Bitter melon (Momordica charantia) attenuates atherosclerosis in apo-E knock-out mice possibly through reducing triglyceride and anti-inflammation

Animals and diets

ApoE-/- mice with the C57BL/6 genetic background provided by Joslin Diabetes Center, Harvard Medical School (Boston, MA, USA ) were breeding in a pathogen-free environment with a 12 h light/dark cycle with free access to food and water and performed according to institutional and governmental guidelines. A high-fat diet containing 21.8% fat providing 42% energy and 1.25% cholesterol was purchased from GDLAC (GDLMC, Guangzhou, China). Twenty four male mice aged 8 weeks were randomized into control group (HFD, n=12) fed with high fat diet (HFD) only and BM -treated group (BM, n=12) fed with HFD mixed with BM at a dose of 1.2%w/w for 16 weeks, based on a previous study [20]..

Analysis of serum metabolic profile

Body weight and food intake of animals were recorded weekly. Intraperitoneal glucose tolerance test (IPGTT), with injection of 20% glucose at a dose of 2g/kg, was administered with tail vein blood at week 14 with One Touch Ultra meter (Lifescan; Johnson & Johnson, USA) at 0, 15, 30, 60 and 120 min. For the measurement of the blood lipids profile, blood was collected from orbital sinus of the animals which already fasting for 8 hours and anesthetized by isoflurane. We centrifuge the blood samples at 1500g 4^oC for 10min to obtain the serums and store at -80oC. The measurement of the serum levels of TG, TC, LDL, HDL, VLDL are operated on BECKMAN AU-5800 automatic biochemical analyzer with Olympus original reagent, calibration and quality control solution(Olympus, Japan). Serum TG levels were measured by Glycerophosphate oxidase - peroxidase (GPO-PAP) method. Serum TC levels were determined by the enzymatic method, HDL levels were determined by chemical modification method. LDL and VLDL levels were determined by selective lysis enzymatic method.

And the serum soluble adhesion molecules were determined by enzyme-linked immune-sorbent assay (R&D Systems, UK). In addition, aortas and other tissues were collected at the end of the study and quickly frozen in liquid nitrogen and then stored at −80°C for later analysis.

Quantification of atherosclerotic lesion area and measurements of atherosclerotic plaques histological composition

The aortas after opening and fixing in 10% formalin for 36h were stained with Sudan IV to detect the atherosclerotic lesion area and then photographed by a digital camera connected to a dissection microscope. The amount of atherosclerosis lesion was evaluated as the ratio of the atherosclerotic lesion area to the whole aorta area by Image-Pro Plus 6.0.

The aorta fixed in 10% formalin after 24h was paraffin embedded and cross-sectioned to analyze its histological composition. Masson’s trichrome stain kit (Maiwei, Xiamen, China) was used to assess the content of collagen fibers in atherosclerotic plaques and immune-histochemistry incubated with goat anti-macrophage-2 antigen (MAC-2) mouse macrophage or anti-alpha-smooth muscle cell (α-SMA) specific actin polyclonal antibody respectively (Bioss, Beijing, China) to qualify the contents of vascular smooth muscle cells and macrophage cells. All cross-sections were analyzed under an upright microscope (Nikon, Tokyo, Japan) .The amounts of macrophage and smooth muscle cells as well as the collagen fibers were quantified by image-processing software (Image Pro-plus 6.0).

Determination of Inflammatory cytokines mRNA levels in aortas

The mRNA levels of MCP-1 and IL-6 were measured by SYBR Green Quantitative Real-time PCR. RNA were ed from aorta tissues with E.Z.N.A Total RNA Kit II (Omega, USA) and then reversed to cDNA with PrimeScript TM RT reagent Kit (Takara biotechnology, Dalian, China) under the following conditions: 37°C for 15 min , 85°C for 5 seconds and 4°C forever. Quantitative Real-time PCR was performed with ABI 7500 (ABI, USA) using SYBR premix Ex Taq (Takara biotechnology, Dalian, China) as follows: 1 cycle at 95°C for 30 minutes; 40 cycles at 95°C for 5 seconds and 60°C for 34 seconds; and 1 cycle 95°C for 15 seconds, 60°C for 1 minute and 95°C for 15 seconds. The primers used as follows: GAPDH F5’-GTG AAGCAGGCATCTGAGGG-3’ and GAPDH R5’-CGAAGGTGGAAG AGTGGGAGT-3’; MCP-1 F5’-GCA GCA GGT GTC CCA AAG AA F-3’ and MCP-1 R5’- ATT TAC GGG TCA ACT TCA CAT TCA A -3’; IL-6 F5’-AGG AGA CTT GCC TGG TGA AAA-3’ and IL-6 R5’- AAA GCT GCG CAG AAT GAG ATG-3’. The relative quantification values for the mRNA expressions of MCP-1 and IL-6 were calculated by ΔΔCT .

Statistical analysis

All data were expressed as mean±SD or mean±SEM. Comparisons of means between the two groups were analyzed by un-paired Student’s t test. Levene's Test was used for Equality of Variances. A p value<0.05 was considered statistically significant. All analysis were performed using SPSS version 13.0 for Windows.

Effects of BM on body weight, food intake and metabolic profiles

There were no differences between the two groups in body weight at baseline (18.02±1.05 and 18.09±1.10 g; HFD vs BM; n=12), but after 16 weeks HFD resulted in a significantly increase of body weight in both groups (32.73±1.43 and 30.95±1.23 g; HFD vs BM; n=12; Table 1). Compared with the control group, the body weight significantly decreased in BM group from 2 to 16 weeks (p <0.05, Fig. 1a).There was no significant difference in food intake and blood glucose levels at 0, 15, 30, 60 and 120 min between the two groups (Fig. 1b and Fig.2a). BM decreased the serum TG level significantly (41.04±11.72 vs 60.73±15.62 mg/dl; p=0.013; Table 1 and Fig. 2b), while with no effect on TC, LDL ,VLDL and HDL (p >0.05; Table 1 and Fig. 2b).

	Control	BM	P value
Daily Food intake(g/w)	37.13±3.42	36.79±3.62	0.79(NS)
Body weight(g) pretrement	18.02±1.05	18.09±1.10	0.87 (NS)
Body weight(g)	32.73±1.43	30.95±1.23b	0.004
Fasting blood-glucose(mg/dl)	109.58±10.65	119.03±10.82	0.10 (NS)
TC(mg/dl)	1211.02±163.79	1281.16±123.93	0.35(NS)
TG(mg/dl)	60.73±15.62	41.04±11.72a	0.013
HDL (mg/dl)	52.78±5.25	50.60±14.32	0.69(NS)
LDL(mg/dl)	919.61±96.49	917.92±70.75	0.97(NS)
VLDL(mg/dl)	789.24±157.62	876.07±78.52	0.19(NS)

Table1 TG as triglycerides, HDL as high-density lipoprotein, LDL as low-density lipoprotein and VLDL as very low-density lipoprotein. Values are expressed as mean±SD; n=8-12 per group; ^ap<0.05, ^bp<0.01; Control versus BM

BM reduced the area of atherosclerotic lesion in ApoE-/- mice

The atherosclerotic plaques were showed by Sudan IV staining as Fig. 3a. In ApoE-/- mice fed with HFD, there were fewer atherosclerotic plaques of the entire aorta in BM group than in control group (12.91±1.15 vs 11.25±1.31 %, p=0.042; Fig. 3b). We found decreasing tendencies exist in the atherosclerotic plaque area of the aortic arch and abdominal aorta under the treatment of BM , although with no statistical significant (p=0.068 and p=0.12; Fig. 3c, d).

Effects of BM on the histological composition of the atherosclerotic plaques

The histological composition of atherosclerotic plaques containing vascular smooth muscle cells, macrophages and collagen fibers were analyzed by immunohistochemistry and Masson’s trichrome staining (Fig. 4a, b, c). The vascular smooth muscle cells and macrophages expressed in aortic plaques tended to decrease in the BM group compared with the control group, although there was no statistical difference (p=0.097 and p=0.134; Fig. 4d, e). There were significantly less collagen fibers in aortic plaques in BM group than in control group (49.48±2.54 vs 59.83±5.82 um²; p=0.03; Fig. 4f).

BM decreased the serum sVCAM-1 and sP-selectin levels and inhibited the expressions of MCP-1 and IL-6 in aortas

The serum levels of adhesion molecules and the expression of inflammatory cytokines in aortas were determined at the end of the study. The serum soluble VCAM-1 and P-selectin significantly decreased in the BM group compared with the control group (1151.92±142.90 vs 1365.18±170.26 mg/dl, p=0.008; 288±44.46 vs 244.43±46.67 mg/dl, p=0047; Fig. 5a, b). BM also significantly reduced the expression of MCP-1 and IL-6 in aorta tissues (p=0.002 and p=0.048; Fig. 5c, d).