Hyperlipidemia, which is closely associated with a fatty diet and aging, is commonly observed in the western and aged society. Therefore, a novel therapeutic approach for this disease is critical, and an immunological view has been suggested as a novel strategy, because hyperlipidemia is closely associated with inflammation and immune dysfunction. In this study, the effects of an aqueous extract of
Our society has currently adopted a westernized diet and lifestyle and, therefore, the prevalence of metabolic diseases has increased. Metabolic diseases are regarded as the leading cause of mortality in the US. In particular, hyperlipidemia is a symptom of metabolic disease and is significantly associated with inflammation. Excessive lipoproteins initiate partial inflammation by modulating leukocyte activity and disturbing cytokine regulation, which worsens over time (Emanuela
The lipid-lowering effect of RO was demonstrated
The RO used in this study was provided by the Berry and Biofood Research Institute (Gochang, Korea). The aqueous extract of the immature RO was prepared in accordance with a previous report (Choi
The ellargic acid content of the lyophilized RO was analyzed as a marker compound by using a standardized high-performance liquid chromatography (HPLC) method (Kim
The male C57BL/6 mice (4-week-old) were purchased from Samtako Inc. (Osan, Korea), and allowed to acclimatize for 1 week before the experiments were conducted. The mice were housed in a laboratory animal facility at a temperature and humidity of approximately 20–24°C and 30–70%, respectively, and a 12-h light-dark cycle, with free access to commercial rodent feed and sterile water. All the experimental procedures were performed in strict compliance with the Guidelines for the Care and Use of Laboratory Animals issued by the Sahmyook University (Seoul, Korea).
To induce hyperlipidemia, the mice were fed a 45-kcal high-fat diet (HFD) for 23 weeks, and the disease progression was confirmed by monitoring the body weight and blood cholesterol levels. Then, RO and rosuvastatin were orally administered for 16 weeks, after which the mice were euthanized under ether anesthesia.
The relevant biomarkers such as body weight and blood cholesterol levels were used as indicators of hyperlipidemia. During the HFD feeding, blood samples from each mouse were collected by retro-orbital bleeding, and a specific amount of serum was isolated for cholesterol analysis by using a biochemical analyzer (AU480, Beckman Coulter, CA, USA). The mice were selected and grouped for further experiments based on their individual cholesterol parameters including total cholesterol, high-density lipoprotein (HDL), LDL, and triglyceride levels. The mice were subsequently euthanized, and their whole blood was collected for analysis.
The mouse splenocytes were prepared in phosphate-buffered saline (PBS). Prior to staining the lymphocytes, non-specific binding was prevented by Fc receptor blocking, and then they were incubated with monoclonal antibodies. The immunostained cells were fixed with 1% paraformaldehyde in PBS, and each sample was analyzed using a fluorescence-activated cell sorting (FACS) system (Beckman Coulter).
The splenocytes were incubated in the presence of lipopolysaccharide (LPS, 100 ng/mL) or concanavalin A (ConA, 1 μg/mL), and the cell proliferation was measured by using the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) cell viability assay. The absorbance values were read at 560 nm using an automated VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA, USA).
The levels of pro-inflammatory cytokines in the lymphocyte culture medium and mouse serum were measured using an enzyme-linked immunosorbent assay (ELISA) kit (eBioscience Inc., San Diego, CA, USA). The experimental process was performed according to the manufacturer’s instructions, and the absorbance values were measured at 450 and 570 nm using a microplate reader (Molecular Devices, Sunnyvale). The values obtained at 570 nm were subtracted from those at 450 nm, and the concentration of each cytokine was calculated according to a standard curve constructed by using each of the recombinant cytokines in the ELISA kit.
The mice in each group were injected subcutaneously with ovalbumin (OVA) peptide suspended in complete Freud’s adjuvant (CFA, Sigma-Aldrich Corp., St. Louis MO, USA), followed by a secondary injection of OVA peptide in incomplete Freud’s adjuvant (IFA, Sigma-Aldrich Corp.). After the secondary immunization, the mice were euthanized, whole blood samples were collected, the serum was separated, and then immunoglobulin G (IgG) levels were measured by using an ELISA. In brief, an immunoplate (NUNC, Roskilde, Denmark) was coated overnight with 300 mg/mL of OVA solution (Sigma-Aldrich Corp.) followed by blocking with 10% fetal bovine serum (FBS) in PBS. The serum samples and standard were dropped onto the plate and incubated, followed by the addition of the horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibodies (Sigma-Aldrich, Corp.) for specific binding, and then incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution to determine the IgG concentration.
According to the method of Im
All the results are presented as mean ± standard error of mean (SEM), and the significant differences between the control and treated groups were statistically analyzed using one-way analysis of variance (ANOVA) followed by a Dunnett’s multiple comparison test for each parameter of interest. This study used a
The major components of the RO were previously elucidated (Choi
To monitor the changes in body weight and lipid parameters of the mice, we measured their body weight every two weeks and blood lipids at the middle and end of experimental period. As shown in Table 1, HFD feeding for 23 weeks successfully induced obesity and hyperlipidemia in mice, but no significant change was observed in the triglyceride level. As shown in Table 2, the oral administration of RO significantly decreased the total cholesterol in the hyperlipidemic mice and improved the other indexes.
We also determined the weights of the spleens and thymuses of the mice as crucial indexes of the host immune state. As shown in Fig. 2A, the weight of the mouse thymuses decreased with age and HFD feeding, but there were no significant differences between the HFD-fed groups. In contrast, the HFD-induced hypertrophy of the spleen was normalized by oral administration of RO and rosuvastatin (Fig. 2B).
We monitored the changes in the ratio of immune cell sub-types to investigate the effects on the balance between them. As shown in Fig. 3A, the numbers of CD4+ and CD8+ T cells in the thymus were dramatically decreased by the fatty diet while the population of CD4+CD8+ double-positive cells was not affected. The oral administration of the RO extract significantly increased the population of these lymphocytes. The same subtypes in the splenocytes were examined, and the calculation of the ratio of CD4+ to CD8+ T cells showed that it was significantly altered by HFD feeding and restored following oral RO treatment (Fig. 3B). However, the absolute number of CD4+ and CD8+ T cells was not significantly modified by RO treatment. The CD11b+ cells in the spleen significantly increased in the HFD-fed mice while this effect was considerably diminished by treatment with both concentrations of RO extract (125 and 250 mg1·kg−1·day−1). No significant difference was observed between the groups in the splenic CD11c+ sub-set analysis.
To confirm the viability and proliferative activity of the lymphocytes, we performed an
We conducted a pro-inflammatory cytokine assay using the mouse host serum samples since inflammation is considered as a key factor in the development of obesity-associated immune dysfunction. The serum cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-6, were measured to determine the degree of systemic inflammation. The level of these cytokines remained high in the serum of the mice that were subjected to prolonged HFD feeding while the TNF-α but not IL-6 level was significantly restored following treatment with the RO extract (Fig. 5).
To determine whether RO extract treatment enhanced the humoral immune function
To determine whether RO extract treatment enhanced the cellular immune function
Metabolic diseases such as obesity, diabetes, and hyper-lipidemia are mutually regulated and accelerated by systemic inflammation (Lumeng
Black raspberry (RO), which is well known for its high content of polyphenols and other antioxidants, is thought to be a prominent therapeutic candidate for metabolic diseases. A series of studies recently elucidated the effect of the
Most importantly, the increased total cholesterol level, which is the metabolic phenotype of hyperlipidemia, was significantly ameliorated by the oral administration of RO extract in our study (Table 2). In addition, the immune-related effect of the RO extract was investigated to demonstrate the improvement in hyperlipidemia in a mouse model of DIO.
The host defense system can be quantitatively and qualitatively evaluated by determining the number of immune cells based on the prevalent subtypes and by assessing the immune cell functions associated with proliferation or cell killing. This can also be evaluated at the protein level by measuring the levels of immune-related proteins such as antibodies or cytokines. Based on these premises, the immunological analysis was designed and performed.
The lymphoid organs, which are the fundamental source of the entire array of immune cells, undergo changes in size based on the host immune status (Pope, 1991), and their weights can be used as a quantitative immunological index. The thymus, which is the site of immature T cell growth and maturation, tends to diminish in size as the host ages, and this effect is reported to be further accelerated by an HFD (Yang
The decline in the lymphocyte functions was determined by their proliferation level during the process of counteracting antigens, LPS- and Con A-targeted T cells. The HFD-fed mice exhibited an impairment in the lymphocyte proliferative activity, which was significantly restored by the oral administration of the RO extract (Fig. 4). Furthermore, the analysis of TNF-α, which was accompanied by a proliferation assay, showed high TNF-α levels in the HFD-fed mice compared to that in the RD-fed mice. This observation suggests that the lymphocytes of the HFD-fed mice were more likely to have initiated the inflammatory process, and the elevated TNF-α production was decreased by the oral administration of the RO extract (Supplementary Fig. 1). The increased levels of serum TNF-α and IL-6 in the HFD-fed mice, which were indicative of systemic inflammation, were decreased by RO extract treatment (Fig. 5), and this was assumed to be a novel finding.
TNF-α is a key regulator of the inflammatory response, and its blockade is used as a potent therapeutic strategy for the treatment of inflammatory diseases such as rheumatoid arthritis (Popa
To evaluate the specific immune function, the mice in each group were immunized with OVA, a model antigen. Since the antigen-specific defense system is associated with cellular and humoral immunity, we designed appropriate experiments, which involved the determination of IgG production level and target cell killing activity, respectively (Fig. 6, 7). The IgG production in B cells, which is generally regarded as the efficient defense strategy, has been found to be fundamentally pathogenic in DIO mice (Winer
These results consistently suggest that the oral administration of the RO extract attenuated the hyperlipidemia associated-immune decline in the HFD-fed mice. Furthermore, the RO extract was not only shown to improve hyperlipidemia, but its immune-enhancing effect was also newly elucidated based on the quantitative and qualitative evidence. In addition, the beneficial effects of the RO extract appeared to be mediated by its potential regulation of the TNF-α level.
This research was financially supported by the Korea Institute of Planning and Evaluation for Technology (IPET) in Food, Agriculture, Forestry, and Fisheries.