Effect of Melatonin and Insulin on Oxidative Stress in Spleen, Impairment of Adherent Leukocytes Activity and Inammatory Response in Lps Treated Diabetic Mice

The present study evaluated the protective effect of melatonin and insulin from LPS and diabetes-induced oxidative stress, impairment of peritoneal leukocyte functions, and inammation in Swiss albino mice. Diabetes was induced in Swiss albino mice by streptozotocin treatment. Experimental mice were divided into two sets. Set-1 contains control, diabetic, melatonin, insulin, and melatonin+insulin groups of mice. In set-II, all groups of mice were challenged with a single dose of LPS (50mg/mice). Lipid peroxidation, antioxidant enzymes (SOD, Catalase, GPx) activity, non-enzymatic GSH level, Nrf2/HO-1 expression, phagocytic index, intracellular ROS generation, and proinammatory cytokines were measured. Diabetic as well as LPS treated diabetic mice showed signicantly increased lipid peroxidation, suppressed antioxidant defence system, and down regulated Nrf2/HO-1 expression in spleen tissues; suppressed phagocytic index and increased ROS generation in adherent peritoneal leukocytes and increased level of circulatory proinammatory cytokines. Either treatment of melatonin or insulin signicantly improved the harmful effects caused by both of LPS treatment and of diabetes in experimental mice. Simultaneous treatment of melatonin and insulin effectively ameliorated the LPS, as well as diabetes, caused oxidative load, impairment of adherent peritoneal leukocyte function, and improved the level of circulatory proinammatory cytokines. In the present study, we have noted that combined treatment of melatonin and insulin was more effective in attenuation of diabetes and LPS induced devastating effects in laboratory mice. Therefore, the present study may suggest a combinatorial approach in the therapeutic use of melatonin and insulin to improve such devastating conditions. rearrangement


Introduction
The bacterial endotoxin lipopolysaccharide (LPS) is a component of cell wall of Gram-negative bacteria.
Exposure of LPS induces generation reactive oxygen species (ROS) which leads to oxidative stress. In healthy conditions ROS are generated in controlled manner and as signalling molecules regulate various functions i.e. cell proliferation, in ammation, immune response and stress response [Ma, 2013;Kallapura et al, 2014]. De ciency of insulin or functional disability of insulin leads to a multifaceted clinical syndrome known as diabetes mellitus (DM) [American Diabetes Association, 2019]. Bacterial lipopolysaccharide caused induction of proin ammatory cascades in diabetic organism which resulted in oxidative stress and hampers bodily immune response.
The pathogenesis of various diabetic complications is said to be contributed by macrophages and other innate immune cells as they are also known to have a pro-in ammatory phenotype [Tesch, 2007]. Antigen induced in ammatory response of an organism leads to an increase in the level of proin ammatory cytokines such as interleukins (IL-1, IL-6 and IL-18) [Dinarello, 2018]. Low level of circulatory cytokines is important to regulate the oxidative damage and interrelated complications in diabetic patients. High blood sugar caused reduced bactericidal function and wound healing capacity of innate immune cells in diabetic patients [Moura et al, 2019]. Spleen is a large lymphatic tissue passed by re-circulating lymphocytes which provoke speci c T or B lymphocyte-mediated immune reactions. The spleen lters bacteria and viruses out of the blood and stores lymphocytes for release when required [Lewis et al, 2019]. Functional and morphological alterations in the spleen result in the pathogenesis of diabetes and obesity-related chronic kidney disease [Buchan et al, 2018].
Insulin is essential for the carbohydrate homeostasis, growth, and development of tissues. It stimulates transportation of glucose into the peripheral tissues such as muscles and adipocytes but inhibits gluconeogenesis and glycogenolysis in the liver [Qaid and Abdelrahman, 2016]. The presence of insulin signalling cascade components in macrophages suggests insulin-mediated alterations in macrophages functions [Ieronymaki, 2019]. Insulin promotes anti-in ammatory Th2 differentiation in CD + lymphocytes mediated by ERK phosphorylation. Insulin receptors are up-regulated in activated both CD4 + and CD8 + T cells [Viardot et al, 2007].
Melatonin is known for its anti-in ammatory, anti-oxidative and immunomodulatory properties. Melatonin acts through sensitization of its G-protein coupled receptors named melatonin receptor 1 (MT1) and melatonin receptor 2 (MT2) [Favero et al, 2017]. Melatonin regulates physiological synchronization of glucose metabolism and stimulates insulin secretion (GSIS) [Simões et al, 2016]. Many in vivo and in vitro studies have suggested that melatonin plays an important role in neuro-immunomodulation. Melatonin possesses great functional versatility exhibiting anti-oxidant, oncostatic, anti-aging, and immunomodulatory effects [Calvo et al, 2013].
Pathogenesis of diabetes caused oxidative stress and impaired immune responses. Reports suggested that both insulin and melatonin are having anti-glycaemic effects and protects the organism from harmful effects of hyperglycaemia. But protective effectiveness of melatonin and insulin from harmful effects of bacterial endotoxin in diabetic condition is less understood. Therefore, in the present study we elucidated the effect of melatonin and insulin on oxidative stress, impairment of peritoneal leukocyte function and in ammatory response in LPS treated diabetic mice.

Methodology
All of the experiments with animals and their maintenance have been done according to the institutional practice and with the framework of CPCSEA (Committee for the Purpose of Control and Supervision of Experimental Animals) and the Act of Government of India (2007) for the animal welfare.

Animal Model
Healthy Swiss albino mice colony was maintained in appropriate laboratory conditions of light (12 h L:12 h D), temperature (25 ± 2°C), and humidity (55 ± 5%). Rice husk was used to make the bed, and mice were permitted ad libitum access to drinking water and typical pellet diet. 10 weeks old healthy male mice (average body weight 25-26g) were selected for the experiment.

Experimental setup
Diabetes in experimental mice was induced by multiple low doses of STZ administration (Sutradhar et al., 2020). Fifty experimental animals were divided into two sets of experimental groups as follows: Set I Set II Group-I: Con Group-I: Con + LPS Group-II: DB Group-II: DB + LPS Group-III: DI Group-III: DI + LPS Group-IV: DM Group-IV: DM + LPS Group-V: DMI Group-V: DMI + LPS In set I-Control (Con) mice were received ethanolic saline (0.01%). Group II represents diabetic (DB) mice. Group III represents diabetic mice received insulin (2IU/100g body weight). Group IV represents diabetic mice received melatonin (100µg/100g body weight). Group V represents diabetic mice received both insulin and melatonin. In Set II mice of all the 5 groups were received single dose of LPS (50µg/mice/ip) 24 hours prior to euthanisation. Peritoneal uid was collected. Blood of each experimental mouse was collected in EDTA vial. Spleen of experimental mice was dissected out and processed for oxidative stress and culture analysis.

Blood glucose determination
Blood glucose was determined with the help of the ACCU-CHEK Active blood glucose monitoring system.

Determination of lipid peroxidation level in spleen
Malondialdehyde is a product of lipid peroxidation and was measured based on its reaction with thiobarbituric acid (TBA) following the method of Ohkawa et al [1979]. 10% homogenate of the spleen was prepared in phosphate buffer. 0.1 ml of tissue homogenate was mixed with 3.3 ml of TBA reagent [containing 8% SDS, 20% acetic acid (pH 3.5), 0.8% TBA and 0.8% butylated hydroxyl-toluene]. Reaction mixtures were boiled, and the optical density of the supernatant was determined at 532 nm. Lipid peroxidation was expressed in nmol TBARS formed/mg protein of experimental tissues.
Evaluation of superoxide dismutase (SOD) activity level in spleen Superoxide dismutase (SOD; EC 1.15.1.1) activity in the spleen of experimental mice was determined by following the method of Das et al [2000]. 10% homogenates of the spleen tissues were prepared in phosphate-buffered saline (pH = 7.4). 0.1 ml of the homogenate was mixed with 1.4 ml of the reaction mixture (containing 50mM phosphate buffer (pH, 7.4), 20mM L-methionine, 1% Triton-X-100, 10mM hydroxylamine hydrochloride, 50 mM EDTA). 50 mM of ribo avin was added to the mixture and exposed to a 20W uorescence lamp. 1ml of Griess reagent was added and optical density was determined at 543 nm. One unit of enzyme activity is de ned as the amount of SOD inhibiting 50% of nitrite formation under assay conditions and was expressed as SOD activity in U/g tissue weight.
Evaluation of catalase (CAT) activity level in spleen Catalase (CAT; EC 1.11.1.6) activity in the spleen of experimental mice was determined by following the method of Sinha [1972], modi ed by Hadwan [2016]. 10% homogenate of the spleen was prepared in phosphate buffer (pH = 7.4) and centrifuged. The supernatant was added to a reaction mixture containing H 2 O 2 and potassium dichromate and boiled in a water bath and centrifuged. The optical density of supernatant was determined at 570 nm and the decrease in the H 2 O 2 content was calculated. The activity of CAT was expressed as the amount of H 2 O 2 degraded per minute.
Evaluation of glutathione peroxidase (GPx) activity level in spleen Glutathione peroxidase (GPx) was determined following the methods of Kusuma and Vedula, [2016]. 50µl of 10% spleen homogenate in PBS (pH = 7.4) was taken in a test tube and mixed with 450µl of water. 2.5ml of assay mixture [75mM Phosphate buffer (pH 7.0), 60mM Glutathione, 30 units/ml Glutathione reductase, 15mM EDTA, 3mM NADPH] was added to the test tube. 7.5mM H 2 O 2 was added to the mixture and absorbance was taken at 340nm immediately for 3 minutes. The glutathione peroxidase activity was expressed as moles of NADP + formed/min/mg protein.

Evaluation of reduced glutathione (GSH) level in spleen
Reduced glutathione (GSH) was determined following the methods of Ellman [1959], modi ed by Gupta et al [2003]. 10% homogenate of the spleen was prepared in phosphate buffer (pH = 7.4) and mixed with 20% trichloroacetic acid in a 1:1 ratio and centrifuged. 200ul of supernatant was mixed with 1.8 ml of Ellman reagent (containing 1% sodium citrate and 0.04% DTNB in 0.1M phosphate buffer (pH = 8.0)). The optical density of the mixture was determined at 412 nm. The concentration of reduced glutathione was expressed as mg/g tissue.

Immunohistochemistry
Immunohistochemical study of Nrf2 and HO-1 in the spleen was done following the modi ed methods of Singh et al (2017). Depara nized and rehydrated sections were placed in phosphate-buffered saline (PBS) for 30 min. Endogenous peroxidase activity was blocked by 0.3% H 2 O 2 in methanol for 30 min at room temperature. Sections were washed with PBS and placed in blocking solution (horse blocking serum, diluted 1:100 in PBS, PK-6200, Vector Laboratories, Burlingame, CA) followed by incubation with primary antibodies [Nrf2; ab31162 and HO-1; ab31163 rabbit polyclonal, Abcam, Cambridge, MA, diluted 1:100] overnight at 4ºC. Sections were washed and incubated with biotinylated secondary antibody (Vectastain ABC Universal Kit, PK-6200, Vector Laboratories, Burlingame, CA, dilution 1:1000). Sections were washed and incubated with preformed AB complex reagent for 30 min. The immune interactions were visualised using the 0.03% peroxidase substrate 3,3-diaminobenzidine (DAB; Sigma-Aldrich Chemicals, St. Louis, MO) and counterstained with Ehrlich's hematoxylin. Sections were dehydrated and mounted with DPX. Microphotographs of the stained sections were taken under 40X objective in Olympus BX-41 Microscope. The speci city of antibodies reactivity was published elsewhere [Sutradhar et al, 2020]. The intensity of immune reactivity was quanti ed by Image J software.

Isolation of peritoneal macrophages
The mouse was euthanized and 70% alcohol was sprayed on its belly region. A small incision was made with a sterilized scissor on the outer skin of the peritoneal cavity and exposes the inner skin of the peritoneal cavity. 5 ml of sterilized PBS was injected in the peritoneal cavity with a 25'gauge needle.
Mouse belly region was gently massaged with hand. Nearly 5ml of peritoneal uid was withdrawn with 22 gauge of the needle and kept in sterilized 15ml of the centrifuge tube. The peritoneal uid was centrifuged and washed with PBS twice and the pellet was resuspended in a culture medium containing 10% FCS.

Spectrophotometric analysis of ROS level
The intracellular ROS level in macrophages was measured through spectrophotometric analysis. In spectrophotometry intracellular ROS level in macrophages were determined following the methods of Majewski et al, [2005]. The Peritoneal leukocytes were plated in 96 well culture plates (10 6 cells/well) and kept for 1h at 37ºC in a 5% CO 2 incubator. After 1h wells were washed with PBS to remove non-adherent cells. 100ul of NBT (1mg/ml) in PBS was added to each well and incubated for another 1h at 37ºC in a 5% CO 2 incubator. After 1h medium was discarded and cells were xed by 70% methanol and then washed with PBS. The formazan crystals were dissolved by adding 120µl of KOH and 140µl of dimethyl sulphoxide (DMSO) and absorbance was taken at 630 nm UV-vis spectrophotometer.

Microscopic analysis of ROS level
Microscopic analysis of cellular ROS was assessed with the uorescent probe H 2 DCFDA. The peritoneal leukocytes were plated on sterilized clean cover glass in a sterilized petri dish (10 6 cells/ml) and kept for 1h at 37ºC in a 5% CO 2 incubator. After 1 hr. of incubation, the cover glass was washed with PBS to remove non-adherent cells. The adherent cells were stained with a 40µM solution of H 2 DCFDA for 30 minutes. Excess dye was washed with PBS and cover glass was mounted on a grease-free slide. The uorescent images were taken by uorescence microscope (Leica 2500) at 488nm of excitation emission. The uorescence generated was measured as corrected total cell uorescence (CTCF), analysed by Image J software, and expressed in terms of mean uorescence intensity [Rastogi and Haldar, 2016].

Evaluation of the change in phagocytic activity of macrophages
The macrophage phagocytic assay was performed following the method of Roy and Rai [2008]. The heat killed yeast cells suspension was ooded on macrophage monolayer and incubated for 90 minutes. The slides were washed with PBS and xed with methanol. The methanol xed slides were stained with Giemsa. Random macrophages were counted and Percent phagocytosis was determined by following formula-the number of phagocytic cells per 100 cells.

Determination of proin ammatory cytokine level
The circulatory level of proin ammatory cytokines (TNFα, IL-1β, and IL-6) were measured by commercial kits. TNFα level was determined by murine TNFα ELISA kit, IL-1β was determined by IL-1β ELISA kit and IL-6 level was determined by murine IL-6 ELISA kit following the manufacturers protocol (Diaclone SAS, France). For TNFα inter-assay and intra-assay coe cient was 9.4% and 5% respectively and sensitivity was 10.7pg/ml. For IL-1β inter-assay and intra-assay coe cient was > 10% and sensitivity was 4pg/ml. For IL-6 inter-assay and intra-assay coe cient was 7.2% and 7.3% respectively and sensitivity was 10pg/ml.

Statistical Analysis
Statistical analysis of the data was performed with one-way analysis of variance (ANOVA) followed by Tukey's honest signi cant difference (HSD) multiple range test. The differences were considered signi cant when p < 0.01. Statistical Package for the Social Sciences (SPSS) and Microsoft Excel program was used for calculation and graph preparation.

Effect of melatonin and insulin on blood glucose level
Insulin is well known for reducing blood glucose level. Insulin treated diabetic mice showed signi cant low glucose level whereas melatonin treatment maintained signi cantly low blood glucose level in comparison to diabetic mice (Fig. 1). The combined administration of melatonin and insulin caused a substantial drop in blood glucose level in diabetic mice. LPS supplementation caused increase of blood glucose level in all treatment groups in comparison with non LPS treated groups. Combined treatment of melatonin and insulin caused strong suppression of blood glucose level in comparison with either treatment.

Effect of melatonin and insulin on lipid peroxidation level in spleen
MDA is a reliable marker for detection of lipid peroxidation of membrane lipids and it was measured in terms of nmol TBARS formed per mg protein of spleen. Diabetic mice showed signi cantly (p < 0.01) increased MDA level in the splenic tissue in comparison with the control mice (Fig. 2). LPS treatment to diabetic mice caused signi cant (p < 0.01) increase of the MDA level in the spleen in comparison with diabetic mice. Treatment of melatonin and insulin individually caused signi cant (p < 0.01) decrease of MDA level in LPS supplemented diabetic mice. Simultaneous treatment of melatonin and insulin to LPS treated diabetic mice showed highly signi cant suppression of MDA level in comparison with other treatment groups.
Effect of melatonin and insulin on superoxide dismutase (SOD) enzyme activity SOD enzyme is an important antioxidant enzyme that responsibly neutralizes superoxide anion formed in the living tissues. Signi cant (p < 0.01) decrease in SOD activity was observed in diabetic mice (Fig. 3). LPS administration to diabetic mice caused a signi cant (p < 0.01) decrease in SOD enzyme activity in comparison with diabetic mice. Melatonin and insulin individually caused signi cant (p < 0.01) increase in the SOD enzyme activity in LPS treated diabetic mice. Co-administration of insulin and melatonin showed additive effect in the restoration of SOD enzyme activity in LPS treated diabetic mice.

Effect of melatonin and insulin on catalase (CAT) enzyme activity
Catalase is an essential antioxidant enzyme that responsibly neutralizes the hydrogen peroxide formed in the living tissues. Signi cant (p < 0.01) suppression of the catalase activity was noted in the spleen of diabetic mice in comparison with the control mice (Fig. 4). LPS treatment to diabetic mice caused signi cant (p < 0.01) suppression of catalase enzyme activity. Signi cant (p < 0.01) increase in the catalase activity was observed in melatonin and insulin treated LPS group of diabetic mice. Concurrent treatment of melatonin and insulin suggestively augmented the catalase activity in LPS treated diabetic mice.
Effect of melatonin and insulin on glutathione peroxidase (GPx) activity in spleen Glutathione peroxidase (GPx), a member of the enzymatic-antioxidant defence armoury, is present ubiquitously in the cytosol and mitochondria. GPx detoxi es lipid peroxides by reducing them to their corresponding alcohol and hydrogen peroxide to water. The present study showed that the diabetic group of mice showed signi cant (p < 0.01) low level of GPx activity (Fig. 5). LPS treatment to diabetic mice signi cantly (p < 0.01) suppressed the GPx activity. The individual supplementation of melatonin and insulin signi cantly (p < 0.01) increased the GPx activity in LPS treated diabetic mice. The combined administration of melatonin and insulin showed an additive effect and signi cantly (p < 0.01) increased the GPx activity in LPS treated diabetic mice.

Effect of melatonin and insulin on reduced glutathione (GSH) level
Glutathione is an important antioxidant and by donating an electron, it reduces hydroperoxide to their corresponding alcohols in living tissues. We observed that diabetic mice showed a signi cant (p < 0.01) reduction in the splenic glutathione level in comparison to the control mice (Fig. 6). However, LPS treatment in diabetic mice further signi cantly (p < 0.01) lowered the splenic GSH level in comparison to diabetic mice. LPS treated diabetic mice showed signi cant (p < 0.01) increase in splenic GSH content after melatonin or insulin supplementation whereas combine treatment of melatonin and insulin alleviated the LPS caused suppression of GSH level in spleen of mice.

Effect of melatonin and insulin on Nrf2/HO-1 reactivity in spleen
Protection against oxidative radicals is given by the expression of antioxidant proteins which are chie y regulated by the nuclear factor Nrf2. HO-1 is one of the genes regulate through Nrf2 in the mammalian system. Decreased reactivity of Nrf2 and HO-1 antisera was noted in spleen of diabetic mice. LPS treatment caused decrease in Nrf2 and HO-1 reactivity (Fig. 7, 8). Either supplementation of melatonin or insulin increased the Nrf2 ad HO-1 reactivity in the spleen tissue of LPS treated diabetic mice. The combine treatment of melatonin and insulin effectively reduced the LPS induced changes and increased the Nrf2 and HO-1 reactivity in spleen and diabetic mice.

Effect of melatonin and insulin on cellular ROS of peritoneal macrophages
Intracellular ROS of peritoneal macrophages can be measured by the amount of NBT converted to waterinsoluble blue formazan crystal by superoxide anions. ROS generation in the diabetic group is signi cantly (p < 0.01) higher in comparison to the control group of mice. LPS treatment to diabetic mice signi cantly increased the intracellular ROS generation in comparison with diabetic mice (Fig. 9). Individual supplementation of insulin and melatonin signi cantly (p < 0.01) suppressed the ROS level in comparison with diabetic mice. The combined treatment of melatonin and insulin neutralized the adverse effects of LPS and diabetes on intracellular ROS generation and minimized the intracellular ROS to control level.
The intracellular ROS, total oxidative stress generated in the cell at any time point can also be measured with uorescent compound H 2 DCFDA. The total cellular ROS was assessed for adherent peritoneal leukocytes of all treatment groups and reported in terms of mean uorescence intensity. Diabetic and LPS treated diabetic group showed signi cant increased (p < 0.01) uorescent intensity in comparison with control mice (Fig. 10). Melatonin and insulin individually reduced the ROS level thus signi cantly (p < 0.01) supressed the uorescent intensity in comparison with diabetic and LPS treated diabetic group as well. Simultaneous supplementation of melatonin and insulin caused signi cant suppression of uorescent intensity in comparison with either treatment groups.

Effect of insulin and melatonin on the phagocytic index of macrophages
Phagocytic activity was assessed by the number of yeast cells phagocytised by the peritoneal macrophages. Signi cant (p < 0.01) decrease in the phagocytic index of macrophage was noted in the diabetic group of mice in comparison to the control group of mice. LPS supplementation to diabetic mice caused signi cant (p < 0.01) suppression of phagocytic index in comparison with diabetic mice (Fig. 11).
Melatonin and insulin signi cantly (p < 0.01) increased the phagocytic index of macrophages as compared to the diabetic group. Simultaneous supplementation of insulin and melatonin to LPS challenged diabetic mice showed a signi cant increase in the phagocytic index of macrophages in comparison with other experimental groups of mice.
Effect of insulin and melatonin on the production of proin ammatory cytokines Diabetes induction caused a signi cant (p < 0.01) increase in the circulatory level of proin ammatory cytokines TNFα (Fig. 12), IL-1β (Fig. 13), and IL-6 ( Fig. 14) in comparison with the control group of mice. LPS treatment to diabetic mice caused a signi cant (p < 0.01) increase in circulatory proin ammatory cytokines level in comparison with diabetic mice. Individual or combined treatment of insulin and melatonin to LPS challenged diabetic mice showed signi cant (p < 0.01) suppression of proin ammatory cytokine levels in comparison with LPS challenged diabetic mice.

Discussion
An e cient and effective immune response requires increased immune cell proliferation and induces high energy-consuming processes such as biosynthetic and secretory activities of immune cells. Glucose is the most effective energy source for these processes [Pearce et al, 2013]. Diabetes indicates ine cient insulin production or a reduction in insulin sensitivity. In the present study, streptozotocin administration caused β-cell destruction and reduced the insulin production which results in the persistent hyperglycaemic condition in the diabetic mice. LPS, a bacterial endotoxin activates Toll-like receptor 4 (TLR4) in immune cells and induced release of proin ammatory cytokines which cause in ammation [Ramachandran, 2014]. Present observation showed that LPS administration to diabetic mice caused signi cant increase in the level of different proin ammatory cytokines. Individual treatment of melatonin and insulin reduced the proin ammatory cytokine levels considerably. Co-implementation of melatonin and insulin caused signi cant reduction of the in ammatory load in the diabetic mice group. Failure of diabetic mice to stand a considerable in ammatory response may lead to sepsis. The increased in ammatory response also leads higher mortality in diabetic mice [Hassan et al, 2020]. Further, exaggerated or unregulated prolonged in ammatory process can induce tissue damage and is the cause of many chronic diseases [Biswas, 2015].
In ammation and oxidative stress are closely related to pathophysiological procedures. A critical component of in ammation is the in ltration of in ammatory cells, like neutrophils, monocytes, and lymphocytes [Abdulkhaleq et al, 2018]. In present investigation, signi cant decreased level of SOD, CAT, GSH, GPx and increased TBARS formation showed high oxidative load in spleen of LPS treated diabetic mice. The treatment of insulin and melatonin simultaneously helped the mice to cope up with this oxidative stress signi cantly. Individual treatment with insulin and melatonin was also effective in lowering the oxidative load but concurrent treatment of both was more effectively reduced the oxidative load in spleen of studied mice. Studies suggested that an intracellular signalling cascade of proin ammatory gene expression can be initiated by free radicals. The report has also suggested that in response to overstated oxidative stress, in ammatory cells can also release reactive species at the site of in ammation [Mittal et al, 2014].
Nrf2 is an important player of cellular redox balance to sustain homeostasis inside the cells. Nrf2 binds to the antioxidant response element (ARE) in the nucleus causing the transcriptional activation of some antioxidant and detoxifying genes [Ishfaq et al, 2019]. GST, HO-1 are few of the antioxidant enzymes which are activated by Nrf2. In the present study we have observed down regulation the Nrf2/HO-1 axis in spleen of LPS treated diabetic mice. Combine treatment of melatonin and insulin effectively minimized the LPS caused suppression of the Nrf2/HO-1 expression in spleen of mice. Recent studies suggested that up-regulation of Nrf2 pathway protects the body from oxidative stress and injuries [Wang et al, 2018]. Diabetes caused down regulation of expression pro le of Nrf2/HO-1 in spleen of mice [Sutradhar et al, 2020].
During the in ammatory process, large amounts of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced by activated macrophages and neutrophils to kill the invading agents [Kalyanaraman, 2013]. The present study showed that LPS treatment increased the ROS generation in peritoneal leukocytes as detected by using the uorescent dye, H 2 DCFDA. The oxidation of H2DCFA emits an intense green uorescence. The uorescence is proportional to the level of ROS generation. Melatonin as antioxidant minimized the level of uorescence emittance in peritoneal leukocytes. Melatonin in presence of insulin showed high signi cant suppression of uorescence. Intracellular ROS generation in peritoneal macrophages can also be measured by the amount of NBT converted to water-insoluble blue formazan crystal by superoxide anions. Diabetes and LPS supplemented diabetes group of mice showed signi cant higher level of ROS in experimental mice. The action of melatonin and insulin signi cantly reduced ROS generation. Antioxidant melatonin in presence of insulin more effectively quenched the free radicals and minimized the oxidative stress. During pathological in ammatory conditions excessive generation of reactive species may be induced and the diffusing out of ROS from the phagocytic cells sometime caused localized oxidative stress and tissue damage [Fialkow et al, 2007].
The ability of pathogens to resist the elimination and power of different macrophage stimulators to combat these pathogens can be determined by phagocytic assay. In the present study, phagocytosis was estimated by the number of yeast cells phagocytised by the macrophages. LPS treatment to diabetic mice signi cantly reduced the phagocytic activity of peritoneal macrophages. Insulin as well as melatonin improved the phagocytic index of peritoneal macrophages while combine treatment highly increased phagocytic activity of peritoneal macrophages. The report suggested that bacterial LPS potentially inhibited phagocytosis of apoptotic neutrophils by peritoneal macrophages [Feng et al, 2011].
Insulin increased the phagocytic activity, H 2 O 2 production, and glucose metabolism of the macrophages [Costa Rosa et al, 1996]. Melatonin potentiates the phagocytic activity of testicular macrophages in diabetic rats by increasing intracellular free Ca 2+ levels . Further, melatonin also inhibited the expression of various genes (i.e. IL-1β, IL-6) induced by LPS in RAW264.7 macrophages [Kadena et al, 2017] and cultured mouse mammary tissue [Yu and Tan, 2019].

Conclusion
Overall experimental data showed that LPS caused aberrations in the diabetic condition led to severe health-related complications. Insulin has been already prescribed as medicine to diabetic patients for glucose uptake by the cells for proper functioning. Increase in the striking role of melatonin's e ciency in amending different abnormalities leads towards its medicinal application in different diseases. The present study showed that melatonin and insulin together effectively restored the LPS induced oxidative stress, impaired peritoneal leukocyte function and in ammatory response in diabetic mice. Therefore, nding of the present study may suggest a combinatorial therapeutic application of insulin and melatonin in such pathological conditions.

Declarations Disclosure Statement
The authors declare that they have no con ict of interest that would prejudice the impartiality of this scienti c work.  Effect of melatonin and insulin treatment on super oxide dismutase (SOD) activity in the spleen of diabetic and LPS treated diabetic mice. Histograms represent Mean + SEM, n=5 for each group. Con = control, DB = diabetic, DI = diabetic mice received insulin, DM = diabetic mice received melatonin, DMI = diabetic mice received insulin and melatonin, DL = LPS treated diabetic mice. ** p<0.01, Con vs respective group; a p<0.01, DB vs respective group; b p<0.01, DL vs respective group; # p<0.01, DB vs DL Figure 4 Effect of melatonin and insulin treatment on catalase activity in the spleen of diabetic and LPS treated diabetic mice. Histograms represent Mean + SEM, n=5 for each group. Con = control, DB = diabetic, DI = diabetic mice received insulin, DM = diabetic mice received melatonin, DMI = diabetic mice received insulin and melatonin, DL = LPS treated diabetic mice. ** p<0.01, Con vs respective group; a p<0.01, DB vs respective group; b p<0.01, DL vs respective group; # p<0.01, DB vs DL Figure 5 Effect of melatonin and insulin treatment on glutathione peroxidase (GPx) activity in the spleen of diabetic and LPS treated diabetic mice. Histograms represent Mean + SEM, n=5 for each group. Con = control, DB = diabetic, DI = diabetic mice received insulin, DM = diabetic mice received melatonin, DMI = diabetic mice received insulin and melatonin, DL = LPS treated diabetic mice. ** p<0.01, Con vs respective group; a p<0.01, DB vs respective group; b p<0.01, DL vs respective group; # p<0.01, DB vs DL Figure 6 Effect of melatonin and insulin treatment on reduced glutathione (GSH) level in the spleen of diabetic and LPS treated diabetic mice. Histograms represent Mean + SEM, n=5 for each group. Con = control, DB = diabetic, DI = diabetic mice received insulin, DM = diabetic mice received melatonin, DMI = diabetic mice received insulin and melatonin, DL = LPS treated diabetic mice. ** p<0.01, Con vs respective group; a p<0.01, DB vs respective group; b p<0.01, DL vs respective group; # p<0.01, DB vs DL  (Mean + SEM, n=5 for each group). Con = control, CL= control + LPS, DB = diabetic, DL = diabetic + LPS, DI = diabetic + insulin, DIL = diabetic + insulin + LPS, DM = diabetic + melatonin, DML = diabetic + melatonin + LPS, DMI = diabetic + melatonin + insulin, DMIL = diabetic + melatonin + insulin + LPS. ** p<0.01, Con vs respective group; a p<0.01, DB vs respective group; b p<0.01, DL vs respective group.  Page 24/29 DI = diabetic + insulin, DIL = diabetic + insulin + LPS, DM = diabetic + melatonin, DML = diabetic + melatonin + LPS, DMI = diabetic + melatonin + insulin, DMIL = diabetic + melatonin + insulin + LPS. ** p<0.01, Con vs respective group; a p<0.01, DB vs respective group; b p<0.01, DL vs respective group.

Figure 11
Effect of melatonin and insulin treatment on phagocytic activity of peritoneal macrophages of diabetic and LPS treated diabetic mice. Histograms represent Mean + SEM, n=5 for each group. Con = control, DB = diabetic, DI = diabetic mice received insulin, DM = diabetic mice received melatonin, DMI = diabetic mice received insulin and melatonin, DL = LPS treated diabetic mice. ** p<0.01, Con vs respective group; a p<0.01, DB vs respective group; b p<0.01, DL vs respective group; # p<0.01, DB vs DL Figure 12 Effect of melatonin and insulin treatment on circulatory TNFα level in diabetic and LPS treated diabetic mice. Histograms represent Mean + SEM, n=5 for each group. Con = control, DB = diabetic, DI = diabetic mice received insulin, DM = diabetic mice received melatonin, DMI = diabetic mice received insulin and melatonin, DL = LPS treated diabetic mice. ** p<0.01, Con vs respective group; a p<0.01, DB vs respective group; b p<0.01, DL vs respective group; # p<0.01, DB vs DL Figure 13 Effect of melatonin and insulin treatment on circulatory IL-1β level in diabetic and LPS treated diabetic mice. Histograms represent Mean + SEM, n=5 for each group. Con = control, DB = diabetic, DI = diabetic mice received insulin, DM = diabetic mice received melatonin, DMI = diabetic mice received insulin and melatonin, DL = LPS treated diabetic mice. ** p<0.01, Con vs respective group; a p<0.01, DB vs respective group; b p<0.01, DL vs respective group; # p<0.01, DB vs DL Figure 14 Effect of melatonin and insulin treatment on circulatory IL-6 level in diabetic and LPS treated diabetic mice. Histograms represent Mean + SEM, n=5 for each group. Con = control, DB = diabetic, DI = diabetic mice received insulin, DM = diabetic mice received melatonin, DMI = diabetic mice received insulin and melatonin, DL = LPS treated diabetic mice. ** p<0.01, Con vs respective group; a p<0.01, DB vs respective group; b p<0.01, DL vs respective group; # p<0.01, DB vs DL