Amelioration of Diabetes-Induced Nephropathy by Loranthus regularis : Implication of Oxidative Stress, Inﬂammation and Hyperlipidaemia

: In traditional Yemeni medicine, various preparations of Loranthus regularis ( L. regularis ), such as powder, decoctions and infusions are commonly used to treat diabetes, kidney stone forma-tions and inﬂammation. In the present study, we evaluated the antinephrotoxic effects of L. regularis extract in experimentally-induced diabetes in male Wistar rats. A single dose (60 mg/kg/day) of Streptozotocin (STZ) was used to induce type 1 diabetes. Animals were then treated for four weeks with L. regularis extract (150 or 300 mg/kg/day) by oral gavage. Renal and blood samples were subsequently harvested. Several biochemical indices, oxidative stress and inﬂammatory markers were assessed. Additionally, histological alterations in the renal tissue were examined. Serum glucose levels were signiﬁcantly ( p < 0.01) lowered while insulin levels were enhanced in L. regularis -treated diabetic animals. The increased renal markers in diabetic rats were decreased by L. regularis treatment. Serum elevated lipid proﬁles were markedly decreased by the plant extract. The serum and renal cytokines that were signiﬁcantly increased ( p < 0.001) by STZ were diminished by L. regularis treatment. Finally, renal tissue antioxidant enzymatic activity was enhanced with L. regularis treatment. Taken together, the data here indicate that L. regularis possesses therapeutic ability to reduce the development of diabetic nephropathy (DN) by minimizing oxidative injury and inﬂammation.


Introduction
Diabetes represents a massive health issue worldwide whose prevalence has only increased over the last decade, particularly in developing countries [1]. Chronic diabetes results in numerous complications, including nephropathy, retinopathy, and neuropathy [2][3][4]. The incidence of diabetic nephropathy (DN) in type 1 diabetes was about 15-25%, contributing to complications and a high mortality rate [5]. One important pathophysiological feature in DN is oxidative stress, which can be caused by hyperglycemia and involves the increased production of reactive oxygen species (ROS) [6,7].
For decades, herbologists have used plants to cure various diseases [14,15]. Herbal medicine can be used to treat various metabolic disorders via different types of phytochemicals, such as flavonoids, tannins, alkaloids, polysaccharides, and hormones [16,17]. The search for new herbal medicines is gaining popularity, as these may typically lack the side effects of chemical medicines [18].
Loranthus regularis Steud. (Loranthaceae), is a shrub that is widely distributed throughout Yemen, Southern Saudi Arabia and several African countries. The Loranthaceae family members are commonly known as mistletoes. Many mistletoe species are extensively used in traditional medicine to treat hypertension, diabetes, inflammatory disorders and gastrointestinal problems [19]. They are also used for general health and cancer therapy [20][21][22][23].
L. regularis is used in the treatment of nephrolithiasis, diabetes and inflammation in various types of preparations such as decoctions, infusions and powders [24]. The antioxidant, anti-inflammatory, antinociceptive, antipyretic and antimicrobial activities in L. regularis were demonstrated previously [25,26]. Despite the aforementioned studies that support the use of L. regularis in different diseases, their impact on the alleviation of DN has not been addressed to date. Therefore, the purpose of this study was to investigate the potential protective effects of L. regularis extract against DN using an animal model of diabetes, with a focus on its antioxidant, anti-inflammatory and antihyperlipidemic effects.

Animals
Male Wistar albino rats (n = 24) weighing 250-270 g were obtained from the Experimental Animal Care Centre, Pharmacy College, University of King Saud, Riyadh. Animals were acclimated to the laboratory conditions for one week prior to the commencement of the experiment. Free access to food and water was provided. The norms of the National Institute of Health Guide for the Care and Use of Laboratory Animals, Institute for Laboratory Animal Research (NIH Publications NO. 80-23; 1996) were strictly followed throughout the experiment. This study was approved by the Research Ethics Committee of King Saud University (SE-19-146).

Induction of Diabetes
A single dose of Streptozotocin (STZ) (65 mg/kg; Sigma-Aldrich, St. Louis, MO, USA) prepared in 0.1 M citrate buffer (pH 4.5) was used to induce type-1 diabetes. The equivalent volume of citrate buffer was injected into control rats. Subsequently, diabetes was confirmed through the measurement of fasting blood glucose levels using Accu-Chek Compact-Plus glucose meter system (Roche Diagnostics, Meylan, France) 48 h after the injection of STZ. A reading > 13.9 mmol/L was considered diabetic, and these animals were included in the study.

Collection and Preparation of Plant Extract
In June 2015, leaves, flowers and twigs of L. regularis were collected from Al-Mahuit governate (Yemen). These were identified at the Pharmacognosy Department, Faculty of Pharmacy, Sana'a University. Voucher specimens (Mo-M05) were deposited at the Pharmacognosy Department, Faculty of Pharmacy, Sana'a University. A crude methanol extract of L. regularis was obtained through grinding one kg of air-dried plant material followed by extraction with 3 L of methanol for 5 h using a Soxhlet apparatus. A rotary evaporator was used to filter and evaporate the extract to obtain 114 g of crude methanol extract.

Experimental Design
Experimental design included 4 groups (n = 6 per group) treated as follows: (i) control (vehicle-treated), (ii) diabetic (STZ-treated), (iii) diabetic plus L. regularis 150 mg/kg/day (Lr150 + STZ − treated) and (iv) diabetic plus L. regularis 300 mg/kg/day (Lr300 + STZ − treated). L. regularis extract was suspended in 0.25% carboxymethyl cellulose sodium (CMC) solution and given once per day orally by gavage for 4 weeks, beginning one week after STZ injection. An equal volume of CMC solution was used as a vehicle for control and STZ-treated animals. Animals were anesthetized through intraperitoneal injection of a ketamine (92 mg/kg, Hikma Pharmaceuticals, Amman, Jordan) and xylazine (10 mg/kg, Bayer, Turkey) mixture followed by the collection of blood samples through cardiac puncture. The collected samples were centrifuged at 1800 g for 10 min. The obtained serum samples were separated and stored at −20 • C. Animals were euthanized, and the kidneys were dissected. A cross-section of the harvested kidneys was fixed in 10% neutral buffer formalin (NBF) (pH 7.4) for histopathological analysis, and the remaining harvest was dipped in liquid nitrogen for one minute and stored at −80 • C.

Tissue Analysis
Kidney tissues were homogenized in physiological buffer (1:10, w/v), and TBARS and GSH levels were subsequently measured using ELISA kits (Cayman Chemical Co., Ann Arbor, MI, USA) according to the manufacturer's protocol. Kidney postmitochondrial supernatants were collected, and enzymatic activities of SOD, CAT and GPx were measured using ELISA kits (R&D systems Inc., Minneapolis, MN, USA). Furthermore, proinflammatory markers such as TNF-α, IL-1β and IL-6 levels were also measured using ELISA kits for rats (R&D systems Inc., Minneapolis, MN, USA) in kidney homogenate.

Histopathological Analysis
Fixed kidney specimens were immersed in paraffin for blocking, and 5-µm sections were obtained by microtomy. Sections were then stained with Hematoxylin and Eosin for microscopic examination. The histopathological changes were examined by a trained pathologist who was blinded to the treatment groups.

Statistical Analysis
Results were presented as the mean and standard error of the mean (mean ± SEM). To determine significant differences between the study groups, One-way ANOVA was used, followed by Newman-Keuls multiple comparisons as a post hoc analysis (Graph Pad Prism version 8). p-values less than 0.05 were considered significant (* p < 0.05, ** p < 0.01, *** p < 0.001).

Results
Blood glucose levels were significantly elevated (p < 0.001) in STZ-treated rats in comparison with control rats. Treatment of these type 1-diabetic rats with of L. regularis (150 or 300 mg/kg/day) for four consecutive weeks significantly lowered blood glucose levels (p < 0.01 and p < 0.001, respectively) as compared to STZ-treated animals ( Figure 1a). Serum insulin levels were also decreased in STZ-injected rats compared to normal control animals (p < 0.05). Furthermore, high taken dose (300 mg/kg/day) of L. regularis treatment significantly (p < 0.01) enhanced the serum insulin levels compared to STZ group.
( Figure 1b). Nephrotoxicity markers such as creatinine and urea levels were significantly increased in the STZ-injected group compared to control rats (p < 0.01 and p < 0.001, respectively) (Figure 1c,d). These increased serum creatinine levels were markedly reduced by treatment with L. regularis (150 or 300 mg/kg/day) in diabetic rats compared to the vehicle-and STZ-treated group (p < 0.05). Similar reductions in serum urea levels were observed in L. regularis-treated (150 mg/kg/day and 300 mg/kg/day) groups when compared to the STZ group (p < 0.05 and p < 0.01, respectively) ( Figure 1c,d).
Appl. Sci. 2021, 11, x FOR PEER REVIEW 4 of treatment significantly (p < 0.01) enhanced the serum insulin levels compared to ST group. ( Figure 1b). Nephrotoxicity markers such as creatinine and urea levels were sign icantly increased in the STZ-injected group compared to control rats (p < 0.01 and p 0.001, respectively) ( Figure 1c,d). These increased serum creatinine levels were marked reduced by treatment with L. regularis (150 or 300 mg/kg/day) in diabetic rats compar to the vehicle-and STZ-treated group (p < 0.05). Similar reductions in serum urea lev were observed in L. regularis-treated (150 mg/kg/day and 300 mg/kg/day) groups wh compared to the STZ group (p < 0.05 and p < 0.01, respectively) (Figure 1c,d).
Oxidative stress biomarkers such as thiobarbituric acid reaction substances (TBAR and glutathione (GSH) levels were estimated in renal tissue homogenate using comm cially available ELISA assay kits. TBARS levels were significantly increased (p < 0.00 whereas GSH levels were significantly decreased (p < 0.001) in STZ-treated rats compar to control animals. Treatment with L. regularis (150 or 300 mg/kg/day) significantly duced TBARS levels (p < 0.01), while GSH levels were significantly increased (p < 0.0 compared to the STZ-and vehicle-treated group ( Figure 5).  Enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST) in renal tissues were measured ( Figure 6). Antioxidative enzymatic activities were significantly reduced in STZ-treated rats compared to the control group (p < 0.001). Treatment with L. regularis (150 or 300 mg/kg/day) significantly increased the enzymatic activities of SOD, CAT and GR compared to the STZ-and vehicle-treated group (p < 0.05 and p < 0.01, respectively) (Figure 6a,b,d). GPx enzymatic activity was significantly increased following treatment with the higher dose of L. regularis (p < 0.05) (Figure 6c). Both doses of L. regularis significantly increased the enzymatic activity of GST compared to the STZ-and vehicletreated group (p < 0.01) (Figure 6e).
Light micrographs of the renal cortex of STZ-induced diabetic rats treated with L. regularis (150 or 300 mg/kg/day) are presented in Figure 7. Normal architecture of Bowman's capsules, glomeruli, proximal convoluted tubules and distal convoluted tubules were observed in sections of the renal cortex from control animals ( Figure 7A). Vacuolar degeneration, necrosis, narrowed glomeruli and mononuclear cell infiltration were observed in sections of the renal cortex from STZ-treated rats ( Figure 7B). However, upon treatment with L. regularis (150 mg/kg/day), sections of renal cortex of diabetic rats demonstrated a moderate improvement of pathologies in the kidney glomeruli and renal tubules ( Figure 7C). Reversal of STZ-induced abnormalities in kidney histology was observed in sections of the renal cortex of diabetic rats treated with the higher dose of L. regularis ( Figure 7D). cantly reduced in STZ-treated rats compared to the control group (p < 0.001). Treatment with L. regularis (150 or 300 mg/kg/day) significantly increased the enzymatic activities of SOD, CAT and GR compared to the STZ-and vehicle-treated group (p < 0.05 and p < 0.01, respectively) (Figure 6a,b,d). GPx enzymatic activity was significantly increased following treatment with the higher dose of L. regularis (p < 0.05) (Figure 6c). Both doses of L. regularis significantly increased the enzymatic activity of GST compared to the STZ-and vehicle-treated group (p < 0.01) (Figure 6e). Data are expressed as means ± SE (n = 6) and were analyzed using one-way ANOVA followed by Student Newman-Keuls as post hoc test. * Control vs. STZ group; # STZ vs. L. regularis (150) or L. regularis (300). # p < 0.05, ## p < 0.01, *** p < 0.001.
Light micrographs of the renal cortex of STZ-induced diabetic rats treated with L. regularis (150 or 300 mg/kg/day) are presented in Figure 7. Normal architecture of Bowman's capsules, glomeruli, proximal convoluted tubules and distal convoluted tubules were observed in sections of the renal cortex from control animals ( Figure 7A). Vacuolar degeneration, necrosis, narrowed glomeruli and mononuclear cell infiltration were observed in sections of the renal cortex from STZ-treated rats ( Figure 7B). However, upon treatment with L. regularis (150 mg/kg/day), sections of renal cortex of diabetic rats demonstrated a moderate improvement of pathologies in the kidney glomeruli and renal Figure 6. Effect of L. regularis (150 or 300 mg/kg for 4 consecutive weeks) on diabetic-induced changes in renal pro-oxidative enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-S-transferase (GST). Data are expressed as means ± SE (n = 6) and were analyzed using one-way ANOVA followed by Student Newman-Keuls as post hoc test. * Control vs. STZ group; # STZ vs. L. regularis (150) or L. regularis (300). # p < 0.05, ## p < 0.01, *** p < 0.001.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 9 of 13 tubules ( Figure 7C). Reversal of STZ-induced abnormalities in kidney histology was observed in sections of the renal cortex of diabetic rats treated with the higher dose of L. regularis ( Figure 7D).

Discussion
Although L. regularis has been used in the management of many diseases such as diabetes, nephrolithiasis and inflammatory diseases [24,26], little is known about its impact in DN. Therefore, we evaluated the effects of L. regularis extract in STZ-induced DN. Following the treatment by L. regularis extract, a dramatic reduction in blood glucose levels, toxic renal markers, proinflammatory cytokines and oxidative stress markers as well as improvement in renal histopathology were observed.
This study found that DN development may be inhibited by L. regularis extract administration. We found that L. regularis extract could increase the antioxidant ability of diabetic rats which displayed high glucose levels. Treatment with L. regularis extract improved the antioxidant potential by increasing SOD and CAT activities, and GSH levels, in diabetic rats. The progression of DN is mainly attributed to oxidative stress generated by hyperglycemia [27,28]. In this context, the generation of ROS results in loss of renal function and elevation of lipid profiles [29,30].
Most medicinal plant-isolated compounds are relatively safe and readily available. The presence of active phenols, flavonoids, and tannins in L. regularis extract may explain its hypoglycemic effect as evidenced by our data. Vessal et al. (2003) have shown that supplementation with flavonoids, specifically quercetin, which is one of the main constituents of L. regularis, had the ability to regenerate pancreatic islets and increase insulin release in STZ-induced diabetic rats [31].  [32]. In addition, Eid and Haddad (2017) attributed the antidiabetic properties of quercetin to various mechanisms including the inhibition of intestinal glucose absorption, insulin secretory and insulin sensitizing activities as well as increased glucose usage in peripheral tissues [33]. The significant increase in serum insulin level after L. regularis treatment could be related to the plant extract ability to protect and regenerate damaged pancreatic β cells resulting in enhancing insulin secretion.
Different diabetes-related complications occur with the progression of disease, including DN, which can further aggravate the patient's health condition [34,35]. In this study, elevation in creatinine and urea levels concomitant with structural and histopathological alterations have confirmed the presence of DN. However, serum creatinine and urea levels were decreased and renal histopathology was alleviated following the treatment of diabetic animals with L. regularis extract, which can be linked to its active phenolic constituents [24]. Hypercholesterolemia and hypertriglyceridemia are also among the complications caused by diabetes, which are well-recognized causative agents of chronic vascular disease [36]. Consistent with many previous reports, STZ-induced diabetes-induced dyslipidemia, as evidenced by increased serum cholesterol, TG and LDL [37,38]. Interestingly, the extract from L. regularis not only lowered TC but also increased HDL. In addition, it reduced TG and LDL in diabetic rats, perhaps due to the presence of quercetin [39,40].
In this study, the most significant finding is that L. regularis extract administration could restrict oxidative damage, lipid peroxidation and inflammation in STZ-induced diabetic rats, which together may account for its renoprotective impact. Chronic hyperglycemiamediated DN induces oxidative damage and inflammation [41]. As a consequence, release of ROS can damage lipids, DNA and other cellular components, leading to cell death. In addition, the generation of ROS results in the elevation of TNF-α, IL-1β, IL-6 and other inflammatory mediators [42]. Thus, hyperglycemia contributes greatly to kidney injury in diabetes. The ability of ROS to cause renal dysfunction and damage has been confirmed by studies that indicated a reduction in renal injury after ROS suppression [43]. Here, TBARS, a byproduct of lipid peroxidation, was increased, while the antioxidant defenses (SOD, CAT and GSH) were markedly diminished in the diabetic treatment group, indicative of a hyperoxidative state. Decreased cellular antioxidant enzymes (SOD, CAT and GSH) in diabetic animals have been previously shown [44]. In addition, TNF-α, IL-1β and IL-6 in both serum and tissue were increased in diabetic rats. Administration of the diabetic rats with L. regularis extract significantly reduced oxidative stress and boosted antioxidant defenses. In addition, L. regularis extract suppressed inflammation as evidenced by the decreased expression of these serum and renal proinflammatory cytokines. These findings demonstrate the potent antioxidant and anti-inflammatory effects of L. regularis extract. The increased insulin levels and suppression of renal damage and lipid accumulation may be specifically linked to antioxidant and anti-inflammatory treatment by L. regularis extract. A previous study by Mothana et al. (2013) showed the in vitro antioxidant and anti-inflammatory activity of crude extract, fractions, and isolated compounds from L. regularis [32]. The strong anti-inflammatory and free radical-scavenging activity evidenced by DPPH assays were associated with the high phenolic content of L. regularis.
However, few studies have investigated the antidiabetic or renoprotective properties of L. regularis extract. Our data present the first evidence that L. regularis extract ameliorated hyperglycemia, oxidative stress, inflammation and renal injury in STZ-induced diabetic rats. These beneficial effects may be directly related to the active constituents of L. regularis extract, specifically quercetin glucosides, which have well documented anti-inflammatory and antidiabetic effects [33,45,46].

Conclusions
Taken together, this work presents strong evidence of the beneficial therapeutic impact of L. Regularis against renal complications associated with diabetes. L. regularis extract improved renal functions and alleviated cellular damage in diabetic rats. Additionally, the capacity of L. regularis to reduce free radical production caused by hyperglycemia and to attenuate inflammation and enhance antioxidative enzyme activities may be underlying mechanisms. Additional clinical trials could support the therapeutic uses of L. regularis in diabetic patients.

Data Availability Statement:
The data used to support the findings of this study are included within the article.