INTRODUCTION:

Diabetes mellitus (DM) is a common disease in the world. The international diabetes federation (IDF) reports that diabetes patient reaches up to 366 million in 2011¹, 382 million in 2013², 415 million in 2015³, and 451 million in 2017⁴. These figures were predicted to increase to 693 million in 2045 if there is no prevention of risk factors and effective treatment⁴. In addition, people with DM have an increased risk of developing some serious health problems to cause death^4,5.

DM was related to the inflammation mechanism, indicated by a high level of pro-inflammatory cytokine production^6,7.

Pro-inflammatory cytokine such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) have played a role in the inflammation mechanism of DM⁸. The elevated level of pro-inflammatory cytokines was triggered by hyperglycemia, which is the main characteristic of DM.

Hyperglycemia refers to an increase in the blood glucose of the human body. It occurs due to the less use of insulin or less insulin production, a hormone that functions to utilize glucose to produce energy⁹. Consequently, the blood glucose increase and ROS production cannot be managed, finally results in an excessive amount of glucose circulates in the blood plasma. Furthermore, the high concentration of ROS causes oxidative stress¹⁰. The highest levels of oxidative stress occur after the formation of advanced glycation end products (AGEs) that induce the synthesis of various pro-inflammatory cytokines^11,12. Therefore, DM treatment can work optimally by mean suppressing pro-inflammatory cytokines.

Treatment of DM until now uses synthetic drugs which have side effects after long-term use. Thus, an alternative medicine from a natural substance is needed to minimize these side effects. For that, traditional medicine is one option. Traditional medicines are believed to be safer and cheaper than synthetic drugs. Traditional medicines are usually made from natural compounds of constituents such as plants and animals. Moringa oleifera Lam (MO), also called “kelor” is a plant in Indonesia beneficial to health. MO is a tree native of Pakistan, India, and Bangladesh, but it is widely distributed in subtropical and tropical of the world^13,14. The use of MO is based on the content of compounds such as flavonoids, phenolic, carotenoids, ascorbic acid, nutrients, minerals, vitamins, and amino acids. In addition, MO has an anti-inflammatory property, antimicrobial, antioxidant, antitumor, anticancer, antihyperglycemic, and diuretic effects^{15,16,17,18,19,20,21}.

Channa micropeltes, also called “ikan toman” in Indonesia, have benefits as a health supplement. The use of toman fish is based on the presence of albumin content. Albumin from fish can suppress pro-inflammatory cytokines so that the damage of tissue and organ can be prevented²². Moreover, albumin has a sulfhydryl (-SH) group that reduces ROS in the body²³. Combination of MO and albumin derived from “toman” fish refers to MA used as a traditional medicine to treat DM. We hypothesize that MA effectively suppresses the overexpression of pro-inflammatory cytokines in DM. Furthermore, the combination of MA as anti-inflammatory properties in DM mice has not been elucidated.

MATERIAL AND METHODS:

Experimental Design:

This study was categorized as experimental research using in vivo procedure. The group consisted of healthy mice (N), untreated DM mice (DM), and DM mice treated by a combination of MO and albumin (MA) with dose 1 (DM-D1), dose 2 (DM-D2), and dose 3 (DM-D3). Each group consisted of 5 replications; thus, we used 25 mice for this experiment.

Animal Model:

A total of 25 female mice (Mus musculus), strain BALB/c with the ages of 8-10 weeks were used in this study. Several studies are known to consider the use of sex/gender to obtain valid data. Most researchers consider male mice to be relatively better used than female mice. The hormonal influence was a consideration in choosing experimental animals. Estrogens, the primary female sex hormone, are suggested to protect against the development of metabolic syndrome such as DM²⁴. However, this does not exclude the possibility of DM complications in women. So that in our study, we specifically wanted to know the effect of MA on female mice experiencing DM. Therefore, the treatment is carried out for 14 days to prevent female mice from experiencing self-repair of the immune system.

The mice were maintained in standard cages at a free-pathogen facility in the Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Malang, Indonesia. All of the study procedures were approved by The Ethical Committee of Brawijaya University (Reg. No. 953-KEP-UB). Diabetic mice were induced by Streptozotocin (STZ) according to DiaComp Protocols²⁵ with modification. STZ at a dose of 145 mg/kg BW was intraperitoneally injected to get the DM mice model. STZ was dissolved in sterile citrate buffer solution pH 4.5. STZ was more stable in pH 4.5, and it will be quickly degraded in alkaline solutions, then it forms alkylating agent, diazomethane²⁶. Therefore, STZ was prepared immediately before injection. Mice fasted for 4 hours before STZ injection.

Measurement of Blood Glucose:

The concentration of mice blood glucose was determined by collecting the blood from the mice's tail. The blood was put on a glucose kit that had been inserted in a glucometer. The blood glucose of all mice was measured before the treatments. Diabetic mice were determined based on blood glucose concentration with the value ≥ 200 mg/dL after 4 days of STZ injection²⁷. Then, the blood glucose was measured once in 3 days.

Treatment of Moringa oleifera - Albumin (MA) in Mice:

MO leaves were obtained from Materia Medica Batu, Malang, Indonesia as powder, called a simplicia. Five grams of simplicia were dissolved in 50 mL of boiling distilled water for 5 minutes. MO solution was filtered to separate filtrate and residue. The extract was evaporated with a freeze dryer to evaporate water from the extract²⁸. MO leaves extract was combined with albumin in 3 different doses. The combination of MO and albumin hereinafter referred to as MA. Albumin was obtained from the Ifalmin product (PT. Ismut Fitomedika Indonesia, Makassar, Indonesia). The dose of MO was determined as recommended by Luqman et al.²⁹ with modifications and the albumin dose was determined by the instructions on the Ifalmin product. The recommended dose of albumin was 416.25 mg/kg BW after conversion of human dose to the animal. Treatment of MA was given orally to all mice from DM-D1, DM-D2, and DM-D3 for 14 days. The comparison of the MA treatment with modification on each dose as follows:

Dose 1 (DM-D1): (100 mg/kg BW MO): (416.25 mg/kg BW A)

Dose 2 (DM-D2): (150 mg/kg BW MO): (208.15 mg/kg BW A)

Dose 3 (DM-D3): (50 mg/kg BW MO): (624.375 mg/kg BW A).

Spleen Isolation, Antibody Staining, and Flow cytometric Analysis:

Mice were sacrificed, and the lymphocyte cell was isolated from a spleen to determine TNF-α and IFN-γ cytokines. The spleen was washed with Phosphate Buffer Saline (PBS) and homogenized with a 10 mL syringe plunger in a petri dish. The homogenate was collected in a tube and added with PBS up to 8 mL. Centrifugation was performed at 2500 rpm, 10 °C for 5 minutes to obtain a pellet. The pellet was added with PBS and placed in a 1 mL microtube containing 500-1000 µL of PBS for antibody staining.

Sample in microtube was centrifuged at 2.500 rpm, 10 °C for 10 minutes. Then, the sample was aspirated and 2 x 10⁶ cells were subjected to antibody staining. According to company protocol, antibodies were applied at a concentration of 0.005 mg/ 100 μL in this experiment. The cell was incubated with PE-conjugated anti-mouse TNF-α (BioLegend, San Diego) and PE/Cy5-conjugated anti-mouse IFN-γ (BioLegend, San Diego) antibodies. The stained cells were moved into cuvet and added 300-500 µL of PBS for flow cytometric analysis.

Data Analysis:

Flow cytometry data were analyzed using BD Cellquest Pro^TM software (BD Biosciences, San Jose, CA, USA). First, the data were statistically analyzed using the SPSS program with one-way variance analysis (ANOVA) with p-values ≤ 0.05. ANOVA analysis was then continued with Tukey Honestly Significant Difference (HSD) test with p-values ≤ 0.05.

RESULTS:

MA Control IFN-γ Expression in the Diabetic Mouse Model:

The expression of IFN-γ based on flow cytometric analysis showed that there were significant differences between normal and DM. The expression of IFN-γ on DM (7.43%) was higher than normal healthy mice (1.92%) (Figure 1). MA administration at dose 1 was significantly able to decrease IFN-γ compared to DM (r≤ 0.05). Dose 1 had the best impact on diabetic treated mice compared with another dose. The expression of IFN-γ on the DM-D1, DM-D2, and DM-D3 were 3.15%, 6.51%, and 4.33%, respectively.

(A)

(B)

Fig.1: MA administration can control the expression of IFN-γ. (A) Diabetic mice were orally administered by MA for two weeks with different concentrations as mentioned in the method. Spleen cells [2x10⁶) were obtained from the treated mice and then subjected to intracellular cells stained with anti- IFN-γ antibody and analyzed by flow cytometry. N (Normal, controls were healthy type mice without manipulation); DM (diabetic mice); DM-D1 as dose 1; DM-D2 as dose 2 and DM-D3 as dose 3, doses of MA mentioned in methods. (B) The bars are a calculation of IFN-γ-expressing cells in splenic cells.

MA Control TNF-α Expression in Diabetic Mouse Model:

The analysis of TNF-α expression was done by flow cytometry. The results showed that the TNF-α-expressing cells in healthy mice (0.99%) were significantly lower compared to the DM groups (4.01%) (r≤ 0.05) (Figure 2). The administration of MA to DM mice gave varying effects depending on the dose. The expressions of TNF-α in the DM-D1, DM-D2, and DM-D3 were 3.55%, 2.27%, and 1.59%, respectively. The DM-D3 group had a lower TNF-α expression than the DM group (ρ≤ 0.05) and not significantly different from the normal healthy group. From these results, we assumed that only dose 3 of MA contributes to reducing the expression of TNF-α significantly.

(A)

(B)

Fig.2: MA administration can control the expression of TNF-α. (A) Diabetic mice were orally administered by MA for two weeks with different concentrations as mentioned in the method. Spleen cells [2x10⁶) were obtained from the treated mice and then subjected to intracellular cells stained with anti-TNF-α antibody and analyzed by flow cytometry. N (Normal, controls were healthy type mice without manipulation); DM (diabetic mice); DM-D1 as dose 1; DM-D2 as dose 2 and DM-D3 as dose 3, doses of MA mentioned in methods. (B) The bars are a calculation of TNF-α expressing cells in splenic cells.

DISCUSSION:

Our study focused on combining bioactive compounds from MO with albumin in toman fish (Channa micropeltes), also known as MA, to treat the DM mice model by suppressing the level expression of IFN-γ and TNF-α. Our study showed that the expression of IFN-γ and TNF-α was significantly higher in DM groups compared to normal healthy mice groups. Thus, it is demonstrated that IFN-γ and TNF-α play an essential role in DM development. According to Reutens and Atkins³⁰, the highest pro-inflammatory cytokines have been linked to caused complications in DM. Based on the results, MA administration at doses 1 and 3 significantly decreased IFN-γ and TNF-α, respectively. Whereas at other doses (Figures 1 and 2), MA administration can increase IFN-γ and TNF-α expression in DM. So, in this case, the variation of MA doses has a different effect in regulating IFN-γ and TNF-α. In this study, we report that MA acted as an immunomodulatory agent. In this study, we showed that a natural substance is needed in a certain amount to increase or decrease the immune response. Immunomodulatory divided into three groups, such as immunosuppressors, immunostimulators, and immunoregulators^31,32.

DM is a metabolic syndrome associated with inflammation. Inflammation is considered a primary physiologic defense mechanism that helps the body protect itself against infection, but under uncontrolled inflammation becomes chronic inflammation. Inflammation occurs when the cytokines overexpression. A higher level of pro-inflammatory cytokines is associated with the incidence of neuropathy³³. Pro-inflammatory cytokines such as TNF-α and IFN-γ are powerful pain-enhancing signals that contribute to neuropathic pain^34,35.

The flow cytometric analysis showed that the decline of IFN-γ and TNF-α (Figures 1 and 2) is assumed due to flavonoids and sulfhydryl (-SH) groups from MA. Every part of MO has a vital nutrient value and serves as a good source of proteins, vitamins, beta-carotene, amino acid, and flavonoids³⁶. Flavonoids such as quercetin are known to have anti-inflammatory effects³⁷. Quercetin works by blocking IκB kinase, so there is no degradation of IκB, which can prevent NFκB activation. Conversely, activation of NFκB can cause a higher level of pro-inflammatory cytokine³⁸.

Furthermore, the albumin in toman fish is believed to function as a food supplement important for human health. Albumin with sulfhydryl (-SH) groups has a function as free-radical scavengers that bind to ROS. These reactions are essential in reducing the level of reactive oxygen species (ROS)³⁹.

Thus, albumin in MA combination may potentially contribute as an antioxidant agent. Furthermore, some studies reported that oxidative stress could induce inflammation and ROS production^40,41,42. Here, we assume that the MA combinations work synergistically and effectively reduces or regulates the expression of pro-inflammatory cytokines by inhibiting ROS. ROS results from normal metabolic processes in cells, which at high concentrations can cause oxidative stress¹⁰. Increased oxidative stress occurs after the formation of advanced glycation end products (AGEs), which can induce the synthesis of various pro-inflammatory cytokines, such as TNF, IL-1α, IL-6, and IFN-γ, resulting in activation of protein kinase-C and nuclear factor {kappa}B, NF-κB^11,12.

CONCLUSION:

MA combination has a role in inhibiting IFN-γ and TNF-α expression in a dose-dependent manner. Therefore, based on the results, we assumed that MA might be one of the biological materials with efficacy to treat DM patients.

ACKNOWLEDGEMENT:

The authors gratefully thank the Ministry of Research, Technology, and Education of the Republic of Indonesia and the Animal Physiology Laboratory Staff for assistance in this research.

CONFLICT OF INTEREST:

The authors have no potential conflict of interest relevant to this article.

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Received on 12.02.2021 Modified on 15.04.2021

Research J. Pharm.and Tech 2022; 15(2):628-632.

DOI: 10.52711/0974-360X.2022.00103