Withania somnifera leaf alleviates cognitive dysfunction by enhancing hippocampal plasticity in high fat diet induced obesity model

The leaves were collected from the seed raised Ashwagandha plant growing at the herbarium of the Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, India. The plant was identified and authenticated by the plant taxonomist and a voucher specimen was deposited in the herbarium for the reference purpose with deposition no. 401-24/07/1982. Our previous lab studies standardized and reported the in vivo dose of water extract of Ashwagandha (ASH-WEX), but in the present study, we used dry leaf powder of ASH. The dose of ASH powder for the current study was calculated based on our previous in vivo report [21, 22] using ASH-WEX (dry weight) at a dose of 35 mg/250 g BW of the animal. Given that 1 mg of dry weight of ASH-WEX is equivalent to 6.80 mg of dry leaf powder, so the dose corresponds to 238 mg/250 g or ~1 g/kg or 1 mg/g BW of the animal as standardized in our previous studies. The composition of ASH-WEX has been characterized and reported in our previous study [23].

Wistar albino young female rats in the age group of 3–4 months with body weight in range of 130–150 g were used for all the experiments. All rats were caged in the group of three under controlled conditions with temperature of 25 ± 2 ̊C and dark/light cycle of 12:12 h with ad libitum access to food and water. Rats were divided into four groups: Low fat diet (LFD) on regular chow, High fat diet (HFD) group on feed containing 30% fat by weight, Low fat diet extract (LFDE) group on regular chow and dry leaf powder of Ashwagandha (ASH) and high fat diet extract (HFDE) group on diet containing high fat and ASH. The leaf powder was mixed in regular chow and high fat diet at a concentration of 1 mg/g of body weight of the animal. All the rats were kept on their respective diet regimen for 12 weeks and they were assessed for the weight gain once after every week. All procedures of animal handling and experiments were conducted in accordance with the guidelines laid down by the Institutional Animal Ethical Committee (IAEC) of Guru Nanak Dev University, Amritsar, Punjab, India and complied with arrived guidelines.

The blood glucose levels of all the rats were evaluated once after every 15 day till the end of the regimen. The rats were subjected to fasting for 12–14 h before the blood glucose test. The glucose level was assessed by using glucometer by collecting small amount of blood from the animal. The corticosterone levels were analyzed from serum collected at the time of experiment post 12 weeks diet regimen, irrespective of the phase of estrous cycle using corticosterone ELISA kit by Cayman chemicals (USA) as per the manufacturer’s protocol.

For immunostaining of PSA-NCAM in dentate gyrus (DG) region of hippocampus and pyriform cortex, experimental rats (n = 3) for each group were transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer saline (PBS). Brains were dissected and incubated in the fixative (4% PFA) for overnight at 4 °C and subsequently cryopreserved in 20% and then 30% sucrose for 24 h each at 4 °C. Coronal sections of 35 μm were cut using cryostat microtome and treated as described in our previous study [22].

Rats (n = 3–4 for each group) were anesthetized using thiopentone injection (1 unit per 10 g) and sacrificed to dissect out brain from the animal. Pyriform cortex (PC) and hippocampus regions were dissected out and lysed in lysing buffer using tissue homogenizer and centrifuged at 5000 rpm for 10 min at 4 ̊C. Supernatant was collected after centrifugation and used for protein estimation by Bradford method. 50 μg of protein from each sample was mixed with 6X sample buffer. Protein samples were resolved in 7–10% SDS-PAGE as per their molecular weight for better resolution, followed by transfer onto a 0.45 μm pore size PVDF membrane (Hybond-P from Amersham Biosciences UK Limited) using the semi-dry Novablot system (Amersham Biosciences UK Limited) at 90 mA for 90 min. Transferred membranes were then blocked with 5% skimmed milk in 0.1% TBS-Tween 20 for 2 h. Further, membranes were probed with mouse monoclonal anti-PSA-NCAM (1:1500), anti-NCAM (1:2000), anti CaMKIIα (1:2000), anti CaN (1:2000) anti-phospho Akt-1 (1:2000), (Sigma-Aldrich St. Louis, MO, USA), anti-c-Jun (1:2000), anti-c-fos (1:2000) (Santa Cruz Biotechnology Inc., USA), for overnight at 4 °C. This was followed by three washing with 0.1% TBS-Triton-X 100 of 10 min each and incubation with HRP-labeled anti-mouse IgM (1:2000) or anti-rabbit IgG (1:5000) or anti-mouse IgG (1:5000) secondary antibodies (Genei) for 2 h at 25 ̊C. The remaining procedure was followed as described in our previous study [22].

Total RNA was extracted from the cells by TRI reagent (Sigma-Aldrich) according to manufacturer’s instructions. For cDNA synthesis, 5 μg of RNA, 1 μl of 250 ng (N6) random hexamer (Applied Biosystem), 1 μl of dNTP mix (10 Mm of each dNTP (Amersham), nuclease free sterile water upto 12 μl, 4 μl 5X first strand buffer, 2 μl of 1 M DTT, 1 μl of of recombinant ribonulease inhibitor (40 units/ μl) (Invitrogen), 1 μl of 200 U of M-MLV reverse transcriptase (Invitrogen) were added for 20 ul reaction volume. 50 μg of cDNA was then used to amplify a gene of interest using StepOne Plus Real Time PCR system (Applied Biosystems). Each 5 μl reaction mixture comprised of 2.5 μl of 2X SYBR green Master Mix, 1 μl of 20× predesigned Primer mix (Applied Biosystem) and of 1 μl water and 1 μl (50 μg) cDNA. GAPDH was used as an endogenous control for each gene of interest. The relative gene expression of each gene was calculated by ‘Livak method’ and represented as 2 − ΔΔCt and final gene expression as 2 − ΔΔCt ± SEM.

A gradual increase in the body weight was observed in rats fed with their respective diets and on completion of diet regimen, HFD rats weighed 24% more than the LFD rats. Interestingly, the percentage weight in LFDE rats was reduced to 15% compared to LFD alone group, whereas, body weight was similar in HFD and HFDE groups (Fig. 1a). Further, the blood glucose levels showed no significant change between the groups (Fig. 1b). Corticosterone levels were significantly higher in HFD rats compared to LFD rats and supplementation of ASH in HFDE group normalized the up regulated levels of corticosterone to basal levels (Fig. 1c).

During locomotor coordination studies, HFD rats showed both lack of interest and strength in performing these tasks as evident from higher number of falls and less time spent on the rotating rod. ASH treated LFDE and HFDE rats (Fig. 1d and e) performed as good as LFD rats, thus indicating their normal neuromuscular coordination. During narrow beam walk test, HFD rats were observed to have poor balance over the narrow beam and showed high percentage of paw slippage compared to LFD and HFDE rats (Fig. 1f). Further HFD rats took longer time to transverse the entire beam, whereas, LFDE and HFDE rats spent time almost equal to LFD group to transverse the beam (Fig. 1g) indicating their intact locomotor functions. These observations from the behavioral studies indicated that HFD regimen caused locomotor and neuro-muscular dysfunction and ASH have the potential to improve motor performance and body balance in rats on HFD regimen.

During novel object recognition task, HFD rats showed impairment in their recognition and working memory as is suggested by higher number of episodes in exploring the old object over the novel one (Fig. 2a) and less time spent in exploring the novel object (Fig. 2c). Both LFDE and HFDE rats performed better as they spent more time in exploring the novel object compared to the old one (Fig. 2c). These observations were strongly supported by the PI score of the rats (Fig. 2b). The HFD rats showed least preference for the novel object (PI ≤ 0.5), whereas ASH supplemented rats showed more preference for the novel object (PI ≥ 0.5) (Fig. 1b). A PI score of less than 0.5 shows no preference for any of the objects and more than 0.5 score shows preference for the novel object [29]. Further, we studied grooming behavior in these rats during NOR test and observed that HFD rats showed higher percentage of grooming bouts, whereas LFDE and HFDE groups showed similar percentage of grooming bouts to LFD animals (Fig. 2d). Grooming is a nonspecific behavior that serves a broad range of purposes [30]. In rodents, grooming is an important behavioral repertoire and is associated with their adaptation to stress conditions as well as to self-cleaning [31]. Rodents generally follow a well ordered and sequential grooming pattern starting from rostrum up to tail under low stress conditions. Conversely, animals with high stress/anxiety levels display fragmented grooming pattern [31, 32]. In other words, self-grooming with non-structured pattern is considered as a marker of increased anxiety like behavior. In our study, HFD rats spent maximum time in grooming with interrupted and fragmented pattern paralleled by high number of grooming bouts, thus indicating their anxiogenic behavior. However, ASH supplemented rats showed least grooming activity coupled to a sequential grooming pattern as compared to HFD rats (Fig. 2e), thus suggesting the anxiolytic potential of ASH powder.

These observations are also supported by the rearing activity of the ASH supplemented rats as these were observed to show maximum rearing episodes compared to LFD and HFD rats (Fig. 2f). Rearing is the innate exploratory behavior found in rodents under most of the environmental conditions and is considered as a marker of environmental novelty that can be used to measure hippocampal learning and memory task [33]. Increased levels of corticosterone also support the higher stress levels and metabolic abnormalities in rats fed with HFD regimen, whereas, ASH feeding restored the basal levels of the stress hormone and the metabolic state in HFD rats (Fig. 1c). Since serum corticosterone levels were analyzed irrespective of the phase of estrous cycle, it may be possible that the different phases of estrous cycle have also contributed to these changes. However, a recent study carried out on high fat diet induced obesity has reported an increase in corticosterone levels in young adult male and female rats [34]. Several other studies in HFD induced obesity model have reported increased levels of corticosterone and their metabolites in obese rats which along with insulin increased the fat stores and contributed to obesity due to high caloric intake [35, 36]. Both basic and clinical research has reported memory retention and memory enhancing role of aqueous and alcoholic formulations from root of Ashwagandha [37‐39]. Aqueous leaf extract of Ashwagandha has been recently reported by our lab to improve memory retention functions by reducing the cellular stress via ameliorating the synaptic plasticity and cell survival pathways in acute sleep deprived rats [23]. Based on these observations, it may be suggested that ASH supplementation reduced the stress levels and exhibit nootropic effect in HFD induced obese rats.

Cognitive impairments are known to be the outcomes of alterations in hippocampal synaptic plasticity thus affecting learning performance and memory functions. Experiments to discern the underlying molecular mechanism of these alterations revealed up regulation in the expression of PSA-NCAM in hippocampus and PC regions of brain with HFD regimen (Fig. 3a and b). It has been reported that chronic restraint stress up-regulates the expression of PSA in DG and pyriform cortex regions of brain while inhibiting the process of neurogenesis [40, 41]. The enhanced polysialylation in HFD animals may be a neuroprotective mechanism in stress vulnerable neuronal circuits promoting the structural recovery and integrity. However, ASH treatment compensated for the stress induced increase in the expression of PSA-NCAM in HFDE rats (Fig. 3a and b). It has been reported that PSA is required for the basal synaptic transmission of new neurons but not in synaptic potentiation, which is solely dependent on NCAM expression [42]. A very recent study has reported that increased expression of NCAM1 with donepezil (acetylcholinesterase inhibitor) and aniracetam (nootropic) improved spatial memory in status epilepticus rats [43]. The poor recognition memory of HFD fed rats might be attributed to decreased expression of NCAM and ASH treatment significantly increased the mRNA and protein expression of NCAM (Fig. 3c, d–g), thereby improving the performance of LFDE and HFDE rats in memory task observed in the current study.

Further, the expression of CaMKIIα was evaluated as it is highly implicated as an initial signaling event in long term memory storage and its constitutive exposure to neurons is known to produce synaptic enhancement. It has been reported that there is reduction in the level of total CaMKIIα in DG and CA1 regions of hippocampus in obese zucker indicating the impairment of synaptic potentiation in hippocampus specifically in the CA1 region [44]. In the present study, the expression of CaMKIIα was significantly down regulated in rats on HFD regimen in both the brain regions (Fig. 3c, d–e) indicating the impairment in calcium mediated synaptic plasticity. ASH supplementation in LFDE and HFDE rats up regulated the expression of CaMKIIα in both the brain regions (Fig. 3c, d–e) indicating the conditions, permissive for synaptic enhancement. Another important regulator of synaptic plasticity is calcineurin and it has been reported that overexpression of calcineurin activate long term depression [45, 46]. In our study, we found no change in the protein expression of CAN between all the treatment groups compared to LFD rats in hippocampus region (Fig. 3c and d), whereas, in PC region, it was decreased in HFD rats and remained unchanged in LFDE and HFDE (Fig. 3c and e). This reduction in PC region could be due to a compensatory mechanism in HFD rats against the reduced expression of total CaMKIIα and due to divergent nature of neurons existing in the two regions.

Based on the changes in the synaptic plasticity molecules in the current study, it may be suggested that poor performance of HFD rats in NOR and locomotor coordination tests could be due to their impaired synaptic function. However, ASH supplementation restored the synaptic function by maintaining the optimal expression of NCAM and its polysialylated form, CaMKII (all implicated in synaptic transmission and learning and memory) thereby improving the performance of rats during the behavioral tasks.

High caloric diet is known to impair learning and memory by modulating synaptic plasticity and neurogenesis accompanied by reduction in the BDNF levels and dendritic spine density in hippocampus of the rats. Long term HFD feeding reduced the BDNF and its tyrosine receptor TRKB levels in hippocampus region of the brain (Fig. 4c) indicating that reduction in the synaptic plasticity in HFD rats may be mediated by the BDNF pathway. Several studies have reported that impaired cognitive functions and hippocampal neurogenesis in middle aged rats and in young offspring’s of the mother under high caloric regimen are mediated by reduced BDNF levels [47, 48]. Interestingly, ASH supplementation recovered the BDNF and TRKB levels in the LFDE and HFDE rats (Fig. 4c), indicating the restoration of the hippocampal function of learning and memory and synaptic plasticity. Treadmill exercise has been recently reported to improve the cognitive functions in high fat diet induced obese mice by improving the levels of BDNF and TRKB in hippocampus region of brain [4]. The better performance of ASH supplemented rats in learning and memory tasks may be due to their enhanced BDNF and TRKB levels in the hippocampus. In PC region, the expression of BDNF was increased in all the groups compared to LFD rats (Fig. 4d) which could be due to differential expression and synthesis of this neurotrophic factor in the two brain regions as supported by previous studies [49, 50]. The expression of TRKB was decreased in HFD rats and ASH supplementation restored the expression of TRKB to normal levels (Fig. 4d) similar to hippocampus region.

Neurotrophins are known to promote neuronal survival by Ras dependent activation of PI3 Kinase which in turn phosphorylates Akt to promote cell survival and inhibit apoptosis. PI3K/AKT is the principal signaling pathway that allows the cell to withstand apoptotic stimulus and promote cell survival. Down-regulation in the expression of PI3K gene in HFD rats (Fig. 4c and d) may be due to the inhibition of cell survival signal, which was restored to LFD levels in ASH supplemented rats (Fig. 4c) thus, suggesting the activation of cell survival signaling events in these rats. Likewise, the gene expression level of total and phosphorylated Akt-1 was down regulated in HFD rats further confirming the suppression of cell survival signal in these animals (Fig. 4a–d). ASH supplementation enhanced the expression of total and phosphorylated Akt-1 in LFDE and HFDE rats in both the brain regions, thereby promoting the survival conditions in the neuronal cells. Similarly, the expression of c-Jun and c-fos (IEGs in the cell survival mechanism) was suppressed in HFD rats (Fig. 4a–d), while ASH supplementation restored the expression levels of these transcription factors, specifically in hippocampus region (Fig. 4a left panel and b). In PC region, we observed activation of cell survival molecules but the changes were more prominent in hippocampus region. The possible reason for this differential response might be due to profound modulation of structural and functional plasticity and subsequent activation of survival molecules in hippocampus region as compared to the PC region. Stress induced activation of glucocorticoids are known to strongly inhibit the hippocampal BDNF expression in HFD rats [51, 52]. So it may be suggested that reduction in the BDNF expression may be linked to enhanced expression of corticosterone in HFD rats. BDNF enhances efficiency of synaptic transmission and hippocampal synaptic plasticity by activating CaMKII in hippocampus region thereby improving the cognitive performance [53]. In conclusion, it may be suggested that ASH treatment improved the memory and learning based functions by promoting the activity dependent hippocampal plasticity and cell survival and by reducing the cellular stress (schematic representation of the possible molecular pathways in ASH mediated activity in HFD rats in Fig. 5).