In our study, we found that a calorically restricted high-fat diet could protect against the adverse effects included by a high-fat diet fed ad libitum, including dyslipidemia, inflammation, oxidative stress and liver function damage in Wistar rats. Additionally, the transcription analysis revealed the major mechanisms for the improvements in inflammation and oxidative stress, which occurred via downregulation of the NF-kappa B signaling pathway and the upregulation of GSTs.
Over the last decade, the effects of a standard diet with CR on improving health and prolong life have been proven in various model species [22, 23]. However, CR has been found to have detrimental effects on bone health, wound healing, and certain immune responses, and excess CR may restrain growth, development and normal physiological processes . To avoid excess CR, we calculated the number of calories based on food intake ingested by rats supplied a normal diet fed ad libitum (NC group) and normalized the number of calories between the NC and EHF groups. Some previous studies have discussed the effects of a high-fat diet with caloric restriction, but the high-fat diets in those studies contained 45% or 60% fat, which are extreme proportions of fat when compared to the Western dietary pattern [25, 26]. Our study aimed to mimic typical Westernized dietary habits and carry out long-term intervention in rats. Therefore, we chose high-fat diet containing 33.8% fat, which is similar in macronutrient composition to the standard AIN-93M diet. As a result, rats in the EHF group gained less weight, accumulated less body fat, and had lower serum TG, serum LDL-C and serum TCHO than rats in the HF group, which is similar to the pattern revealed in previous studies.
Inflammatory and oxidative stress are thought to be the key contributors to the pathogenesis of metabolic dysfunctions caused by a high-fat diet, while CR can delay the aging process via reducing inflammatory and oxidative stress [23, 27]. For example, rats under long-term CR are protected against inflammatory and oxidative stress in multiple tissues, including the brain, lung, liver, kidney and heart[28, 29]. Simultaneously, CR reduced the inflammatory factor levels in tissues and the circulatory system and decreased ROS, and animals under a CR modulated their antioxidant defenses to more effectively detoxify the generated radicals . In the present study, compared with the NC group, the serum levels of ROS and IL-6 significantly increased in the HF group, and the serum levels of TNF-α and MDA were slightly increased. No significant differences in these factors were observed between the EHF and the NC groups. Compared with the HF group, the serum levels of IL-6 and ROS were significantly decreased, while the serum levels of TNF-α and MDA were slightly decreased. Moreover, the mRNA expression level of IL-6 and the mRNA level and protein expression level of TNF-α in the liver were significantly decreased in the EHF group. The activities of SOD and GSH were assayed as indices of antioxidant capacity. Rats in the EHF group expressed a higher level of both indices in the serum and the mRNAs in the liver in comparison with the HF group, while the serum levels of SOD and GSH were only slightly increased in the NC group compared to the HF group. These data indicate that an EHF maintained the oxidoreductive force in tissues and organs, which was similar to, or even higher than that in the NC group and revealed reduced inflammatory and oxidative damage induced by a high-fat diet fed ad libitum.
The liver is the most important metabolic and secretory organ and participates in various physiological activities. A high-fat diet was reported to aggravate liver lipid accumulation, resulting in hepatic steatosis or pathological changes [5, 31]. In our study, the EHF group showed lower serum levels of AST and ALT in serum compared to the HF group. In addition, histological examination of liver tissues showed decreased macrovesicular steatosis, inflammatory cell infiltration and structural damage in the EHF group compared to the HF group. Therefore, these findings suggest that an EHF reduces liver lipid accumulation, relieves functional damage and improves inflammatory and oxidative stress.
Whole genome expression analysis was conducted in liver tissues, where the gene transcription level was distinctly higher compared with that in other organ types . GSEA analysis and DEG enrichment analysis with KEGG pathway were both executed to avoid deviations due to single analysis. Members of Toll-like receptors (TLRs) family act as primary sensors that detect a wide variety of microbial components and elicit immune responses. All TLR signaling pathways culminate in activation of the transcription factor NF-κB . NF-κB, a primary regulator of inflammatory responses, plays a critical role in a variety of physiological and pathologic processes, and the NF-kappa B signaling pathway is involved in both inflammation and oxidative stress and participates in the development of various metabolic diseases . Recent studies have demonstrated that a high-fat diet can affect peripheral tissues, and cause systematic inflammation by directly or indirectly activating the TLR4 and nuclear factor-kappa B NF-κB pathways [35, 36]. Our transcription data obtained through KEGG pathway analysis indicated that the NF-kappa B signaling pathway was significantly inhibited by an EHF, and TLR4, the most pronounced hub gene according to both PPI and MDOCE analyses, was significantly downregulated in the EHF group. Furthermore, the mRNA and protein levels of TRL4 and NF-κB in liver tissues were significantly decreased in the EHF group versus the HF group in the present study. These data proved that a major component of the mechanism by which the EHF ameliorates inflammation and oxidative damage is via a reduction in TRL4 to inhibit the NF-kappa B signaling pathway. In addition, the Glutathione metabolism pathway was significantly upregulated in the EHF group, and the GSTA2, GSTA3 and GSTA5 genes were also significantly upregulated, as shown by RNA-seq analysis and verified by Q-PCR. Glutathione S-transferases (GSTs) are a major group of detoxification enzymes and the main line of defense against the spectrum of highly toxic substances produced by ROS-mediated oxidative reactions . The increased levels of GSTs induced by an EHF probably participate in antioxidant processes and mitigate oxidative stress.