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ARTEMISIA ANNUA EXTRACT AMELIORATES HEPATO-RENAL DYSFUNCTIONS IN OBESE RATS
A.F. Hasan1*, A.A. Alankooshi2, M.N. Modher1, S.A. El-Naggar3, H.M. El-Wahsh4 A.E. El-Bagoury5, S.I. Sakr5, R.S. El-Deen5, E. El-Nahas3, D.I. KabiP
1 Al-Nahrain University-Biotechnology Research Center, Iraq;
2 Department of Human Physiology, Faculty of Medicine, Jabir Ibn Hayyan Medical University, Iraq;
3 Zoology Department, Faculty of Science, Tanta University, Egypt;
4 King Abdulaziz University, Faculty of Marine Sciences, Marine Biology Department, Saudi Arabia;
5 Home Economics Department, Faculty of Specific Education, Tanta University, Tanta, Egypt.
* Corresponding author: ahmed_flayyih@nahrainuniv.edu.iq
Abstract. Obesity is recognized as a multifactorial health condition characterized by excess body fat accumulation. Orlistat is a well-known effective anti-obesity therapeutic drug, however, like many other medications on the market, it has certain unpleasant side effects. Medical herbs have recently acquired popularity in the treatment of obesity. The current project's intention was to evaluate the effect of treatment with Artemisia annua extract (AAE) to ameliorate hepato-renal dysfunction in obese rats. 40 male Sprague Dawley (SD) rats were divided into 4 groups (n = 10). The 1st group (Gp1) served as a negative control, and Gp2 was used as a positive control and given a high-fat diet (HFD) for a period of 12 weeks. For a period of 8 weeks, Gp3 and Gp4 received HFD and daily treatments of orlistat (30 mg/kg) or AAE (150 mg/kg), respectively. Hematological, biochemical, and histopathological parameters were determined. The results demonstrated that obese rats, Gp2, had hepato-renal impairment. Moreover, hepato-renal dysfunctions were exacerbated when orlistat was administered to obese rats of Gp3. In contrast, AAE-treated obese rats, Gp4, have shown alleviated hematological changes and resulted in considerable improvements in hepato-renal function. Taken together, AAE administration demonstrated potential ameliorative effects against hepato-renal dysfunctions in obese rats when compared to treatment with orlistat.
Keywords: obese, Artemisia annua, Orlistat, hepato-renal, antioxidants.
List of Abbreviations
AAE - Artemisia annua extract SD - Sprague Dawley BD - balanced diet HFD - high-fat diet T2-DM - type 2 diabetes mellitus NFLD - non-alcoholic fatty liver disease TC - total cholesterol TG - triglyceride HDLs - high density lipoproteins LDLs - low density lipoproteins HDL-C - high-density lipoprotein cholesterol
LDL-C - low-density lipoprotein cholesterol levels
VLDL-C - very low density lipoproteins cholesterol
ALT - alanine amino transferase AST - aspartate amino transferase SOD - superoxide dismutase CAT - catalase
MDA - malondialdehyde RBCs - red blood cells WBCs - white blood cells Hb - hemoglobin Hct - hematocrit Ctrl - control Orli - orlistat
Introduction
Obesity is an international health issue with a prevalence that is anticipated to climb from ~300 million in 2005 to more than 500 million by 2030 (Kelly et al., 2008). A body mass index of more than 30 kg/m2 indicates the presence of this nutritional metabolic disorder (WHO, 2020). It has become an international health issue, wreaking havoc on the body's many systems (Suleiman et al., 2020). Increased oxida-tive stress levels are associated with obesity as well as malfunctioning antioxidant mecha-
nisms, and excessive lipid buildup in adipose and liver tissues (Okla et al., 2015). There were also several metabolic dysfunctions linked with obesity, such as resistance to insulin, dyslipidemia, type 2 diabetes mellitus (T2-DM), hypertension, non-alcoholic fatty liver disease (NFLD), and some forms of cancer (Eckel et al., 2011; Jung & Choi, 2014; Sanchez et al.., 2014; Hsin et al.., 2020). It has been suggested that the cornerstone of obesity control is lifestyle modification (Heymsfield & Wadden, 2017).
Increases in total cholesterol (TC), triglyceride (TG) low-density lipoprotein cholesterol levels (LDL-C) have been associated with an increase in body fat mass and/or visceral fat, with a decrease in high-density lipoprotein cholesterol (HDL-C) (Shabana et al., 2020). Modern treatment techniques primarily target the inhibition or stimulation of numerous biomolecules and enzymes involved in fat metabolism (Andrej et al., 2010). Several synthetic medications, such as orlistat, lorcaserin, liraglutide, phentermine-topiramate, and naltrexone-bupropion, have been used to treat obesity (Hsin et al., 2020; Grilo et al.., 2021; Aaseth et al.., 2021).
Orlistat is a lipase inhibitor that alters gastrointestinal nutrient absorption by inhibiting gastric and pancreatic lipases and lowering TG digestion and absorption (Bansal & Al Khalili, 2022). Fat malabsorption, diarrhea, dyspepsia, flatulence, sleeplessness, occasional high blood pressure, pancreatitis, irregular menstrual cycle, upper respiratory tract infections, choles-tatic hepatitis, and tubular necrosis have all been documented as side effects of orlistat (Ballinger, 2000; Ahmad & Mahmud, 2016). Additionally, there is a high chance of developing breast cancer, acute pancreatitis, and gallstone disease as well (Heck et al., 2000). The use of orlistat has reportedly been linked to an increased risk of hepatic impairment (Sall et al., 2014). Moreover, earlier research showed that orlistat causes apoptosis, DNA damage, and kidney toxicity in both normal and obese female rats (Beyea et al., 2012; Al-Safo & Al-Dulaimi, 2022; Hameed et al., 2022).
Since they are less expensive, more readily available, and less harmful than synthetic drugs, medicinal herbs have drawn interest as
alternatives to conventional therapies for obesity (Hasani-Ranjbar et al., 2013; Saad, 2022). Artemisia annua L. (A. annua), a member of the Asteraceae family, is a well-known medicinal herb that has been used to treat malaria, anthelmintics, diabetes, bronchitis, ulcers, and tuberculosis (Choi et al., 2021; Sadiq et al., 2013; Nurlybekova et al., 2022). Moreover, anti-obesity properties of A. annua have recently been reported (Harvey & Jacquelin, 2021). Furthermore, A. annua extract therapy decreased adi-pogenesis and suppressed appetite (Baek et al., 2015). As a result, the current study sought to assess AAE's hepato-renal ameliorative effect in obese rats.
Materials and Methods
Tanta city's local market provided the herbs for A. annua. A specialist identified A. annua leaves, and a specimen was placed in the Tanta University Herbarium (code #TAN-147). Orlistat (Xenical) was obtained from Sigma Pharmaceutical Industries in Egypt; each capsule contains 120 mg of orlistat. The dosage used was 30 mg/kg weight according to Mopuri et al., (2015). Biochemical kits for lipid profile, alanine amino transferase (ALT), aspartate amino transferase (AST), urea, creatinine, total protein, albumin, globulin, glucose, superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were purchased from Bio-diagnostic Company, Cairo, Egypt.
Preparation of A. annua extract
A. annua leaves were rinsed with distilled water, dried in the shade, and ground into powder in a motorized mortar. 50 g of the powder were steeped in 500 mL of 70% ethanol for 3 days at room temperature before being filtered and dried using a rotary evaporator. The final powder, AAE, was kept at 4 °C until it was used.
Balanced and high fat diets
The balanced diet (BD) and the high-fat diet (HFD) were the 2 types of diets adopted. The BD was purchased from Helwan University's Animal Research and Service Center in Helwan, Egypt. 10% protein, 10% fat, 74.4% carbs, 3.5% mineral mixture, 0.1% methionine,
1% vitamin mixture and 1% fiber made up the BD used to feed healthy control rats (Pugh et al., 1999). A mixture of 64 g of normal chow, 32 g of animal-sourced saturated fat, 300 IU of vitamin D3, and 15% and 12% of cholesterol powder was used to create the HFD diet. Before being kept in a refrigerator at 4 °C, the mixture was rolled into handball size. To shield lipids from oxidative processes, the HFD was made every other day (Altunkaynak, 2005).
Rats and experimental design
Forty male SD rats (145 ± 5 g) were bought from the animal husbandry department at Helwan University in Egypt. All rats were given a week to adapt in the animal housing at Tanta University's Faculty of Science. The study design was approved by the institutional animal care committee at Tanta University-Egypt's Zoology Department, Faculty of Science (IACUC-SCI-TU-0217). The light-dark (day/night) cycle was achieved, with target values for temperature and relative humidity of about 22 ± 1 °C and 55 5%, respectively. The rats were divided into four groups of ten (n = 10): The first group (Gp1) was used as a negative control and was fed BD. The second group (Gp2) was fed an HFD for 12 weeks as a positive control (obesity group). The third group (Gp3) was fed the same HFD as Gp2 and was given orlistat (30 mg/kg) orally every day for two months. The fourth group (Gp4) were fed HFD and given AAE (150 mg/kg) for two months (Fig. 1).
In addition, the initial body weight (I.B.wt) and the final body weight (F.B.wt) were calculated. The percentage of body weight change (% b.wt) was computed as (F.B.wt - I.B.wt / I.B.wt) x 100.
Determination of the hematological and biochemical parameters
The portal vein was used to collect blood samples. A complete blood count was performed on whole blood, while sera were extracted for biochemical analysis. The Mindary automatic blood counter (Guangzhou, Guangdong, China) was utilized to count the whole blood picture. Kostener (1977) colorimetric
methods were used to determine sera TC, TG, and HDL-C. According to Fruchart (1982), low density lipoprotein cholesterol (LDL-C) was computed as [LDL-C = TC - HDL-C - VLDL-C]. VLDL-C (very low-density lipoprotein cholesterol) was computed as follows: [VLDL-C = = TG / 5] (Friedewald et al., 1972).
The ALT and AST activities of Sera were calculated using the Reitman and Frankel method (1957). Tietz (1995) was used to calculate serum glucose. Total protein was determined using Thomas' (1998) procedures, while albumin and globulin were done using Doumas et al.'s (1971) methods. The activities of SOD and CAT have been estimated using Flohe & Gunzler (1984). MDA was determined according to Ohkawa et al. (1979).
Histopathological investigations
Sections of liver and kidney tissues were taken and kept at -80 °C. The liver and kidney tissues from separate groups were fixed in 10% formalin for histological examination. The process of dehydration was carried out using a series of mild alcohol solutions. Following the completion of the tissue processing in various degrees of alcohol and xylene, paraffin blocks were created. To investigate cellular damage, sections of 5 |im were cut from paraffin blocks using a microtome, stained with hematoxylin and eosin, and visualized using a light microscope (Optika light microscope (B-350)) (Bancroft & Gamble 2002).
Ethical approval
The housing conditions were randomly selected and included ambient temperature (22 to 25 °C), relative humidity, a 12-hour light/ 12hour dark cycle, and a two-week supply of commercial food and water that was always accessible. Our Institutional Animal Care and Use Committee has given it the approval number IACUC-SCI-TU-0233.
Statistical analysis
The study's findings were presented as mean ± standard error means (SEM). For data analysis, the SPSS application was chosen. The data was evaluated using one-way analysis of vari-
Fig. 1. Timelines showing the experimental groups under the study (A), and representative photomicrographs of different groups on the day of dissection (B)
ance (ANOVA), followed by the Tukey test for multiple comparisons. Values with a (P < 0.05) were statistically significant.
Results
AAE treatment reduced the fluctuations in body weight in obese rats
From week 1 (W-1) to week-20 (W-20), the body weight changes in the various experimental groups were determined kinetically. When compared to the negative control (Gp1), the obese group (Gp2) body weight increased significantly (P < 0.05). Obese rats treated with orlistat (Gp3) or AAE (Gp4) had considerably higher body weights (P < 0.05) until week-12 (W-12), but after treatment with orlistat or AEE, the body weight was significantly lower
until W-20 (Fig. 2A). Rats treated with orlistat (Gp3) or AAE (Gp4) demonstrated significant decreases in their percentage of body weight changes (P < 0.05) when compared to obese rats only (Gp2) (Fig. 2B).
AAE treatment and its impact on hematological parameters
Gp2 obese rats had significantly lower total red blood cell (RBC) count, hemoglobin (Hb) concentration, and hematocrit percentage (Hct%) as compared to Gp1 (P < 0.05). As obese rats were given orlistat (Gp3), the prior metrics decreased significantly as compared to their values in the fat group (Gp2). Furthermore, AAE treatment of obese rats restored these parameters to values comparable to Gp1.
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Fig. 2. Body weight kinetic changes of the different groups (A). Percentages of body weight changes (B). The values represented mean < SD; Ctrl: Control; Orli: orlistat; AAE: Artemisia annua extract. (P < 0.05) was considered to be statistically significant. Means that do not share a letter are significantly different
Table 1a
The total red blood cells count, haemoglobin concentration, haematocrit, and platelets count in the different groups
Groups RBCs (x 103/uL) Hb (mg/dL) Hct (%) Platelets (x 103/uL)
Ctrl 8.52 ± 0.39 c 13.44 ± 1.2 c 39.59 ± 2.3 c 478.3 ± 19.0 c
Obese 6.25 ± 0.45 b 10.06 ± 1.5 b 33.31 ± 2.0 b 444.8 ± 23.9 c
Obese/orli 5.54 ± 0.54 c 9.96 ± 1.2 c 30.75 ± 2.7 508.3 ± 28.6 a
Obese/AAE 7.95 ± 0.26 c 13.69 ± 1.2 c 39.5 ± 2.1 553.5 ± 21.4 a,b
Note: the values represented mean ± SD; RBCs: Red blood cells; Hb: Hemoglobin; Hct: Hematocrit; Ctrl: Control; Orli: orlistat; AAE: Artemisia annua extract. P value < 0.05 was considered to be statistically significant. Means in each column that do not share a letter are significantly different
Table 1b
The total white blood cells count and relative differential percentages in the different groups
Groups W.B.Cs x 103/uL Monocytes (%) Lymphocytes (%) Neutrophils (%)
Ctrl 8.4 ± 1.07 c 3.9 ± 0.36 a 85.0 ± 2 a 10.5 ± 0.85 b
Obese 13.0 ± 1.82 a 5.0 ± 0.57 b 74.1 ± 2.28 b 21.0 ± 1.33 b
Obese/orli 14.8 ± 1.03 a 4.5 ± 0.44 b 73.0 ± 2.65 b 24.1 ± 1.75 b
Obese/AAE 9.2 ± 1.26 b'c 3.9 ± 0.4 b 86.9 ± 2.12 a 11.1 ± 1.03 a
Note: the values represented mean ± SD; W.B.Cs: Artemisia annua extract. P value < 0.05 was considere that do not share a letter are significantly different
In terms of platelet count, the findings revealed no significant changes between Gp2 and Gp1. Unlike Gp3 and Gp4, which caused large increases in platelet counts (Table 1a).
Moreover, the total count of white blood cells (WBCs) was considerably higher in Gp2 and Gp3 when compared to Gp1 (P < 0.05). However, when compared to the Gp2 WBCs count (13.0 ± 1.82 x 103/|l), AAE therapy in Gp4 resulted in a considerable drop in WBCs count (9.2 ± 1.26 103/|l). Additionally, the percentage of monocytes in Gp2 and Gp3 was significantly lower when compared to Gp1. Conversely, Gp4 monocyte percentages recovered to near-normal levels. The percentages of lymphocytes and neutrophils were determined to be comparable (Table 1b). Conversely, Gp4 displayed a recovery in their percentages to near-normal levels. The percentages of lymphocytes and neutrophils were found to be similar (Table 1b).
In obese rats, AAE treatment improved the hepatorenal dysfunctions
When compared to Gp1, there were significant increases in ALT, AST, urea, and creatinine levels in the Gp2 (Gp2: 71.28 ± 3.16 U/L, 175.47 ± 12.21 U/L, 49.5 ± 3.42 mg/dL, and 0.74± 0.04, respectively; Gp1: 44.01 ± 2.21 U/L, 114.2 ± 11.58 U/L, 23.54 ± 3.42 mg/dL, and 0.44 ± 0.04, respectively). However, when compared to Gp2, Gp3 exhibited substantial rises in the values of the previous parameters, but Gp4 showed significant declines in these
White blood cells; Ctrl: Control; Orli: orlistat; AAE: d to be statistically significant. Means in each column
hepatorenal biomarkers (P < 0.05) (Fig. 3 and 4).
Restoration of albumin, globulin, total proteins, and glucose levels in obese rats treated with AAE
When compared to Gp1, the levels of albumin, globulin, and total proteins were considerably lower in Gp2 and Gp3 (P < 0.05). However, when compared to Gp2, Gp4 showed a significant increase in these parameters (Fig. 5 and 6A). Moreover, when compared to Gp1, Gp2 had a substantial increase in serum glucose levels. In contrast, when compared to Gp3, orlistat medication resulted in a considerable drop in serum glucose levels (200.7 ± ± 5.2 mg/dL). Additionally, the administration of AAE to obese rats (Gp3) resulted in a significant decrease in glucose levels (167.6 ± ± 4.4 mg/dL) (Fig. 6B).
Lipid disturbance improved in obese rats treated with orlistat or AAE
When compared to Gp1, Gp2 had significant increases in total cholesterol, TG, LDL-C, VLDL-C, and atherogenic index (Gp2: 148.64 ± 13.05, 200.93 ± 7.22, 32.1 ± 2.01,39.83 ± 3.01, and 2.47 ± 0.15 mg/dL, respectively; Gp1: 79.48 ± 2.67, 71.18 ± 2.53, 18.4 ± 1.63, 20.32 ± 2.43, 20.32 ± 2.43 and 0.74 ± 0.05, respectively; P < 0.05). In contrast, when compared to Gp1(43.1 ± 2.73 mg/dL), Gp2 (28.0 ± ± 2.04 mg/dL) had a considerable drop (P < < 0.05) in HDL-C levels. However, when com-
Fig. 3. Serum alanine transaminase (ALT) activity (A). Aspartate transaminase (AST) activity (B) of the different groups under the study. Ctri: Control; Orli: orlistat; AAE: Artemisia annua extract. (P < 0.05) was considered to be statistically significant. Means that do not share a letter are significantly different
Fig. 4. Sea urea (A), and creatinine (B) levels of the different groups under the study. Ctri: Control; Orli: orlistat; AAE: Artemisia annua extract. (P < 0.05) was considered to be statistically significant. Means that do not share a letter are significantly different
Fig. 5. Albumin (A), and globulin (B) levels of the different groups under the study. Ctrl: Control; Orli: orlistat; AAE: Artemisia annua extract. (P < 0.05) was considered to be statistically significant. Means that do not share a letter are significantly different
Fig. 6. The total protein (A), and glucose (B) levels of the different groups under the study. Ctril: Control; Orli: orlistat; AAE: Artemisia annua extract. (P < 0.05) was considered to be statistically significant. Means that do not share a letter are significantly different
pared to the Gp2 group, treatments with orlistat (Gp3) or AAE (Gp4) resulted in significant reductions in total cholesterol, TG, LDL-C, VLDL-C levels, and atherogenic index (Table 2 a and b).
Attenuation of hepatic oxidative stress in obese rats when treated with AAE
When Gp2 was compared to Gp1, there were remarkable drops in the hepatic SOD
and CAT activity (Gp1: 3.16 ± 0.15 and 54.58 ± 2.13 U/g tissue, respectively; P < < 0.05). In contrast, significant increases in MDA levels of Gp2 (75.97 ± 4.2 nmol/g tissue) were seen as compared to Gp1(48.62 ± ± 1.17 nmol/g tissue) (Table 3). When compared to Gp2, Gp3 did exhibit significant declines in the hepatic SOD and CAT activities together with a large increase in the MDA level (P < 0.05). However, Gp4 demonstrated
improved antioxi-dant/oxidant hemostat-sis as indicated by a large rise in hepatic SOD and CAT activity (4.52 ± 0.33 and 73.61 ±
± 2.05, U/mg tissue, respectively) and a considerable drop in MDA levels in tissue (54.54 ± 1.22 nmol/g; P < 0.05) (Table 3).
Table 2a
Total cholesterol, triglycerides, and HDL-C levels in the different groups
Groups Total Cholesterol (mg/dL) Triglycerides (mg/dL) HDL-C (mg/dL)
Ctrl 79.48 ± 2.67 c 71.18 ± 2.53 d 43.1 ± 2.73 a
Obese 148.64 ± 13.05 a 200.93 ± 7.22 a 28.0 ± 2.04 a,b
Obese/orli 92.13 ± 3.42 b,c 109.42 ± 3.16 cd 35.2 ± 2.65 b
Obese/AAE 84.43 ± 3.78 c 93.96 ± 3.26 c 40.6 ± 3.15 a
Note: the values represented mean ± SD; HDL: High density lipoproteins; Ctrl: Control; Orli: orlistat; AAE: Artemisia annua extract. P value < 0.05 was considered to be statistically significant. Means in each column that do not share a letter are significantly different.
Table 2b
LDL-C, VLDL-C levels, and atherogenic index in the different groups
Groups LDL-C (mg/dL) VLDL -C (mg/dL) Atherogenic index
Ctrl 18.4 ± 1.63 b 20.32 ± 2.43 b 0.74 ± 0.05 d
Obese 32.1 ± 2.01 a 39.83 ± 3.01 a 2.47 ± 0.15 a
Obese/orli 24.3 ± 3.04 b,c 26.48 ± 2.29 b,c 1.82 ± 0.14 c
Obese/AAE 22.4 ± 2.05 b 24.06 ± 2.01 b 0.95 ± 0.12 d
Note: the values represented mean ± SD; LDL-C: Low density lipoproteins cholesterol; VLDL-C: Very low density lipoproteins cholesterol; Ctrl: Control; Orli: orlistat; AAE: Artemisia annua extract. P value < 0.05 was considered to be statistically significant. Means in each column that do not share a letter are significantly different.
Table 3
Hepatic SOD, CAT activities, and MDA levels in the different groups
Groups SOD (U/mg protein) CAT (U/mg protein) MDA (nmol/g tissue)
Ctrl 6.21 ± 0.43 a 82.94 ± 2.61 a 48.62 ± 1.17 d
Obese 3.16 ± 0.15 c 54.58 ± 2.13 d 75.97 ± 4.2 b
Obese/orli 1.46 ± 0.27 d 33.86 ± 1.39 f 94.16 ± 3.8 a
Obese/AAE 4.52 ± 0.33 b 73.61 ± 2.05 b 54.54 ± 1.22 d
Note: the values represented mean ± SD; SOD: Superoxide dismutase; CAT: Catalase; MDA: Malondialdehyde; Ctrl: Control; Orli: orlistat; AAE: Artemisia annua extract. P value < 0.05 was considered to be statistically significant. Means in each column that do not share a letter are significantly different.
Improvement of hepato-renal histopatholog-ical alterations with AAE treatment
Gp1 rat liver slices revealed an ordinary hepatic structure with polygonal hepatocytes with conspicuous nuclei set up in a radial fashion.
Figure 7a shows the hepatic strands alternating with narrow blood sinusoids bordered by an en-dothelial cell layer containing Kupffer cells. Histological examination of Gp2 rats' liver sections revealed significant hepatic disorganiza-
Fig. 7. Photomicrographs of liver sections stained with hematoxylin and eosin from the different groups. Liver section of control group shows normal hepatic structure with radial arrangement of polygonal hepato-cyte containing pronounced nuclei (H), central vein (CV), blood vessels (BV) lined by endothelial cells and distinct phagocytic Kupffer cells (K) (a). Liver section of obese rats shows hepatocytes with cellular infiltration (IF), hemorrhage in the central vein (HR), Kupffer cells (K), pyknotic nuclei (P) and karyolitic ones (arrow) appear (b). Liver section of orlistat treated obese mice shows hepatic degeneration with irregular congested central vein (CV), widening of blood vessels (BV), pyknotic nuclei (P), Kupffer cells (K), and area of infiltration around central vein (IF), binucleated (BH) and vacuolated hepatocytes (VH) appear (c). Liver section of obese rats treated with AAE shows improvement in the hepatic tissues with slight widening of central vein (CV), Kupffer cells (K) and binucleated hepatocytes also appear (BH) (d). (X=400)
Fig. 8. Photomicrographs of kidney sections stained with hematoxylin and eosin from the different groups. Kidney section of control group shows normal structure of the cortex, normal glomeruli (G) with normal bowman's capsule (BC), bowman's space (*), and normal renal tubules (R) lined by cuboidal epithelium (a). Kidney's section of obese rats shows disorganized glomeruli (G) with irregular Bowman's space (*), mild degeneration of renal epithelial cells of renal tubules (RT), their lining epithelium were undistinguished with cellular degeneration and widening of renal lumen, hemorrhage area may appear (arrowhead) (b). Kidney's section of obese rats treated with orlistat shows destructed, shrunken, and congested glomeruli (G), irregular destructed bowman's capsule (DBC), wide bowman's space (*), damaged renal tubules (DRT) with distinct vacuolated and highly degenerated lining epithelium; cellular infiltration at the intertubular spaces was observed (arrow) and appearance of cellular hemorrhage at the intertubular spaces (arrowhead) (c). Kidney's section of obese rats treated with AAE shows normal like architecture of glomeruli with normal bowman's capsule (BC), normal bowman's space (*), and normal renal tubules (RT) (d). (X = 400)
tion, congested central veins with hemorrhage, intensive degeneration of its epithelial lining, the area around the central vein was associated with cellular infiltration, Kupffer cells, pyk-notic nuclei, and karyolitic nuclei (Fig. 7b). Gp3 liver sections revealed hepatic degeneration with an irregularly congested central vein,
blood vessel widening, and a region of infiltration around the central vein. Kupffer cells, pyk-notic nuclei, and binucleated, vacuolated hepatocytes have also been observed (Fig. 7c). Gp4 rat liver slices revealed an improvement in hepatic tissue with normal hepatocytes, normal architecture, a small widening of the central
vein, Kupffer cells, and binucleated hepato-cytes (Fig. 7d).
The renal cortex of Gp1 rats appeared normal, with a normal glomerulus, proximal and distal convoluted tubules surrounded by cu-boidal epithelium, and Bowman's capsule, and no evidence of inflammation (Fig. 8a). Gp2 kidney sections revealed disorganized glomeruli with abnormal Bowman's space, expansion of some renal tubules that look blocked with hyaline casts, slight degeneration of the lining renal epithelial cells, their contents intermixed with each other, and a hemorrhage area may appear (Fig. 8b).
Kidney sections from Gp3 rats displayed de-structed, shrunken, and congested glomeruli with irregular destructed bowman's capsule, wide bowman's space, and damaged renal tubules with distinct vacuolated and highly degenerated lining epithelium; cellular infiltration at the intertubular spaces was also observed, as was the appearance of cellular hemorrhage (Fig. 8c). However, kidney sections from Gp4 rats revealed normal-like glomeruli architecture with normal bowman's capsule, normal bowman's space, and normal renal tubules (Fig. 8d).
Discussion
Obesity is recognized as one of the world's most serious public health issues. Obesity promotes the development of metabolic disorders including diabetes, hypertension, and cardiovascular disease, as well as chronic diseases like strokes, osteoarthritis, sleep apnea, certain malignancies, and inflammation-based pathologies (Gonzalez-Castejon & Rodriguez-Casado, 2011). In a prior investigation, orlistat administration resulted in a significant drop in body weight in an obese rat model (Othman et al., 2021). Furthermore, a prior study found a significant drop in the percentages of body weight after AAE treatment of obese rats exhibited a considerable decrease in their percentages of body weight (Choi et al., 2021). As a result, the purpose of this study was to examine the effects of orlistat and AAE on hepato-renal functioning in obese rats. The current study found that treating obese rats with orlistat or AAE reduced body weight, which is consistent with earlier re-
search (Othman et al., 2021; Choi et al., 2021). Obesity has previously been linked to an increase in total WBC count (Nagamma et al.,
2019). Similarly, the concentrations of Hb and Hct% in obese rats tended to decrease (Monte-omo et al., 2018; Alia et al., 2019). Obesity was found to cause hematological alterations in the current study. Orlistat therapy had little effect on the hematological alterations generated in obese rats. These side effects could be attributable to orlistat's harmful effect on blood components. The AAE treatment, on the other hand, resulted in an improvement in the total blood count. Furthermore, the effects of A. annua L. herbal extract on the improvement of hematological parameters in obese animals have been previously reported (Choi et al., 2021).
Obesity is directly related to hepatic dysfunctions, as a recent study found that obese rats had a significant fatty liver and metabolic abnormalities (Fotschki et al., 2020). The findings were consistent with previous research that found significant increases in liver transaminases in the sera of obese rats (El-Naggar et al., 2021; Opyd & Jurgoski, 2021). Obesity caused by HFD is accompanied with increased oxida-tive stress in the hepatic and renal organs, as evidenced by a decrease in antioxidant enzyme activity (Noeman et al., 2011). The current study found that obese rats and obese rats treated with orlistat had higher levels of liver transaminases; however, obese rats treated with AAE (Gp4) had lower levels of hepatic trans-aminases.
Furthermore, obese rats also had lower amounts of albumin, globulin, and total proteins. These findings were consistent with a prior study, which found that obesity is related to an increased risk of hypoalbuminemia (Mosli & Mosli, 2017). AAE therapy enhanced protein levels in obese mice (Gp4) in this study. Additionally, total protein, albumin, and globulin levels in alloxan-induced diabetic rats were dramatically improved by A. annua treatment (Helal, 2014). Obesity is a risk factor for insulin resistance and type 2 diabetes (Wondmkun,
2020). Orlistat has been shown to promote body weight loss and glucose homeostasis in obese rats (Vickers et al., 2014). Also, AAE was
shown to ameliorate insulin resistance and glucose levels in HFD-diabetic mice (Ghanbari & Sadeghimahalli, 2022). Likewise, it has been shown that A. annua leaf extract has ameliorative effects on hepatic dysfunctions and inflammation in HFD-fedfed mice (Kim et al., 2016).
Obesity is linked to elevated levels of plasma total cholesterol, TG, and LDL-C, which is one of the leading causes of death worldwide (Jia et al., 2013; El-Naggar et al., 2021). In obese rats that have undergone HFD induction, orlistat 10 mg/kg/day for six weeks may have preventive benefits against changes in the lipid profiles and obesity index (Othman 2019). In HFD-diabetic mice, the serum lipid profile was reportedly improved by AAE extracts (Choi et al., 2021; Ghanbari & Sadeghimahalli, 2022).
According to the literature, HFD administration experimentally produced obesity and resulted in significant variations in the blood pro-teome profile of rodents due to hepatic dysfunctions; these effects can be recovered by natural products (Duan et al., 2022; Janoschek et al., 2023). Prior work with obese rats showed HFD induced metabolic syndrome and oxidative stress consequences (Lasker et al.., 2019). Obesity causes an oversupply of free radicals, which can react with macromolecules in animals or humans, changing proteins, carbohydrates, lipids, and DNA sequences, and debilitating normal cell activities (Saha & Tamrakar, 2011). In this investigation, the HFD group had considerably lower SOD activity than the Gp1 group possibly due to the presence of cardiac oxidative stress. Additionally, when compared to Gp2, orlistat-treated obese rats (Gp3) revealed significant reductions in hepatic SOD and CAT activity as well as a significant rise in MDA levels. Therefore, to avoid health risks, it is recommended that orlistat be used under prescription and not in high doses or for an extended period of time. In contrast, another study found that administering orlistat for 6 weeks resulted in significant increases in SOD and CAT activities as compared to the HFD group (Bharati et al., 2018).
Natural defense antioxidant enzymes have been proven to counteract oxidative stress by lowering the lipid peroxidation process in
HFD-induced obese mice (Charradi et al., 2013). Previous research has shown that AAE reduces hepatic stress and inflammation in HFD-fed animal models, which is consistent with our findings (Park et al., 2020). A recent study found that consuming HFD for 14 weeks in a row causes obesity and changes the normal morphological function of the liver in male rats due to the lipid accumulation process (Pad-manabhan and Arumugam 2014). In the current study, orlistat increased hepatic disorganization, degeneration, and necrosis of many hepatocytes, cytoplasmic vacuolation, and blood vessel congestion more than obese rats. Joyce et al. (2020) reported a similar finding. The administration of AAE to obese rats improved the hepatic architecture, central vein, modest broadening of blood sinusoids, Kup-ffer cells, and pyknotic nuclei. These findings were consistent with those of Lim et al. (2013), who demonstrated that Artemisia was beneficial in preventing obese-induced liver damage in rats.
Obesity generated histological changes in kidney tissues that included destructed, shrunken, and congested glomeruli with irregular bowman's space, showed that most renal tubules that were damaged and lost their characteristic look, and intertubular bleeding was observed. This increase in disorder and destruction was observed in orlistat-treated rats (Gp3) more than in control rats (Gp1). Solomon et al. (2017) reported similar findings to the latter. Furthermore, AAE treatment in obese rats (Gp4) improved the glomeruli and renal tubules. These findings indicate that AAE was beneficial in avoiding kidney impairment in obese mice. When compared to orlistat-treated obese mice (Gp3), AAE treatment (Gp4) revealed potential ameliorative effects against hepato-renal dysfunctions.
Conclusion
The study concluded that treating orlistat to obese rats increased their hepato-renal dysfunctions; however, treating with AAE resulted in significant weight loss and potential ameliorative effects on liver and kidney dysfunction, as shown by improvements in hema-
tological, biochemical, and histopathological alterations. More research is needed to discover AAEs most active constituent and to
mechanistically understand the anti-obesity benefits, which could aid in the development of more effective pharmaceutical derivatives.
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