Effects of Karela (Bitter Melon; Momordica charantia) on genes of lipids and carbohydrates metabolism in experimental hypercholesterolemia: biochemical, molecular and histopathological study

Ethidium bromide and agarose were purchased from Sigma-Aldrich, St. Louis, MO, USA). The Wistar albino rats were purchased from King Fahd center for Scientific Research, King Abdel-Aziz University, Jeddah, Saudi Arabia. Serologic kits for HDLC, Cholesterol and triglycerides (TG) HUMAN Gesellschaft für Biochemica und Diagnostica mbH (Wiesbaden, Germany). The deoxyribonucleic acid (DNA) ladder was purchased from MBI, Fermentas, Thermo Fisher Scientific, USA. Qiazol for RNA extraction and oligo dT primer were purchased from QIAGEN (Valencia, CA, USA). Kits for antioxidants such as superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and malondialdehyde (MDA) were bought from Bio-diagnostic Co., Dokki, Giza, Egypt.

Ethical Committee Office of the scientific Deans of Taif University, Saudi Arabia approved all procedures of this study for the project #4860–437-1. Forty male Wistar rats, 2 months old (200–222 g) were used for this study. Animals were kept under observation for 2 weeks to ensure complete acclimatization before the onset of the experiment. The animals were kept at equal light–dark cycle (12/12 h) with free access to food and water. Four groups each containing 10 healthy Wistar rats were used for the study as follows: Group 1, served as negative control with free access to food and water. Group 2 served as positive hypercholesterolemic group and was given orally cholesterol in a dose of 40 mg/kg body weight daily for 6 weeks. Group 3 was administered orally Karela in a dose of 5 g/kg body weight daily based on a previous study [28]. Group 4 administered orally cholesterol in a dose of 40 mg/kg body weight daily for 10 weeks plus karela in a dose of 5 g/ kg bw daily at week 6 and continued for 4 weeks later. Dose of cholesterol was used based on the finding of Co and To [29]. After 10 weeks, rats were inhaled dimethyl ether and decapitated after overnight fasting. Liver and adipose tissue were preserved in Bouin’s solution for histopathological examination and in Qiazol reagent for RNA extraction for gene expression.

Fresh karela fruits (Bitter melon) was purchased from commercial local markets in Taif governate (Panda, Taif), Saudi Arabia. The plant fruits was identified by botanist in College of Science, Taif University, Saudi Arabia. Karela was washed thoroughly with water, and dried after cutting into small pieces, dried and powdered using a blender. The dose used was 5 g /kg BW by intragastric tube based on previous reports [28].

Glucose was measured colormetrically using commercial available kits. Antioxidants such as superoxide dismutase, SOD, GSH, MDA and catalase were measured spectrophotometrically using commercial ELISA kits based on manufacturer’s instruction manual. Serum triacylglycerol, total cholesterol and high density lipoproteins-cholesterol (HDLC) were measured spectrophotometrically according to the manufacturer’s protocol.

The collected specimens of liver were fixed in 10% buffered neutral formalin solution and then routinely processed. Paraffin sections of 5 μm thickness were prepared, stained with Hematoxylin and eosin stain (H&E) as described before [30]. By using avidin-biotin-peroxidase method, the liver samples were embedded in paraffin and cut into 3 μm sections. Samples were mounted on positively charged slides for caspase 3 immunohistochemical examination. Sections were dewaxed, rehydrated and autoclaved at 95 °C for 20 min in antigen retrieval buffer (10 mM citrate buffer, pH 6). After washing with phosphate buffered saline (PBS), endogenous peroxidase was blocked using 3% H₂O₂ in methanol for 15 min. A primary rat specific antibody for caspase 3 (cat.no. RB 1197 B0, B1; Thermo Fisher Scientific Inc) was diluted in PBS (1:100), and incubated for 30 min. The slides were then rinsed three times with PBS. Horseradish peroxidase conjugated goat anti mouse IgG secondary antibody (Cat # 32230; Thermo Fisher Scientific Inc.) was incubated for 30 min with tissue sections. Extra rinsing for 3 times with PBS was done. Samples were visualized after 10 min from adding metal enhanced diaminobenzidine (DAB) substrate (Sigma-Aldrich, St. Louis, MO, USA) as a working solution (Thermo Fisher Scientific Inc.) as stated before [31]. The immune reactivity score was used to evaluate the intensity of immunohistochemical staining and the proportion of the stained cells [31].

Total RNA was extracted from liver and epididymal adipose tissue samples (50 mg per sample) as stated before [32]. In short, samples were flash frozen in liquid nitrogen and subsequently stored at −70 °C in 1 ml Qiazol (QIAGEN, Valencia, CA, USA). Frozen samples were homogenized using a Polytron 300 D homogenizer (Brinkman Instruments, Westbury, NY, USA). Then, 0.3 ml chloroform was added to the homogenate. The mixtures after shaking for 30 s, centrifuged at 4 °C and 16,400 x g for 15 min. The supernatant was transferred to new tubes. Equal volume of isopropanol was added to the samples and centrifuged at 4 °C and 16,400 x g for 15 min. The RNA pellets were washed with 70% ethanol, briefly dries up, and then dissolved in diethylpyrocarbonate (DEPC) water. RNA concentration and purity were determined spectrophotometrically at 260 nm. The RNA integrity was confirmed in 1.5% denaturated agarose gel stained with ethidium bromide. The ratio of the 260/280 optical density of all RNA samples was 1.7–1.9. For cDNA synthesis, a mixture of 3 μg total RNA and 0.5 ng oligo dT primer (Qiagen Valencia, CA, USA) in a total volume of 11 μl sterilized DEPC water was incubated in the Bio-Rad T100™ Thermal cycle at 65 °C for 10 min for denaturation. Then, 2 μl of 10X RT-buffer, 2 μl of 10 mM dNTPs and 100 U Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase (SibEnzyme. Ak, Novosibirsk, Russia) were added and the total volume was completed up to 20 μl by DEPC water. The mixture was then re-incubated in BIO-RAD thermal cycler at 37 °C for one hour, then at 90 °C for 10 min to inactivate the enzyme. For semi-quantitative RT-PCR analysis, specific primers for examined genes (Table 1) were designed using Oligo-4 computer program and synthesized by Macrogen (Macrogen Company, GAsa-dong, Geumcheon-gu. Korea). PCR was conducted in a final volume of 25 μl consisting of 1 μl cDNA, 1 μl of 10 pM of each primer (forward and reverse), and 12.5 μl PCR master mix (Promega Corporation, Madison, WI, USA), the volume was brought up to 25 μl using sterilized, deionized water. PCR was carried out using Bio-Rad T100™ Thermal Cycle machine with the cycle sequence at 94 °C for 5 min one cycle, followed by variable cycles (Table 1) each of which consists of denaturation at 94 °C for one minute, annealing at the specific temperature corresponding to each primer (Table 1) and extension at 72 °C for one minute with an additional final extension at 72 °C for 7 min. As a reference, expression of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was examined (Table 1). PCR products underwent electrophoresis on 1.5% agarose (Bio Basic, Markham, ON, Canada) gel stained with ethidium bromide in TBE (Tris-Borate-EDTA) buffer. PCR products were visualized under UV light and photographed using gel documentation system. The intensities of the bands from four different rats per group and three independent experiments were quantified densitometrically using Image J software version 1.47 (https://​imagej.​en.​softonic.​com/​).

Table 2 shows that experimental hypercholesterolemia was associated with an increase in serum total cholesterol levels, triglycerides and glucose. In parallel there was a decrease in high density lipoproteins (HDL). Administration of Karela normalized and ameliorated this altered parameters and increased HDL levels (Table 2). Hypercholesterolemia as seen in Table 3, significantly increased the serum levels of malondialdehyde (MDA) and decreased serum levels of super oxide dismutase (SOD), reduced glutathione (GSH) and catalase. Administration of karela significantly improved the decrease in antioxidant activity and normalized the increase in MDA (Table 3). Of note, administration of karela to control rats induced high antioxidants potency.

The histological examination of liver in control and karela administered rats had a normal architecture of hepatic lobules with plates of polygonal hepatocytes radially arranged around the central vein and separated by blood sinusoids, the hepatocytes revealed an acidophilic cytoplasm and rounded nuclei (Fig. 1a, b). In hypercholesterolemic rats revealed loss of normal hepatic architecture with deposition of cholesterol crystals are found in many radicular cysts forming cholesterol clefts and necrosis of hepatocytes (Fig. 1c). Also, sever congestion of portal blood vessel with extensive portal fibrosis, hyperplasia of epithelial lining of the bile duct and karyolysis of hepatocytes nuclei (Fig. 1d, e). In liver of hypercholesterolemic rats administered karela, liver showed apparent normal hepatic parenchyma with few cholesterol clefts (Fig. 1f). In Immunohistochemical examination for caspase-3 expression in liver. The control and karela administered rats showed negative immunohistochemical staining for caspase-3 immunoreactivity (Fig. 2a, b). Liver of hypercholesterolemic rats showed strong immunohistochemical staining of caspase 3 (Fig. 2c). Liver of hypercholesterolemic rats administered karela showed mild immunohistochemical staining of Caspase-3 (Fig. 2d).

Figure 3, shows that hypercholesterolemia decreased significantly glutathione-S-transferase (GST) and superoxide dismutase mRNA expression in liver. Administration of karela for cholesterol administered rats normalized mRNA expression pattern compared to control rats. On the other hand, hypercholesterolemia decreased pyruvate kinase (PK) mRNA expression that were ameliorated when karela co-administered for hypercholesterolemic rats (Fig. 4). In contrast, karela down regulated phosphoenolpyruvate carboxykinase (PEPCK) that was upregulated in hypercholesterolemic rats (Fig. 4). Co-administration of karela with cholesterol ameliorated this increase in PEPCK expression in hypercholesterolemic rats.

To examine the expression of genes for fatty acids oxidation such as acyl CoA oxidase (ACO) and carnitine palmitoyltransferase-1 (CPT-1), RT-PCR was carried done on liver tissues. Figure 5, shows that karela alone partially increased mRNA expression of ACO and CPT-1 while cholesterol administered rats showed downregulation in their mRNA expression. When karela co-administered with cholesterol it ameliorated this downregulation. In parallel, the enzyme essential for fatty acids synthesis (fatty acids synthase; FAS) showed a decrease in karela administered rats and upregulated in hypercholesterolemic rats. It was downregulated when karela co-administered with cholesterol (Fig. 5). Next, we examined the expression of PPAR-α and PPAR-γ in the liver and adipose tissue respectively as a transcriptional regulator of lipid metabolism and glucose homeostasis. As seen in Fig. 6, Karela activated PPAR-α and PPAR-γ expression in liver and adipose tissue respectively that were downregulated significantly in hypercholesterolemic groups.

Finally, we examined the effect of karela on genes associated with cholesterol metabolism such as sterol responsible element binding protein-1c (SREBP-1c), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR) and cholesterol 7α-hydroxylase (CYP7A1). Karela alone showed downregulation in SREBP-1c and HMG-CoAR expression that were upregulated in cholesterol administered rats (Fig. 7). Administration of karela to hypercholesterolemic rats normalized significantly this increase in mRNA expression of SREBP-1c and HMG-CoAR. Regarding the expression of CYP7A1, Karela showed partial increase in CYP7A1 and feeding cholesterol alone induced more and clear stimulatory effect in CYP7A1 mRNA compared to karela. When karela administered to hypercholesterolemic rats an additive upregulation effect was reported (Fig. 7).