free radical scavenger to prevent the hepatotoxicity effects of STZ-diabetic rats. I recommend applying future studies on another natural mixture, recent synthetic organic materials, and provide continuous biomarkers related to antioxidants, inflammatory biomarkers, and its related gene expression to treat the risk of diabetes.

Conclusion

The results showed that CUR+ZnCl₂ is effective in protecting rats against STZ-induced diabetes in serum and tissue. The results showed the additive effects of this combination on decreasing oxidative stress and dyslipidemia in diabetic rats. They may be used as good therapeutic agents for a diabetic. The conclusion that CUR and ZnCl₂ as non-toxic and cheap sources of diabetes treatment can be applied on a large scale to protect the liver in diabetic rats. As far as we know, by targeting hyperglycemia, oxidative stress, and dyslipidemia, this study provides the first evidence about a promising therapeutic strategy by combined CUR with ZnCl₂ for managing diabetes complications, especially heart and blood vessels diseases. Further research is necessary to understand the mechanisms of actions of this combination therapy that result in helpful effects observed in diabetes.

Acknowledgments: The author is grateful for the assistance of the Imam Abdulrahman Bin Faisal University. Also, warmest thanks to Professor Soheir, N. Abd El-Rahman, who made this work possible. Her friendly guidance and expert advice have been valuable throughout all stages of the work. Also, for helping with the figures and the final preparation of the manuscript. Moreover, special thanks to Professor Kamal A. Amin for his extended discussions and valuable suggestions, which have contributed greatly to the improvement of the manuscript.

Conflict of interest: None

Financial support: None

Ethics statement: The animal experiments were carried out by the National Institutes of Health guidelines for the care and use of laboratory animals. I certify that this work was performed following local ethical guidelines, with the approval of Imam Abdulrahman Bin Faisal University's Institutional Review Board (IRB). The IRB number for this study was lRB-2018-10-216.