“Regulation of Obesity and Fatty Liver by Moringa oleifera: Insights into Inflammatory Pathways”

Obesity and fatty liver are relatively benign states but continued inflammatory stress and its metabolic implications turn them into one of the most devastating diseases of humankind. Generally, obesity and fatty liver precede diabetes mellitus, cardiovascular problems and malignant growths. The present research aimed to explore the efficacy of methanolic extract of Moringa Olifera (Me.MO) for the management of obesity and fatty liver and related inflammatory state that prime the body for devastating effects. A series of in-vitro and in-vivo studies were employed. Data from HPLC analysis confirmed the presence of flavonoids and phenolic acids. Rats were fed on either normal diet (ND) or high fat diet (HFD and streptozocin (STZ) in the presence or absence of Me.Mo (250 mg/kg & 500 mg/kg) or metformin (70 mg/kg). Findings showed that rats received 500 mg/kg Me.MO showed a significant (p > 0.01) decrease in body weights, liver weights, and plasma glucose level. Laboratory data exhibited a significant (p < 0.05) inhibitory effect on Me.MO on pro-inflammatory mediators (IL-1B and TNF) and caused a sharp increase in anti-inflammatory cytokines levels (IL-10, IL-6 and COX-2) in all treatment groups. Histopathological analysis exhibited no structural and functional alteration in the liver and adipose tissues. Altogether, Me.MO ameliorates experimentally induced obesity accompanying fatty liver and inflammatory stress. However, further investigations are still needed to confirm the safety and efficacy of Moringaβ olifera (MO) for clinical application.


Introduction
The view that obesity and related metabolic complications are not merely lipid overload conditions but a chronic low-grade inflammatory state with metabolic repercussions has largely changed our understanding of the disease.It is now understood that most obese states incite the immune system [1] that themselves undergo metabolic changes pressed by fats [2][3][4] to initiate a vicious immunometabolic dysregulation cycle [5,6], often culminating in complicated disorders [7,8].
Obesity with a low-grade inflammatory state is often accompanied by fatty liver [9], serum lipid dysregulations [10] and fatal cardiovascular complications [11].Currently, there are five FDAapproved drugs for chronic obesity namely orlistat, phentermine-topiramate, naltrexone-bupropion, liraglutide, and semaglutide [12] but these medications primarily focus on suppressing appetite by targeting GLP-1 [13] or work by reducing the absorption of fat from intestine [14].The lack of anti-inflammatory actions, inability to systemically regulate lipids, varied pharmacokinetic profile and spectrum of adverse drug reactions leave a room for better future choices.
Moringa Olifera (MO) is a tree that belongs to the family Moringaceae and is commonly known as the "Miracle Tree".It is native to Asia and Africa and widely grown in tropical and subtropical climates.The leaves, roots, pods, flowers and seeds of MO possess both nutritional as well as medicinal properties [15].Traditionally, MO has been employed extensively for the treatment of malaria, typhoid, hematological, cardiovascular and gastrointestinal disorders [16].Several studies have demonstrated the presence of secondary metabolites vitamins (A, B, C), alkaloids, glycosides, flavonoids, tannins, saponins and terpenoids in MO tree [17].Some flavonoids (myricetin, quercetin, kaempferol, isorhamnetin and rutin) present in MO leaves and seeds showed excellent antimicrobial and antiproliferative (inhibits S and G2M pathways) and anti-apoptotic effects (downregulate nuclear factor kappa-B) [18,19].Recently, various pre-clinical and clinical studies have evidenced multiple biological activities of MO including gastro-protectant, hypotensive, antidiabetic, hepatoprotective, antimicrobial, antihyperlipidemic, anticancer and antiinflammatory.It has also been exhibited therapeutic effects on renal injury, and thyroid hormone regulation [20].The anti-inflammatory potential of MO are already reported in cancer [21] and renal damage [22] but how it exerts its anti-inflammatory actions under lipid overload states is yet to be discovered.
This study aims to investigate the MO potential in obesity-induced fatty liver and accompanied inflammatory state.This study forms the basis for further experimentation of MO on the immunometabolic axis, which is pivotal for holistic obesity treatment.

Collection and Extraction
The whole plant was collected from Benessere Health Company (BHC), Multan, Pakistan in June 2020 and authenticated by Dr. Altaf Dashti and voucher specimen 3456AS was deposited at the herbarium of the Department of Botany, University of Punjab, Lahore, Pakistan.Firstly, the plant was dried under shade for 14 days then crushed into fine powder and soaked in analytical grade methanol for maceration.The rotary evaporator was under for excessive solvent evaporation under reduced pressure and temperature (45°C).The percentage yield of extract was calculated to be 5% w/w and labeled as Me-MO.

Induction of obesity and lipid dysregulation
At the start of the 1 st week, the body weight and blood glucose level of all animals were measured with the help of glucose oxidase reagent strips (Accu-Chek ® ).The diabetogenic agent streptozotocin (STZ) (30 mg/kg) was injected intraperitoneally in addition to HFD to all animals except group-I [23].After 48 hours, glucose level was again measured and defined doses of plant extract as well as standard was administrated for the period of the next 8 weeks.

Physical parameters
The glucose level and body weight of all animals were measured after a constant interval (1 week).
All rats were maintained on HFD throughout the study except the normal group.On the final day, animals were slaughtered, and blood was drawn for biochemical and hematological analysis.
Changes in weight of liver and adipose tissue were also estimated through Microsoft excel software®.

Histopathological analysis
Liver and adipose tissues were excised and preserved in formalin solution for histopathological examination.Slides were prepared by using hematoxylin and eosin dyes and analyzed under a microscope for any pathological change [26].

High-Performance Liquid Chromatography (HPLC)
To quantify phytoconstituents in Me.MO, high-pressure liquid chromatography was performed.
By using two different types of mobile phases having a composition as phase-A (water and acetic acid in a ratio of 94:6 and pH = 2.27) and phase-B including acetonitrile 15% from 0-15 minutes followed by 45% from 15-30 minutes and 100% from 30-45 minutes.Shim-pack HPLC (CLC-ODS; C-18; Shimadzu, Japan) column having length/height dimensions of 25 cm × 4.6 mm and 5 µm diameter was used to isolate the compounds.Analysis of samples was done with help of an ultraviolet detector (280 nm wavelength).In the end, a chromatogram was drawn between voltage (x-axis) and time (y-axis) units respectively, and compared with standards [27].

Statistical analysis
The analysis of data was done through Graph-Pad Prism Software and Microsoft Excel.Results are presented as mean ± SEM after applying the one-way analysis of variance.The value of "p" below 0.05 is assumed to be significant.

HPLC analysis
Data in Table 1 shows five compounds including quercetin, vanillic acid, chlorogenic acid, synergic acid and m-coumaric acid obtained after HPLC analysis of Me.MO.The retention time, percentage areas and concentration of all phenolic acids in parts per million are also presented.
Moreover, peaks of all identified compounds are shown in the chromatogram (Figure 1).Me.MO 250 mg/kg, Me.MO 500 mg/kg and metformin (70 mg/kg) was calculated to be 205.0 ± 0.55, 291.0 ± 1.00, 206.0 ± 0.15, 215.0 ± 0.30 and 186.0 ± 0.90 respectively.In addition, macroscopic analysis with a naked eye also showed a marked increase and decrease in body weight of rats at the initial (4 th week) and final stage (12 th week) of the study (Figure 3).Similarly, liver weight was also increased in HFD induced group while administration of Me.Mo significantly reversed the raised liver weights (Figure 4).

Assessment of lipid profile
Data in figure 5B

Histopathological analysis
Hepatic tissues showed fatty changes, hyperplasia, hypertrophy and macrophages inflammations in M-HFD and 500 mg/kg Me.MO treatment groups.While no specific changes were observed in 250 mg/kg Me.MO and metformin (70 mg/kg) treatment group except mild inflammation (green arrow).Moreover, relevant modifications in adipose tissues of model-HFD was also seen in form of adiponecrosis (blue arrow) (Figure 6).

Discussion
Weight gain and related fatty liver are considered early risk factors for much larger metabolic complications [28].Obesity and accompanied fatty liver are regarded as benign states but often precede cardiovascular complications, chronic heart disease, stroke, type 2 diabetes, chronic inflammation and osteoarthritis [29].The best-devised clinical strategy would be to counter the benign states (obesity and fatty liver) much earlier that avoid the major onslaught of destructive metabolic states.The current clinical regimen for these states are unable to provide the desired results or lead to adverse effects that make the therapy too unsuitable.Despite the widespread use of synthetic agents (such as antidiabetics, anti-hyperlipidemia and anti-obesity) treatment with herbal extracts has demonstrated more significant effects and safe drug profile in multiple pre-and clinical studies.Therefore, the development of innovative plant-based medicines that can mitigate obesity and accompanying complications are of higher priority for management [30].
The intake of HFD and STZ mimics the obese and high plasma glucose states with considerable inflammation [31,32].Obesity and related abnormalities can be prevented by consuming the plants extracts having an abundance of potential polyphenolic compounds [33].In the present study, the result exhibited the provision of the promising therapeutic option of Me.Mo produced a remarkable decrease in body weight gain, fatty liver and related inflammation.Besides this, the accumulation of fats and insulin resistance in hepatic tissues could assume to be responsible for increasing rats liver weight in current research.
Moreover, lipid profiles including low-density lipoproteins (LDL), cholesterol and triglycerides are keynotes in the development of fatty deposits and plaques in vascular walls ultimately leading to a series of atherosclerosis, coronary heart disease and diabetes mellitus.As insulin has the inhibitory action on HMG-CoA reductase enzymes (primary contributor in cholesterol-rich LDL particles) thus, deficiency of insulin abnormally increases levels of free fatty acids and decreases their utilization in the body consequently developing diabetic conditions [34,35].On the other hand, high-density lipoproteins (HDL) play a primary contribution in lowering the risk of cardiovascular disease and stroke by scrubbing the inner walls of blood vessels from bad cholesterol [36].The present work showed that in HFD-induced diabetic rats, Me.MO exerted inhibitory action on cholesterol, triglycerides, and LDL levels and cause upregulation of HDL concentration in the blood.These findings highlight the anti-obesity and lipid-lowering effects of Me.MO which are inconsistent with previous scientific studies [37].
Besides this, HFD induces significant insulin resistance and hyperinsulinemia in animals [38].
Similarly, in present research work oral administration of Me.Mo for consecutive 60 days significantly decreased HFD-induced glucose concentration, however, no marked changes were observed in the model-HFD group.The insulin-sensitizing effect of Me.MO may be due to stimulatory action on receptor insulin signaling pathways as attributed in previously published inline studies [39,40].
Chronic inflammatory stress is the fundamental feature of obesity [41].The inflammatory mediators serve as a magnet in attracting more fats to tissues that can store them.In turn, inflammatory cells themselves undergo metabolic rewiring in response to excess lipids [42] and often turn more active favoring autoimmune disease [43][44][45][46].In the present work, results of RT-PCR analysis demonstrated that treatment with Me.MO for continuous two months decreased gene expression of chemo-cytokines.Therefore, it is speculated that Me.MO can prevent induction and propagation of inflammatory mediators release and have beneficial anti-inflammatory spectrum.
In addition, histopathological analysis of liver and adipose tissues of Me.MO and metformintreated diabetic rats showed a definitive reduction in adipocytes hypertrophy/hyperplasia and microvascular fatty changes with mild lymphocytes recruitment and no necrotic/apoptotic bodies.
However, in M-HFD group adipocytes hyperplasia, fatty bodies, adiponecrosis and inflammatory infiltration was clearly seen.
HPLC analysis confirmed that Me.MO extract contains high content of polyphenolic compounds which have well-documented metabolic effects [47].Quercetin plays a vital role to attenuate symptoms of the metabolic disease by decreasing oxidative stress and associated inflammation [48,49].Vanillic acid cut down hyperinsulinemia and improves antioxidants status in the body resultantly reducing the risk of cardiac problems [50].Chlorogenic acid plays a dual role not only blocking the expression of glucose-6-phospatse but enhancing glucose uptake by skeletal muscles as well.Consequently, alleviates insulin sensitivity and glucose tolerance and dyslipidemia [51].
Synirgic acid downregulates altered levels of metabolic enzymes and stimulates β-cells regeneration to prevent pancreatic damage [52].m-coumaric acid inhibits the overproduction of reactive oxygen species thus suppressing hyperglycemia-induced vascular damage [53].Hence, in the present study, it can be attributed to the presence of these phytochemicals in Me.MO may be responsible for its anti-inflammatory and anti-obesity effects.We also believe that antiinflammatory activities are responsible for lowering lipid load in experimental rats.

Conclusion
This study revealed that Me.Mo ameliorated body weights and related liver weight gains in HFD induced model.Interestingly, the reduction in body and liver weights was accompanied with inflammatory regulation.The study highlights the possible role of the immunometabolic axis in the manifestation of the anti-obesity potentials of Me.Mo.

Figure 2
Figure2and figure3represent the effect of Me.MO on body weight of animals before, during and after the study.On zero-week, no significant (p > 0.05) difference was observed in body weights of all groups statistically.While at 4 th week, the weight of animals drastically rose after the administration of HFD.Statistical analysis showed a significant (p < 0.001) increase in the weight of animals after receiving the HFD.On the 12 th week, weight variation for control, model (HFD),

Figure 2 :
Figure 2: Macroscopic analysis of body weight variations on 0, 4 th and 12 th week in different study groups shown in representative pictographic form.A visitor, not knowing the background of study, chose subjects for macroscopic analysis randomly.Animals were weigh after the pictures were taken and found consistent with the macroscopic analysis.

Figure 3 :
Figure 3: Shows the variation in body weights at 0, 4 th and 12 th week of study.The data shown represent the means ± SEM. ***P < 0.01.

Figure 4 :
Figure 4: Macroscopic analysis and weight measurement of rats.The average liver weight of that group (n-=5) is given above the respective representative images.

Figure 5 :
Figure 5: Section A shows graphical representation of random blood glucose levels in HFD and STZ induced model at 0, 4 th and 12 th week of treatment respectively.While section B shows graphical representation of lipid profile after twelve-week study.The data shown represent the means ± SEM. ***P < 0.01.

Figure 7 :
Figure 7: Graphical representation of random blood glucose levels in HFD and STZ induced diabetes mellitus model at 0, 4 th and 12 th week of treatment respectively.Red color stars show comparison of control with model-HFD while brown and violet color stars show comparison of model-HFD with Me.MO (250 and 500 mg/kg) and metformin treatment groups respectively.The data shown represent the means ± SEM. ***P < 0.01.

Table 1 :
Compounds identified in HPLC

Mo reduces body weights and liver weights
Me.Mo raised the plasma HDL levels significantly.This elevation was significant (p < 0.001) in Me.MO treated groups as compared to model-HFD.