ASH was instrumental in management of body weight
A gradual increase in the body weight was observed in the rats of LFD, HFD, and HFDE group over the period of 12 weeks. On the completion of respective regimen for 12 weeks, LFD and HFD rats weighed 16.7% and 40.1%, respectively, more than their initial weight (Fig. 1a). HFDE rats also weighed 23.9% more than their initial weight, but on the contrary, LFDE rats showed 10.2% reduction of body weight after 12 weeks of feeding ASH-supplemented normal chow diet. Further, the amount of calories from fat was also calculated for each week. Highest calorie intake was observed in the HFDE group over the period of 12 weeks (Fig. 1b), though it was reduced by 40.63% at the end of the 12th week as compared to initial intake. The HFD group also showed gradual reduction in calorie intake over the 12-week period. However, LFD and LFDE groups showed consistent calorie intake from fat during the respective regimens of 12 weeks.
ASH suppressed anxiety-like behavior in diet-induced obese rats
Prior to the EPM test, the animals were not given any training. Among the four groups, HFD rats spent minimum time in the open arm and maximum time in the closed arm as compared to LFD, LFDE, or HFDE rats (Fig. 1c). The HFDE group showed EPM profile comparable to LFD group. Further, HFD animals also showed less number of entries in both open and closed arms as compared to LFD animals (Fig. 1d). The number of crossings in the open arm was also reduced in HFD animals as compared to LFD animals (Fig. 1e). This behavior was reversed in HFDE animals, which showed highest number of entries and significantly high number of crossings (p ≤ 0.05) into the closed arm. Further, the number of head dips, which is an indicator of anxiety in rodents, was also reduced in HFD animals as compared to LFD animals, while HFDE animals showed head dips comparable to LFD animals (Fig. 1f). Overall, the behavior of LFDE animals was comparable to LFD animals.
ASH suppressed reactive gliosis and modulated inflammatory response
Glial cells play an indispensable role during generation of immune response under various stressed conditions. High fat diet feeding led to upregulation in the expression of GFAP, an astroglial marker, in both hippocampus and PC regions of the brain (Fig. 2a). Western blotting and real-time PCR data also supported the immunostaining data of upregulation of GFAP in hippocampus and PC regions of HFD animals (Fig. 2b–d). The supplementation of high fat diet with ASH suppressed the change in GFAP expression in HFDE animals as observed by immunostaining, Western blotting, and real-time PCR study (Fig. 2). The expression of GFAP in the LFDE group was not different from the LFD group. These results are suggestive of reactive gliosis in HFD animals, which was effectively suppressed in ASH-fed HFDE animals. Reactive gliosis may be one of the initial events leading to chronic immune activation and inflammatory response. So, we further studied the expression of some inflammatory markers by both Western blotting and real-time PCR.
The expression of Iba1, a microglial cell-specific marker, was upregulated by 19.9% in the hippocampus region (p ≤ 0.05) as compared to LFD animals (Fig. 2b, c). The treatment with ASH suppressed the expression of Iba1 in HFDE group. PPARγ, which is a nuclear receptor controlling the transcription of genes responsible for growth and differentiation of adipocytes, showed significant upregulation (p ≤ 0.05) in the hippocampus region, whereas its expression was alleviated in the HFDE group (Fig. 2b, c).
Further, the mRNA levels of various inflammatory markers were studied by quantitative real-time PCR (Fig. 2d). ITGAM, which is a gene coding for Cd11b on macrophages/microglia, showed 1.6-fold upregulation in the hippocampus region of HFD animals, which was reduced to 1.2-fold in the HFDE group as compared to LFD animals. The mRNA levels of other inflammatory markers, such as iNOS (inducible nitric oxide synthase), MCP-1 (monocyte chemoattractant protein-1), and COX2 (Cyclooxygenase-2) also showed upregulation in the hippocampus region of HFD animals, which was alleviated in the HFDE group. Overall, the mRNA levels of different markers in the LFDE group were similar to the LFD group.
Further, the expression of Iba1 showed 23.5% increase in the PC region of HFD animals (p ≤ 0.05), which was suppressed with ASH supplementation in HFDE animals (Fig. 2b, c). The expression of PPARγ was not different in the PC region between the LFD and HFD group animals; however, it was reduced significantly (p ≤ 0.05) in HFDE animals (Fig. 2b, c). However, no difference in the mRNA levels of ITGAM, iNOS, MCP-1, and COX2 was observed in the PC region among the different groups of animals (Fig. 2d).
ASH modulated the expression of pro-inflammatory cytokines
Since the preliminary data showed induction of inflammation in HFD animals, we further extended the work to study the expression of pro-inflammatory cytokines TNFα, IL-1β, and IL-6 by ELISA, Western blotting, and quantitative real-time PCR (Fig. 3). In ELISA-based assays, the circulating levels of TNFα, IL-1β, and IL-6 showed significant increase (p ≤ 0.001) in the HFD group animals as compared to the LFD group, which were alleviated in the HFDE animals (Fig. 3a). Further, the expression of TNFα, IL-1β, and IL-6 also showed significant increase (p ≤ 0.05) in the hippocampus region of the HFD group as compared to the LFD group at both translational (Western blotting Fig. 3b, c) and transcriptional levels (quantitative real-time PCR data Fig. 3d). However, the supplementation of ASH suppressed the HFD-induced increase of pro-inflammatory cytokines in the HFDE group (Fig. 3b–d). The expression of TNFα also showed upregulation in HFD animals as compared to LFD animals in the PC region (p ≤ 0.05), but it was reduced to near-control (LFD) level in both LFDE and HFDE animals as is evident from Western blotting (Fig. 3b, c) and real-time PCR results (Fig. 3d). No change was seen in the mRNA expression of IL-1β between the LFD and HFD groups, but it showed significant recovery in the LFDE and HFDE groups in the PC region at transcriptional level (Fig. 3d).
ASH ameliorated hyperleptinemia and hyperinsulinemia caused by HFD
Further, we estimated the circulating levels of leptin and insulin in these groups using ELISA-based assays. The level of leptin significantly increased in the HFD group (p ≤ 0.01) as compared to LFD animals and was partially recovered in the HFDE group (Fig. 4a, left panel). The level of leptin in the LFDE group was not different from the LFD group. Along with the increase in circulating level of leptin, the expression of leptin receptor OB-Rb showed decrease in HFD animals in both the hippocampus and PC regions of the brain as shown by both Western blot (Fig. 4b) and real-time PCR analysis (Fig. 4c, d), whereas, the expression of OB-Rb in HFDE and LFDE animals was similar to the LFD group. The circulating level of insulin also showed increase in the HFD animals (p = 0.06), which was recovered to near-control (LFD) level in HFDE animals (Fig. 4a, right panel).
Since SOCS1 and SOCS3 have been implicated in the development of leptin and insulin resistance, we further evaluated the mRNA expression level of these genes. Real-time PCR analysis showed significant upregulation (p ≤ 0.05) of both SOCS1 and SOCS3 in the hippocampus and PC regions of the brain, which was alleviated in HFDE animals (Fig. 4c, d). Leptin is known to activate JAK2-STAT3 signaling pathway, which regulates the expression of SOCS3. So, we further elucidated the differences in mRNA expression of JAK2 and STAT3. The expression of both JAK2 and STAT3 showed significant upregulation (p ≤ 0.05) in the hippocampus region of HFD animals, which was ameliorated by ASH in the HFDE group (Fig. 4c). However, the PC region did not show any change in the expression of JAK2 and STAT3 (Fig. 4d). We further studied the mRNA expression level of insulin receptor substrates IRS1 and IRS2. Consistent with the high level of circulating insulin in the serum, the mRNA levels of IRS1 and IRS2 showed downregulation in HFD animals in both the hippocampus and PC regions, which was restored to the normal level with ASH supplementation in HFDE animals (Fig. 4c, d).
ASH modulated NF-κB pathway and prevented apoptosis
We further extended the study to elucidate the effect of ASH on NF-κB pathway by both Western blot and quantitative real-time PCR (Fig. 5). The phosphorylated form of IKKα/β showed slight upregulation in the hippocampus region of the HFD group as compared to the LFD group, which was partially reduced with ASH supplementation in the HFDE group (Fig. 5a, c). Further, IKKα, which is known to be activated during CNS insult, showed significant (p ≤ 0.05) increase in the hippocampus region of the HFD group animals which was ameliorated in ASH-fed HFDE animals (Fig. 5a, c). IKβα, which is responsible for transcriptional activation of NF-κB, showed upregulation in the hippocampus region of HFD animals in both protein (Fig. 5a, c) and mRNA expression (Fig. 5e), which was suppressed in ASH-fed HFDE animals. However, the PC region of HFD animals showed upregulation only in mRNA expression (Fig. 5e), which was ameliorated in HFDE animals. NF-κB did not show any significant change in hippocampus region among the four groups (Fig. 5a, c, and e). However, its mRNA expression was significantly (p ≤ 0.05) downregulated in the PC region of the LFDE and HFDE groups (Fig. 5e). TLR4, which activates NF-κB pathway via MyD88 protein, showed significant (p ≤ 0.05) upregulation in the hippocampus region of HFD animals (Fig. 5e), but its expression was alleviated in HFDE animals. TLR4 also showed significant (p ≤ 0.05) downregulation in the PC region of HFDE animals (Fig. 5e). MyD88 showed slight upregulation in the HFD animals in the PC region only (Fig. 5e). LFDE animals also showed slight upregulation in the expression of MyD88 in both the hippocampus and PC regions (Fig. 5e) of the brain, but the change was not statistically significant.
Since NF-κB is also known to be instrumental in mediating cell survival pathway, we further studied the expression of apoptotic marker proteins. AP-1 showed significant upregulation in HFD animals as compared to the LFD group in both the brain regions at translational level (Fig. 5b, d), but its expression showed near-control level in ASH-supplemented HFDE animals. However, no change in mRNA expression of AP-1 was observed in HFD animals in either of the brain regions at transcriptional level (Fig. 5e), but it was reduced to 0.6-fold in the hippocampus region in the HFDE group. The PC region did not show any change in mRNA expression of AP-1 (Fig. 5e). Further, Bcl-xL, which is an anti-apoptotic protein, showed downregulation in the HFD animals as compared to LFD animals in both Western blot (Fig. 5b, d) and real-time PCR analysis (Fig. 5e), but the expression of Bcl-xL increased significantly in both the brain regions in LFDE and HFDE animals after supplementation with ASH (Fig. 5b, d, and e). BAD, the pro-apoptotic marker, did not show any change in either of the brain regions (Fig. 5e) in the HFD group, but it was reduced to 0.6-fold in the hippocampus region in the HFDE group. The PC region showed slight upregulation in mRNA expression of BAD in the HFDE group, but it was not statistically significant (Fig. 5e).
ASH suppressed HFD-induced inflammatory response in the hypothalamus region
As the hypothalamus is most affected by obesity-associated neuroinflammation, so we also evaluated the expression of some inflammatory markers in this region. The expression of Iba1 showed significant increase by 24.9% in the HFD fed group as compared to the LFD group (Fig. 6a, b), while it was significantly reduced to 47.7% with ASH supplementation in HFDE group animals. The increase in expression of Iba1 in the HFD group was also complemented by significant upregulation in the expression of pro-inflammatory cytokines TNFα and IL-1β (p ≤ 0.05) (Fig. 6a, b). However, ASH supplementation effectively suppressed the HFD-induced expression of these cytokines as evident from their near-control expression in the HFDE group.
Further, mRNA expression of iNOS significantly increased by 12.2-fold in the HFD fed group (p ≤ 0.05) as compared to LFD fed rats (Fig. 6c), but it was notably reduced to near-control level with ASH supplementation in the HFDE group. The mRNA levels of other inflammatory markers, such as COX2, TNFα, and IL-1β also showed upregulation in the hypothalamus region of HFD fed animals (p ≤ 0.05) (Fig. 6c). The transcriptional expression of COX2 was suppressed to some extent only in the HFDE group, but TNFα and IL-1β showed near-control levels with HFD+ASH regimen (Fig. 6c). The mRNA expressions of iNOS, COX2, and IL-1β in the LFDE group were not different from the LFD group, but TNFα showed 78% reduction in the LFDE group animals as compared to the LFD group.
ASH modulated leptin signaling pathway in hypothalamus
Since hypothalamus is the main site for leptin action, we evaluated the effect of HFD on leptin signaling pathway in this region. The expression of OB-Rb, the long form of leptin receptor, showed reduced expression in the HFD fed group as compared to the LFD fed group at both transcriptional (Fig. 7c) and translational (Fig. 7a, b) levels. This also corresponded with the higher circulating level of leptin in this group. However, the expression of OB-Rb was restored to near-control level with ASH supplementation in the HFDE group animals. Since leptin is known to activate JAK2-STAT3-SOCS3 pathway [27], we further elucidated the mRNA expression level of these markers in the hypothalamus region. All the three markers, viz., JAK2, STAT3, and SOCS3, showed significant upregulation in the HFD-fed animals (p ≤ 0.05) as compared to the LFD group (Fig. 7c). However, ASH supplementation effectively suppressed the stress-induced expression of these markers. The LFDE group also showed significant reduction in expression of STAT3 and SOCS3 as compared to the LFD group, but expression of JAK2 was enhanced significantly (Fig. 7c).
ASH modulated NF-κB pathway in hypothalamus
Considering the significant inflammatory changes observed in the hypothalamus region of the HFD group animals, we further analyzed the expression level of NF-κB pathway marker proteins. The upstream markers in NF-κB signaling pathway, IKKα and IKβα, showed significant upregulation by 50 and 130%, respectively, in HFD group animals as compared to the LFD group (Fig. 8a, b). The expression of these marker proteins was attenuated to some extent in the HFDE group. NF-κB also showed upregulated expression in the HFD fed group at both transcriptional (Fig. 8c) and translational (Fig. 8a, b) levels. However, it was only reduced to some extent at the translational level (Fig. 8a, b), but mRNA level of NF-κB showed significant reduction in the HFDE group (Fig. 8c). The expression of NF-κB in the LFDE group was similar to the LFD group. Further, the mRNA level of Bcl-xL, the anti-apoptotic marker protein, showed 24% reduction in the HFD-fed group as compared to the LFD group (Fig. 8c), but it was restored to near-control level in the HFDE group. The LFDE group animals also showed higher expression of Bcl-xL as compared to the LFD group animals.