Protective Effect of Nimbolide against High Fat Diet-induced Obesity in Rats via Nrf2/HO-1 Pathway

having the kidney disease along with the obesity, increase the 60 ％ of mortality 6 ） . During the COVID-19 pandemic, the report suggested the increases evidence of obesity, increases the risk factor to develop the more complication of COVID-19. As per the report of Public Health England, BMI 35-45 could enhance the chance of dying due to the COVID 19 （ 40 ％） and when the BMI reach above the 40 could enhance the risk factor 90 ％ 9, 10 ） . During the pandemic COVID-19, the obese patient having the higher risk and Abstract: Current time obesity is the major challenges globally and the incidence of the obesity has raised dramatically in current years. The obesity enhanced the various metabolic diseases such as diabetes, cardiac, cancer and steatohepatitis. Natural drug having the long history to ameliorate the obesity and its related metabolic disorder. In this experimental study, we scrutinized the anti-obesity effect of nimbolide against high fat diet (HFD) induced obesity in rats. Wistar rats were divided into 5 groups and each group contains 10 rats. The body weight, tissue weight was estimated at regular time. Carbohydrate, lipid, hepatic, inflammatory cytokines, antioxidant and inflammatory parameters were estimated. The mRNA expression was also estimated. Nimbolide treated groups significantly ( p < 0.001) suppressed the body weight at dose dependent manner. Nimbolide significantly ( p < 0.001) reduced the hepatic parameters and altered the antioxidant parameters such as thiobarbituric acid reactive substances (TBARS), glutathione (GSH), catalase (CAT), glutathione peroxidase (GPx), superoxide mutase (SOD), glutathione S transferase (GST); decreased the level of inflammatory cytokines (IL-1β, IL-6, TNF-α). Nimbolide suppressed the mRNA expression of glucose-6-phosphatase HO-1 and nuclear factor erythroid-2 related factor-2 (Nrf2). Collectively, we can say that nimbolide having the capability to suppressed the HFD induced obesity via Nrf2/HO-1 pathway.

more chance for the death 6 .
Previous studies already proved the link between the HFD and oxidative stress 11 . Long term taken the high saturated fat diet play an important role as inducer for the oxidative stress and suppressed the endogenous antioxidant defence system 12,13 . During the increase level of oxidative stress, enhances the level of lipid peroxidative LPO in the hepatic tissue and the circulation 13,14 . Report suggests that the oxidative stress pathogenesis of metabolic derangements leadings the obesity, insulin resistance and TIIDM 15 . It is proved that increase the oxidative stress precedes the expansion of obesity and metabolic dysregulation there are induced by an HFD.
Natural product having the long history to treat the various disease due to less side effects and more protective effect 16 . Recent years natural product gain more attraction for researcher due to effective therapies against the various human diseases. Various research investigation have been directed and scrutinize for the biological effects 17,18 . Azadirachta indica A. is very popular tree in China 19 . The bark, flower, leaves, root, seed and bark of the plant used against the diseases and also used in the industrial products 20,21 . The leaves of the plant commonly used for the treatment of diabetes, reducing fever and eczema. The bark of the plant used as toothbrush and its root help to heal the disease and mosquito repellent 17,22 . Nimbolide is the active phyto-constituent of plant and commonly used as anti-diabetic, antimalarial, antitumor, antiulcer, anti-inflammatory and hepatoprotective 20 22 . Due to its antidiabetic and anti-inflammatory and antioxidant effect of nimbolide, in this investigation scrutinize the anti-obesity effect of nimbolide against HFD induced obesity in rats and explore the possible mechanism.

Animals
Sprague Dawley SD rats sex-male, weight 200 20 g; aged 3 weeks were used for the current experimental study. The rats were kept in the standard laboratory condition such as maintained the temperature 20 5 ; relative humidity 65 with 12/12 h dark/light. The rats were kept in the laboratory condition for 7 days before the experimental study.

High fat diet
After the acclimatization, the rats were received the HFD mention in Table 1 during the whole animal experiment. The normal control group rats received the standard diet throughout the experimental period. Throughout the whole animal protocol, the rats were received the water ad libitum.

Experimental group
The rats were divided into different groups and each group contains 10 rats as follows Group I: normal control Group II: HFD control Group III: HFD Nimbolide 5 mg/kg , Group IV: HFD Nimbolide 10 mg/kg and Group V: HFD Nimbolide 15 mg/kg , respectively. The body weight of all group rats were estimated at regular time 6 . After the 10 weeks, all group rats were fasted overnight 16 h , before the sacrificed. Ketamine 90 mg/kg and xylazine 10 mg/kg were used for anesthetized the all-group rats. The blood samples were collected from all group rats via puncturing the retro orbital and centrifuged at 15,000 rpm at 4 for 15 min. The serum sample were separated out and kept in the sterile cryovials at 80 for further biochemical analysis.

Organ weight
After scarified the rodent, immediately removed the organ such as kidney, heart, pancreas, aorta, brain and liver within 30 min and the organ washed with the ice-cold phosphate buffered saline to remove the excessive blood clot 23 . After that, the absorbent paper was used for remove the remaining fluids before weighing on electronic balance for getting the constant weight. The ratio of organs hepatic and renal to final body weight was also estimated.

Antioxidant parameters
The antioxidant parameters such as MDA, TAC, enzymatic antioxidant parameters GSH, GPx, SOD and CAT were estimated using the previous reported method with

Lipid pro le
Total cholesterol TC , low density lipoprotein LDL , high density lipoprotein HDL and triacylglycerols TG were analyzed using the colorimetric assay via following the manufacture instruction Bioclin, Belo Horizonte, Brazil . The very low density lipoprotein VLDL was estimated using the following formula VLDL Triacylglycerol 5 2.7 Non-esteri ed fatty acids NEFA The NEFA level was estimated in the serum using the manufacture protocol Elabscience calorimetric kit, USA .

Hepatic parameters
Alanine transaminase ALT , albumin ALB , aspartate transaminase AST and alkaline phosphatase ALP was estimated using the ELISA kits following the manufacture protocol Nanjing Jiancheng Bioengineering Institute, Nanjing, China.

Renal parameters
The level of total protein TP , creatinine and albumin were determined using the previous reported method with minor modification 23, 24 .

Hematological parameters
Hematological parameters such as hematocrit HCT , white blood cell WBC , haemoglobin Hb , red blood carpules RBC , mean cell volume MCV , lymphocytes LYM , platelet PLT and mean corpuscular hemoglobin concentration MCHV were determined using readymade diagnostic reagent kits Sunlong Biotech Co., Ltd. Zhejiang, China .

mRNA expression
The total RNA was isolated from the DNA using the Trizol Invitrogen, Carlsbad, USA using the manufacture protocol. PrimeScript RT reagent kit was used for synthesized the complementary DNA cDNA with gDNA Eraser. 7300 real time PCR detection system was used for conducting the RT-PCR Applied Biosystems, CA, USA . 1 µg total RNA was used as templates with corresponding gene primers Table 2 . The mRNA expression of insulin receptor, Nrf2, phosphoenolpyruvate carboxykinase, glucose-6-phosphatase and HO-1 were estimated using the qRT-PCR. Hypoxanthine guanine phosphoribosyltransferase HPRT1 was used as the internal gene. The data were presented as relative expression levels and were calculated via comparative Ct method ΔΔCt .

Statistical analysis
The data of the current study was analyzed using the GraphPad Prism Software GraphPad Prism 8, St Louis, USA . One way ANOVA with Dunnett s test was used for comparison between the groups. All the result of this experimental study was presented as mean SEM. P 0.05 was considered as the significant. Table 3 exhibited the water intake of all group rats. Normal rats showed the normal pattern for the water consumption throughout the experimental period. Obese rats demonstrated the increased water intake. Nimbolide treatment significantly p 0.001 suppressed the water intake. Nimbolide 15 mg/kg treated rats showed the water intake pattern similar to the normal control rats.

Water intake, body weight and organ weight
During the obesity, increased the body weight commonly observed. HFD induced obese rats exhibited the increased Table 3 The effect of nimbolide on the diet pattern of HFD induced obesity in rats.

S. No
Parameters All the values are expressed as mean SD for six animals. Significant levels are *p < 0.05, **p < 0.01, ***p < 0.001 when compared with DMBA control group.  Fig. 1a and suppressed the kidney relative weight Fig. 1c and hepatic relative weight Fig. 1d . Nimbolide treated rats significantly p 0.001 suppressed the body weight and increased the kidney relative weight and hepatic relative weight. Figure 1b showed the feed intake of all group rats. HFD induced rats exhibited the increased feed intake and nimbolide treated group rats exhibited the suppression of the feed intake and nimbolide 15 mg/kg treated rats exhibited the feed intake similar to the normal patter. Figure 2a demonstrated glucose level of all group rats. HFD induced obese rats demonstrated the increased glucose level and suggest the impairment of the glucose adsorption. Nimbolide treatment significantly p 0.001 suppressed the glucose level and 15 mg/kg treated group rats bring the glucose level almost near to the normal level.

Glucose and NEFA
The level of NEFA was considerably boosted in the HFD induced obesity rats. The similar result was observed in the HFD induced obesity rats. Nimbolide treated rats significantly p 0.001 suppressed the level of NEFA Fig. 2b .

Lipid parameters
The alteration of lipid parameters is commonly observed during the obesity. In this experimental study, HFD induced obese rats exhibited the boosted level of TC, TG, LDL, VLDL and reduced level of HDL. Nimbolide treatment significantly p 0.001 altered the lipid parameters Fig.  3 .

Resistin, HFABP and leptin
HFD induced obese rats showed the boosted level of resistin, HFABP and leptin as compared to other group rats. Fig. 1 Showed the effect of nimbolide on the body weight, relative organ and fed intake of HFD induced obesity in rats. a: body weight, b: fed intake, c: kidney relative weight and d: hepatic relative weight. Statically significant different are presented via asterisks. Where *p 0.05, **p 0.01 and ***p 0.001 was considered significant, more significant and extreme significant. Each group contain the 10 rats. Nimbolide treatment significantly p 0.001 reduced the level of resistin Fig. 4a , HFABP Fig. 4b and leptin Fig.  4c .

Hepatic parameter
During the obesity, the alteration of hepatic parameter is commonly observed due to deposition of fat in the hepatic tissue. HFD induced obese rats showed the increased level of AST Fig. 5a , ALT Fig. 5b and ALP Fig. 5c and nimbolide treatment significantly p 0.001 decreased the level of hepatic parameter.

Non-hepatic parameter
HFD induced obese rats showed the suppressed level of albumin, TP and increased level of creatinine as compared to other treated or non-treated rats. Nimbolide treated rats significantly p 0.001 increased the level of albumin Fig.  6a , TP Fig. 6b and reduced the level of creatinine Fig.  6c .

Antioxidant parameters
HFD induced obese rats showed the increased level of MDA and suppressed level of CAT, SOD, GPx, GSH, TAC. Nimbolide treatment significantly p 0.001 decreased the Fig. 3 Showed the effect of nimbolide on the lipid parameters of HFD induced obesity in rats. Statically significant different are presented via asterisks. Where *p 0.05, **p 0.01 and ***p 0.001 was considered significant, more significant and extreme significant. Each group contain the 10 rats. level of MDA Fig. 7a and boosted the level of CAT Fig.  7b , SOD Fig. 7c , GPx Fig. 7d , GSH Fig. 7e , TAC Fig.  7f .

In ammatory cytokines
HFD induced obese rats demonstrated the increased level of inflammatory cytokines such as TNF-α Fig. 8a , IL-1β Fig. 8b and IL-6 Fig. 8c . Nimbolide treatment significantly p 0.001 suppressed the level of inflammatory cytokines at dose dependent manner. Table 4 showed the effect of nimbolide on the HFD induced obese rats. HFD induced rats showed the modulated level of Hb, RBC, WBC, MCV, PCV, MCH, MCHC, lym-phocytes, neutrophils, basophils, monocytes and nimbolide treatment altered the level of hematological parameters at dose dependent manner. Figure 9 exhibited the mRNA expression level of all group rats. HFD induced group rats exhibited the reduced mRNA expression of HO-1, Nrf2 and increased expression of G6Pase. Nimbolide treatment significantly p 0.001 boosted the expression of HO-1, Nrf2 and suppressed the expression of G6Pase. Statically significant different are presented via asterisks. Where *p 0.05, **p 0.01 and ***p 0.001 was considered significant, more significant and extreme significant. Each group contain the 10 rats. Fig. 6 Showed the effect of nimbolide on the non-hepatic parameters of HFD induced obesity in rats. a: albumin, b: total protein and c: creatinine. Statically significant different are presented via asterisks. Where *p 0.05, **p 0.01 and ***p 0.001 was considered significant, more significant and extreme significant. Each group contain the 10 rats.

Discussion
Metabolic syndrome is a multifactorial condition of fasting hyperglycaemia, insulin resistance, dyslipidaemia, hypertension and obesity 25,26 . Previous research suggests that presence of metabolic syndrome enhance the risk for the development of the type 2 diabetes mellitus T2DM 24 . HFD proficiently induced the metabolic syndrome which further showed via significant changes in the body characteristics, insulin resistance, dyslipidaemia and fasting hyperglycaemia 13,27 . Previous study showed the HFD indued obesity is a valid animal model for the determination of protective effect of tested drug against the obesity 28 . Due to limitation of the available treatment for the obesity, in this experimental study we try to explore the anti-obesity effect of nimbolide active phyto-constituent of Azadirachta indica against the HFD induced obesity in the rats and explore the underlying mechanism.
In this experimental study, we fed the rats with the HFD presented in Table 1 and HFD group rats exhibited the increased body weight as well as boosted the relative fat mass due to consumption of high dietary fat content. The body weight of HFD group rats boosted possibly due to taken the high fat mass. HFD rats treated with the nimbolide significantly reduced the body weight. This result  showed the ability of nimbolide to suppress the fat mass as well as body weight. Previous research showed the similar result and provide the strength of our experimental study 23 . The hematological parameters were altered during the obesity. In this study, the level of Hb, RBC, MCV, PCV, MCHC, MCH, WBC, eosinophils, basophiles and monocytes were altered. The previous study exhibited the similar result 23 . HFD group rats exhibited the increased level of neutrophil count and suppressed level of lymphocytes which in occurred due to the fact that obesity can start the production of glucocorticoids as well as IL-6 that play an important role in bone marrow granulopoiesis 29 . They further boost the neutrophils mobilization from bone marrow and also induces the prolongation of their intravascular half-life 23,30 . Previous study suggests that lymphocytopenia is commonly observed in the obese rats due to systemic inflammatory response due to suppression of T-cells in thymus, peripheral blood and spleen 31 . Furthermore, the production of the corticosteroids increases during the obesity that further shown the increase production of lymphocytopenia 23,30 . Targeting the lymphopenia and neutrophilia is the best approaches to treated the obesity 23 . Both parameters can be attributed for their antioxidant and anti- G6Pase. Statically significant different are presented via asterisks. Where *p 0.05, **p 0.01 and ***p 0.001 was considered significant, more significant and extreme significant. Each group contain the 10 rats. Table 4 The effect of nimbolide on the hematological parameters against HFD induced obesity in rats. All the values are expressed as mean SD for six animals. Significant levels are *p < 0.05, **p < 0.01, ***p < 0.001 when compared with DMBA control group.

S. No Parameters
obesity effect that can suppress the production of oxidative stress via corticosteroids and cytokines. Nimbolide treatment considerably modulated the haematological parameters at dose dependent.
The current investigation has showed the alteration of lipid profile in the HFD rats that suggesting the dyslipidaemia along with the considerably boosting the glucose level 32,33 . Previous investigation exhibited the similar types of results 32,34 . Obesity is the hallmark of the dyslipidaemia and increased the level of TGs along with the increased level of LDL and suppressed the level of HDL 23 . The dyslipidaemia mainly attributed to enhanced the NEFA level that resultant excessive spill over due to HFD 35,36 . Previous research suggests that the NEFA can affect the insulin signaling pathway, suppress the glucose uptake taken by muscle, increased TGs synthesis and finally induces the gluconeogenesis in the liver 36,37 . All the alteration commonly observed during the hyperglycemia in HFD group and nimbolide modulated the all parameters and suggesting the anti-obesity effect 23 . It is well known that oxidative stress plays an important role in the hyperglycemia and obesity or both. Oxidative stress starts the production of free radicals that can delay glucose utilization via peripheral tissue and insulin function 38,39 .
Previous study suggest that the increased fat mass led to increase the adipokines production include resistin, leptin and inflammatory cytokines 40,41 . The observed hyperleptinemia, as well as the observed decrease in food intake, are suggestive of leptin resistance, a situation that resulted in decreased energy expenditure. Resistin is directly responsible for the expansion of insulin resistance 42 . In this study, we have observed that HFD induced rats exhibited the increase level of resistin and leptin and nimbolide treatment considerably suppressed the resistin and leptin. This result coincided with the previous result 23,43 . However, NEFA can act not only as a source of energy, but it can also play a considerable role in regulation of intracellular protein kinases such as JNK and pKC, which resultant its induces the dysfunction in insulin signaling 42,44,45 . HFD induced rats exhibited the increased level of NEFA and nimbolide treatment considerably suppressed the NEFA level.
IL-6 and TNF-α are considered adipokines marker and both parameter level boosted during the obesity. In this experimental study, we observed the increased level of inflammatory cytokines includes IL-6, TNF-α, IL-1β which can function to boost the inflammatory reaction as well as boosted the obesity 23,46 . During the obesity, start the accumulation of fat in the tissue due to dysfunctional of lipid metabolism such as lipolysis which in turn boost the production and secretion of free fatty acids FFAs 23 . Increased FFAs level can boost the inflammatory cytokines and expansion of insulin resistance. Additionally, reduction of intracellular antioxidant in fat tissue, boosted the production of reactive oxygen species ROS which further induced the oxidative stress. The increased oxidative stress enhanced the production of insulin resistance and inflammatory cytokines 38,41 . Enhancing the NEFA in the obese rat potentially enhanced the inflammatory cytokines and oxidative stress, due to induces the electrophysiological remodelling in the cardiac muscle 44,46 . In this study, HFD group rats exhibited the increased level of lipid peroxidation, glucose and reduced level of endogenous antioxidant enzymes and nimbolide treated rats significantly p 0.001 suppressed the level of glucose and altered the lipid parameters. Nimbolide exhibited these effects due to suppressing the adipokines as well as lipid metabolism and its treatment restores the antioxidant enzymes and suppressed the inflammatory cytokines.
Previous study suggest that Nrf2 affect the various antioxidant protein expression includes NAD P H quinine oxidoreductase 1 NQO1 , glutathione peroxidase and HO-1 phase II detoxifying enzyme 6 . In this experimental study, we scrutinized the anti-obesity effect of nimbolide against HFD induced obesity in rats via alteration of Nrf2/ HO-1 pathway. The result suggests that nimbolide considerably boosted the expression of Nrf2 and HO-1 and previous research suggest that the Nrf2/HO-1 pathway play an important role in induction of obesity and oxidative stress. Definitely, nimbolide administration increased the HO-1 and Nrf2 mRNA expression in hepatic tissue.

Conclusion
The HFD induced obesity is related with the metabolic perturbations that can be arise due to induction of inflammatory reaction and oxidative stress which further exhibited the effect on the kidney, endothelial blood vessels and liver function. In this study, nimbolide considerably reduced the body weight, relative liver, kidney and feed intake. Nimbolide also suppressed the blood glucose level along with the alteration of hepatic and non-hepatic parameters. Nimbolide treatment significantly p 0.001 altered the lipid and antioxidant parameters along with suppression of inflammatory cytokines. The result suggests that the nimbolide having the beneficial effect in the treatment and prevention of obesity, oxidative stress, dyslipidemia, gluconeogenesis and inflammation induced by HFD via Nrf2/HO-1 pathway. These findings point to the possibility of using nimbolide to prevent and treat metabolic syndrome.

Author Contribution
Lin Zhang performed research, Yujun Li Daqing Sun and Feng Bai contributed analytic tools, analyzed data. Feng Bai designed research. All authors wrote the manuscript and corrected the proof.