ARPs improved glucose and lipid metabolism disorders caused by HFD
In order to study investigated whether ARPs can regulate body weight, glucose and lipids homeostasis, we measured the following indicators. As illustrated in Fig. 1a, mice fed with a HFD were visibly heavier than the CD and HARPs. Compared with HFD, the mice in HARPs markedly decreased the serum glucose, triglyceride (TG) and total cholesterol (TC) levels (Fig. 1b-d). At week 12, the mice in HFD emerged insulin resistance accompanied by glucose intolerance compared with that in CD, while ARPs reversed these changes (Fig. 1e-h). These results suggest that ARPs effectively improved obesity and abnormal levels of serum lipids and blood glucose caused by HFD.
ARPs ameliorated memory impairments triggered by HFD
Spatial learning is often assessed using maze tasks such as the MWM or Y maze test [22, 23]. In this study, we use the MWM to assess the learning and memory ability. During the period of the MWM test, mice in HARPs group produced a decrease in the escape latency when compared with HFD (Fig. 2a). Simultaneously, during the probe trial (Fig. 2b), HARPs mice exhibited a significant increase in the average time spent in the target quadrant and exhibited greater numbers of platform crossings than HFD mice, which indicates that ARPs could improve HFD-induced memory impairments (Fig. 2c-f). Consistently, Y maze test also indicated that ARPs ameliorated memory impairments triggered by HFD significantly (Fig. 2c-f). The above results suggest that dietary ARPs supplementation effectively inhibited the impairment of learning and memory in obese mice induced by HFD.
ARPs improved cognitive disorder induced by HFD
BDNF is essential in maintaining neural cells survival and promoting neurite outgrowth, synaptogenesis, memory and learning . In addition, microtubule-associated protein Tau is a key molecule in the pathogenesis of Alzheimer’s disease . In this study, the morphology of hippocampal neurons was observed by H&E staining. The results showed that 12-week HFD increased the number of nuclear pyknosis of hippocampal neurons, and ARPs could improve this phenomenon (Fig.3a). Furthermore, immunofluorescence and RT-PCR analysis were used to analyze the content of neurotrophic factors in hippocampal. The immunofluorescence results showed that 12-week HFD decreased BDNF levels in hippocampal, and ARPs effectively reversed this trend (Fig.3b). RT-PCR analysis of BDNF expression in hippocampal also reached the similar result, meanwhile, Tau protein expression were decreased by ARPs treatment. Moreover, the mRNA expression of quinone oxidoreductase-1 (NQO1), neurotrophin-3 (NT3), postsynaptic density protein-95 (PSD95) and recombinant human fibroblast growth factor-21 (FGF21) in mice hippocampus were decreased in HFD mice, and dietary supplementation of ARPs reversed the trend (Fig.3c-d). Taken together, these results suggest that ARPs promotes the survival of nerve cells and improve cognitive impairment.
ARPs attenuated HFD-induced neuroinflammation in hippocampal
Inflammation is thought to be the most important pathophysiological mechanism of cognitive disorders , and Tumor necrosis factor-α (TNF-α) is reported to be closely associated with these impairments. In this study, the results of immunohistochemistry showed that ARPs improved the abnormal increased TNF- α induced by HFD. RT-PCR results revealed that compared with CD group, the mRNA expression of TNF-α, monocyte chemoattractant protein-1(MCP-1), interleukin- 6 (IL-6) and mouse EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80) in hippocampal were increased in mice on HFD, while ARPs effectively abolished these effects. These data demonstrate that dietary supplementation of ARPs ameliorates hippocampal inflammation caused by HFD.
ARPs attenuated HFD-induced colon inflammation
Gut microorganisms and metabolites can stimulate inflammatory factors production and secretion, which triggers or exacerbates central inflammation and even induces cognitive dysfunction . In this study, immunohistochemistry results showed that supplementation of ARPs attenuated the increased TNF- α expression in colon induced by HFD. Meanwhile, RT-PCR analysis revealed that increased mRNA expression of TNF- α, MCP-1, IL-6 and F4/80 in colon were increased by HFD, and dietary supplementation of ARPs effectively reversed this trend.
ARPs ameliorated intestinal barrier and microbial metabolite disorder induced by HFD
The integrity of intestinal epithelium is considered to be the first defense line of the gastrointestinal tract . Studies have shown that HFD lead to damaged intestinal epithelial barrier by increasing inflammation . In this study, H&E staining results demonstrated that the vacuoles of lipid accumulation in colonic villi from HFD mice were increased, and ARPs significantly improved these phenomenons (Fig.6a). Moreover, PAS staining showed that the number of mucus-secreting goblet cells, intestinal wall thickness and villus height were decreased by HFD (Fig.6a), and RT-PCR analysis further revealed the expression of MUC-2 were down-regulated (Fig.6b). By contrast, dietary supplementation of ARPs reduced the damage of epithelial integrity caused by HFD and increased the number of goblet cells.
Related studies have shown that long-term HFD can destroy the integrity of intestinal barrier and cause intestinal leakage . As shown in (Fig. 6c), the expression of tight junction protein zonula occludens-1 (ZO-1) in colon from HFD mice was down-regulated. Moreover, mRNA levels of recombinant regenerating islet derived protein 3g (Reg3g), mucoprotein-2 (MUC-2) and Occludin-1 in colon (Fig. 6b) also reached the same result. while ARPs reversed these effects.
HFD has been reported to induce intestinal microbial disorder and increase LPS production, which lead to increased LPS and inflammatory factors levels in the circulatory system, causing or aggravating systemic inflammation . In this study, the contents of serum LPS and TNF- α in mice on HFD were significantly increased, and ARPs could effectively ameliorate the phenomenon (Fig.6d). The above results suggest that dietary supplementation of ARPs can improve the integrity of intestinal barrier induced by HFD, and has a certain ability to regulate intestinal microbial metabolites.
ARPs ameliorated HFD-induced intestinal microbial disturbance
In has been suggested that gut microbiota influence brain plasticity and cognitive function . In this study, 16S rDNA sequencing analysis was used to detect whether ARPs could affect the composition of intestinal microflora. Firstly, principal component analysis (PCA) showed significant differences in microflora structure among the three groups (Fig.7a), and ɑ-diversity and β-diversity of the microbiota from mice on HARPs were similar to mice on CD (Fig.7b). In order to further study the specific changes of bacterial communities, we compared the relative abundance of dominant phyla and genera in the three groups, especially those that responded to ARPs. The results showed that HFD led to a significant increase in the abundance of eight bacterial genus including Parabacteroides, Enterorhabdus, Lactoccus, Bacteroides, Lactocbacllus, Erysipelatoclostridium, Dubosiella and Faecalibaculum, while ARPs supplementation significantly decreased the relative abundance of the above bacteria genus (Fig.7c).