Antioxidant and Hepatoprotective Effects of Moringa oleifera-mediated Selenium Nanoparticles in Diabetic Rats.

Materials

Streptozotocin (Cat #HY-13753) were purchased from MedChemExpress, Monmouth, NJ, USA. Metformin tablet (500 mg) was purchased from BIG Pharmacy, Malaysia. Insulin (Cat #E-EL-R3034), GSH-Px (Cat #E-BC-K096-M), CAT (Cat #E-BC-K031-M), T-SOD (Cat #E-BC-K020-M), MDA (Cat #E-BC-K025-M), IL-6 (Cat #E-EL-R0015), TNF-α (Cat #E-EL-R2856), ΙL-1β (Cat #E-EL-R0012), iNOS (Cat #E-EL-R0520) and BCA Protein (Cat #E-BC-K318-M) ELISA assay kit were purchased from Elabscience, Houston, Texas USA. AGE (Cat #CSB-E09413r) ELISA assay kit was obtained from CusaBio, Houston, TX, USA.

Moringa oleifera-mediated selenium nanoparticles (MO-SeNPs) preparation

The chemicals used in this study included sodium selenite (Na₂SeO₃, ≥98%, CAS No. 10102-18-8), 2,2-diphenyl-1-picrylhydrazyl (DPPH, CAS No. 1898-66-4), sodium hydroxide (NaOH, CAS No. 1310-73-2), hydrochloric acid (HCl, CAS No. 7647-01-0), potassium ferricyanide (K₃[Fe (CN)₆], CAS No. 13746-66-2), ferric chloride (FeCl₃, CAS No. 7705-08-0), trichloroacetic acid (C₂HCl₃O_2, CAS No. 76-03-9), phosphate buffer solution (PBS, P4417) and ascorbic acid (C₆H₈O₆, CAS No. 50-81-7). All the chemicals are analytically graded and sourced from Merck (West Point, PA). Fresh Moringa oleifera leaves were collected from a local supplier in Sungai Petani, Kedah, Malaysia, and identified by a botanist from Universiti Putra Malaysia (Voucher No. MFI 0213/21). The leaves were thoroughly washed, oven-dried at 40 °C for 72 hours, and ground into a fine powder. For the aqueous extract, 20 g of powdered leaves were homogenized in 800 mL of boiling distilled water, shaken at 150 rpm for 4 hours, centrifuged at 4000 rpm for 20 minutes, and filtered using Whatman filter paper No. 1 (Cat No. 1001 125) from GE Healthcare (New Jersey, USA). Selenium nanoparticles (MO-SeNPs) were synthesized by adding 5 mL of a 50 mM sodium selenite solution dropwise to 20 mL of Moringa oleifera extract under magnetic stirring, followed by incubation at 37 °C for 48 hours at pH 8 to facilitate the green synthesis. All of these green synthesis of MO-SeNPs was adapted from our previous work.¹⁷ The leaves extract of M. oleifera was employed to reduce 50 mM sodium selenite solution under magnetic stirring condition. Optimisation of the synthesis led to production of red amorphous selenium nanoparticle deposits and the suspension were collected for cleaning and characterization. MO-SeNPs imaging showed that it has a spherical structure and have the mean sizes of 95.36 nm.

Experimental animals

Since animals may experience pain, researchers must evaluate the expected consequences on laboratory animals to minimise injury. Researchers must minimise animal pain and foster well-being. Suffering encompasses pain, hunger, thirst, starvation, extreme temperatures, anxiety, stress, injuries, infections, and inability to behave normally. Trapping, labelling, anaesthetising, breeding, transporting, and stabilising might cause suffering before and after the experiment, therefore researchers must examine all of it. Researchers must also consider pre- and post-experiment adaption periods.

Hence, in this study, the in vivo rat study was conducted on male Sprague-Dawley rats in Animal House, Science Lab, Level 9, located in Management and Science University (MSU) after ethic approval for animal experiment from MSU Ethic Committee (EA-L3-01-SGS-2024-03-0002). A total of 30 male rats, with the weight ranged around 180-220 g was purchased and stationed in Animal House at MSU. The animals were placed in common conditions of the animal house in polypropylene cages, supplied with water and food ad libitum. The rats were kept in a conducive environment with 12:12 h light and dark cycle with air-conditioned at 21-24°C to maintain the relative humidity between 30–70% throughout the experimental process. All the rats were admitted with continuous access of food and water without showing distress such as diarrhea, restlessness or dulling of fur during the observational period. After a period of adaptation (± 72 hours), the animals were randomly divided into five groups. Induction of T2DM was done through high-fat diet (HFD) with Streptozotocin (STZ) injection following the method of previous study with slight modification,¹⁸ whereby the rats were randomly selected and normal control group was fed with common chow diet (standard) while the rest of the diabetic groups were placed on a HFD for inducing prediabetes condition for two weeks period as desired. The HFD was prepared using a recipe formulation from previous study where it was made using 50% commercial chow pellet, 24% ghee, 20% full cream milk powder and 6% corn starch flour.¹⁹ After two weeks, single intraperitoneal injection of low dose freshly prepared STZ (45 mg/kg body weight, BW) diluted in 0.1M sodium citrate buffer (pH 4.5) were administered to the overnight fasted rats from the diabetic group. Fasting blood glucose level (FBG) was observed with a blood glucometer device (On Call Plus, ACON, USA) by tail-pricked method after a week of induction with STZ. Rats with FBG level more than 16.7 mmol/L were considered as diabetic and selected for the study.²⁰ The rats selected as the normal control were administered with a vehicle citrate buffer (0.25 ml/kg BW) at the same time. All the rats were fed with a normal chow diet after the induction of STZ. The FBG level and body weights of the rats were observed weekly, before and after injection of STZ while food and water intake were measured post STZ administration.

Experimental design

After the confirmation of T2DM induction, the experimental rats were grouped as follows (n=6): Group 1: Normal control (NC) rats with vehicle (sodium carboxymethylcellulose, Na-CMC)

Group 2: Diabetic control (DC) rats with vehicle

Group 3: Positive control (PC) diabetic rats treated with 100 mg/kg BW of MET

Group 4: Diabetic rats treated with 0.25 mg/kg BW of MO-SeNPs (Tx 0.25)

Group 5: Diabetic rats treated with 0.5 mg/kg BW of MO-SeNPs (Tx 0.5)

The selection of doses for selenium nanoparticle and metformin (MET) were according to previous study. High dosage of 4 mg/kg BW for selenium nanoparticles have been reported to produce therapeutic effect. However, high doses of selenium nanoparticle have been associated with insulin resistance.²¹ Thus, low and medium doses of selenium nanoparticle were chosen for this study with comparison to standard antidiabetic drug. After of four weeks of daily oral administration of treatment finished, all the rats were weighed, anesthetized with a mixture of ketamine and xylazine and the blood was extracted from the abdominal aorta via cardiac puncture and was collected into blood collection vacutainer tube. Serum was isolated from blood sample by centrifuging at 4000 rpm for 15 min. The liver tissues were excised and weighed, washed with phosphate buffer solution (pH 7.4, 4 °C) and blood serum and liver tissue were kept at -80 ° C for further analyses.

Assessment of insulin resistance and β-cell function

Homoeostasis model assessment (HOMA) of β-cell function (HOMA-β%) and insulin resistance (HOMA-IR) was performed using fasting serum insulin (μIU/mL) and fasting blood glucose (mg/dL) using the following formula: IR (HOMA-IR) = [fasting glucose (mg/dl) × fasting insulin (μIU/ml)]/405.²² For β-cell function, it will be calculated according to this formula: (HOMA-β) % = [360 × fasting insulin (μIU/mL)]/(fasting glucose (mg/dL)– 63).²³ The measurement of insulin level was done using ELISA kit according to the manufacturer’s instructions.

Serum biochemical analysis

The liver function was studied by measuring liver enzymes such as ALT, AST, ALP while lipid profile was analysed by observing the level of lipid biomarkers such as LDL, HDL, cholesterol and TGL. The biochemical serum biomarkers were analysed based on standard method using Biosystem BA400 chemistry analyser (Biosystem SA, Barcelona, Spain).

Assessment of antioxidant enzyme and inflammatory biomarker status

The serum samples and liver tissue were subjected to analysis of antioxidant enzyme activities and inflammatory cytokine level. The samples and supernatants were prepared according to the instruction from the manufacturer of ELISA kits. Serum antioxidants enzyme activities were evaluated by testing the glutathione peroxidase (GSH-Px), catalase (CAT), total superoxide dismutase (T-SOD) and whereas the lipid peroxidation in the liver were determined by malondialdehyde (MDA) level using hepatic tissue sample. Analysis of pro-inflammatory cytokine levels were done towards AGE, IL-6, IL-1β, iNOS and TGF-α using the hepatic tissue sample.

Statistical analysis

Statistical analysis of the results was done using GraphPad Prism 10 software (version 10.2.1)(RRID:SCR_002798)(GraphPad Software Inc., San Diego, CA, USA). All the data acquired throughout the experimental period were recorded in Microsoft Excel. Recorded body weight, biochemical parameter analysis, antioxidant enzyme assays, inflammatory biomarkers analysis, were conducted with analysis of variance (ANOVA) with Tukey’s post hoc test to compare the differences among the treatment groups. The differences were considered as statistically significant when p-value < 0.05.

Food and water intake changes

Based on Figure 1, the consumption of normal chow diet and water intake after HFD/STZ induction was measured and observed to be distinct between each group even though the animal groups received same amount of food and water source every day. From Figure 1a, animal groups that received HFD/STZ generally consumed almost 2-fold of the amount from the NC group initially. With treatments commencement, their food intake gradually decreases in level while DC group showed steady increase in food intake. Similar pattern can be seen for the animals’ water intake based from Figure 1b, where at first the rats with diabetic induction showed higher intake of water at more than 3 fold from NC group. Throughout the treatment, treated rats showed reduction in water intake while the DC group maintain its increase until the end of experiment.

Figure 1. Changes in food (a) and water (b) intake pattern of all experiment groups during four weeks of treatment.

Graph points are expressed as mean with SEM as error bar (n=6).

Body weight analysis

Figure 2 showed that animal groups that received HFD at the first two weeks of experimental period gained substantial body weight as compared to NC rats that consumed only normal chow diet. After a week of STZ injection, all the subjected rats showed a reduction in body weight. However, all treatment groups exhibited increase in body weight throughout the four weeks of treatment. Rats from NC group showed a steady pattern of body weight gain while DC group appeared to gradually lose body weight after STZ administration. As shown in Table 1, at the end of treatment period, rats treated with MET and both MO-SeNPs dosage showed noticeable increase from DC group with 0.25 mg/kg MO-SeNPs treatment showed statistically significant changes.

Figure 2. Body weight changes of the treatment groups during the experimental stage.

Graph points are expressed as mean with SEM as error bar (n=6).

Fasting blood glucose level

Based on Figure. 3, animal groups that received HFD showed a slightly higher FBG throughout the first two week compared to the NC group. STZ injection to the diabetic groups post-HFD showed a remarkable increase in FBG level, passing the benchmark for confirmation of diabetes (16.7 mmol/L). During four weeks of treatment, rats treated with metformin, 0.25 mg/kg and 0.5 mg/kg MO-SeNPs showed a decreasing trend of FBG level but with different efficiency. Interestingly, based on Table 1, both MO-SeNPs treated groups showed significant reduction of FBG level after four weeks of treatment compared to DC group with Tx 0.25 group values nearly reaching the normal rats.

Figure 3. Pattern of FBG level of the animals groups throughout the experimental period.

Graph points are expressed as mean with SEM as error bar (n=6).

Serum insulin, HOMA-IR and HOMA-β% profile

In this experiment, DC group showed the highest insulin level, HOMA-IR index and low values of HOMA-β%. Table 1 demonstrated that rats that receive both dosage treatment of MO-SeNPs and MET after four weeks, showed much lower serum insulin level as compared to DC rats and with MET and 0.25 mg/kg MO-SeNPs administration exhibit statistically significant different of the insulin values. Furthermore, HOMA-IR index showed that both MO-SeNPs dosage and MET treatment produced a significant reduced from DC values with lower dose of MO-SeNPs (0.25 mg/kg) showed lower index than NC group. However, the β-cell function from HOMA-β% values showed that each treatment group did not reach a significant improvement in β-cell function comparing to DC rats.

Serum liver and lipid biochemical profiles

Based on Table 2, induction of diabetes by HFD/STZ showed to cause elevated level of serum hepatic enzyme like ALT, AST, ALP which reflected by DC group through the experiment. Moreover, it also showed a highest value for serum lipid biomarkers such as LDL, TGL and cholesterol with lowest value of HDL among other experimental groups. After 4 weeks of treatment period, MO-SeNPs and MET administration have shown to alleviate the values for the serum liver and lipid profiles. Tx 0.25 group have shown to exhibit significant changes on the level of ALT, AST and ALP level while Tx 0.5 group showed significant improvement on AST, HDL and LDL level as compared to DC group. Effect of metformin treatment exhibited a significant change towards TGL level as compared to diabetic control rats. However, cholesterol had shown no noteworthy differences among every treatment groups.

Serum antioxidant enzyme activity and hepatic lipid peroxidation analysis

In order to explore the capacity of MO-SeNPs treatment on its ability to defend against oxidative damage, the level of serum enzymatic antioxidant was assessed in this research. Referring to Figure. 4, throughout the four weeks of treatment with MO-SeNPs, the treated rats showed a significant alleviation all enzymatic antioxidant such as GSH-Px, CAT, T-SOD as compared to the diabetic control rats. Moreover, both MO-SeNPs dosage did not showed a staggering difference of values when being compared with each other with 0.25 mg/kg MO-SeNPs to be slightly higher at GSH-Px (3449 ± 371.6 U) and CAT (92.32 ±14.09 U/mL) while 0.5 mg/kg MO-SeNPs being vaguely higher at T-SOD activity level (252.8 ± 14.98 U/mL). Meanwhile, MDA level for hepatic lipid peroxidation assessment proved that both dosage of MO-SeNPs treatments to be significantly effective for the protection against oxidative damage towards the level with 0.25 mg/kg MO-SeNPs to be most effective with values much lower than NC group. Our results also exhibited that DC groups possess lowest enzymatic antioxidant activity and highest impact on lipid peroxidation.

Figure 4. Visualisation of MO-SeNPs' effects on enzymatic antioxidant activity and lipid peroxidation after four weeks.

The graphs are expressed as mean + SEM (n=6). Significant value (p<0.05) is noted as letters (a) and (b) which expressed significant different to NC and DC respectively.

Hepatic inflammatory cytokine level analysis

To study the efficacy of MO-SeNPs influence towards cytokine that are pro-inflammation, assessment towards biomarkers level such as AGE, TNF-α, IL-6, IL-1β and iNOS were done on the liver tissue of experimental rats. From Figure 5, the untreated DC group showed the highest level of inflammatory cytokines tested in this study. On the other hand, experimental groups that received both MO-SeNPs and MET have shown improvements and decreases in values. Similar to antioxidant activity status, the lower dosage of MO-SeNPs (0.25 mg/kg) exhibited much significant changes towards hepatic AGE, IL-6, IL-1β and iNOS as compared to DC group while another MO-SeNPs treatment (0.5 mg/kg) managed to cause significant improvement on hepatic AGE level. However, both dosage treatments of MO-SeNPs and MET did not show a significance improvement from TNF-α despite having a lower value as compared to diabetic control rats.

Figure 5. The effect of inflammatory cytokine level of liver tissue after four weeks of daily MO-SeNPs treatments.

The graphs are expressed as mean + SEM (n=6). Significant value (p<0.05) is noted as letters (a) and (b) which expressed significant different to NC and DC respectively.