Beta-hydroxybutyrate, an endogenic NLRP3 inflammasome inhibitor, attenuates stress-induced behavioral and inflammatory responses

Abstract

Neuro-inflammation has been shown to play a critical role in the development of depression. Beta-hydroxybutyrate (BHB) is a ketone body and has recently been reported to exert anti-inflammatory effects via inhibition of NLRP3 inflammasome. Here, we investigated the potential antidepressant and anti-inflammatory effects of BHB on rats exposed to acute and chronic stress. We examined the influence of repeated BHB administration on depressive and anxiety behaviors in a rodent model of chronic unpredictable stress (CUS). Additionally, the influence of acute immobilization (IMM) stress and single BHB administration on hippocampal interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were assessed. Repeated administration of BHB attenuated CUS-induced depressive- and anxiety-related behaviors. IMM stress increased levels of IL-1β in the hippocampus, while a single pre-administration of BHB attenuated this increase. Although no effect was observed on hippocampal TNF-α levels after 1 h of IMM stress, a single BHB pre-administration reduced hippocampal TNF-α. Our previous report showed that the release of IL-1β and TNF-α caused by stress is tightly regulated by NLRP3 inflammasome. These findings demonstrate that BHB exerts antidepressant-like effects, possibly by inhibiting NLRP3-induced neuro-inflammation in the hippocampus, and that BHB may be a novel therapeutic candidate for the treatment of stress-related mood disorders.

Figure 1

Elevated levels of hippocampal BHB after subcutaneous administration of BHB. Levels of hippocampal BHB measured after subcutaneous injection of BHB are shown (n = 4). The concentration of BHB increased at 1 h after administration, after which gradual decreases in hippocampal BHB levels were observed. BHB: beta-hydroxybutyrate, Hip: hippocampus.

Figure 2

BHB treatment blocks chronic unpredictable stress-induced depressive-like behaviors. (a) The BWs of the rats during the CUS procedure are shown. CUS rats exhibited a decreased rate of BW gain at 1 week after starting the CUS procedure, as compared to the non-CUS rats. (b) Time spent immobile during the FST is shown. CUS rats showed a trend toward more immobility time relative to rats of the non-CUS control group. BHB significantly decreased the duration of immobility in CUS model rats. (c) The results of the SPT are shown. BHB significantly increased the preference for sucrose. (d) Latency to feed in the NSFT is shown. BHB significantly reduced the latency to feed in CUS rats. (e) Time spent in the open arms in the EPM is shown. CUS tended to reduce time spent in the open arms. BHB significantly attenuated this effect. (f) Numbers of open-arm entries in the EPM are shown. CUS tended to decrease the number of open-arm entries, although BHB administration significantly attenuated this effect. (g) Numbers of closed-arm entries in the EPM are shown. There was no statistically significant difference. CUS: chronic unpredictable stress, PBS: phosphate buffered saline, BHB: beta-hydroxybutyrate, BW: body weight, FST: forced swim test, SPT: sucrose preference test, NSFT: novelty suppressed feeding test, EPM: elevated plus maze test. *p < 0.05, **p < 0.01, ****p < 0.0001, Error bars: standard error of the mean (SEM).

Figure 3

Pretreatment with BHB reduces stress-induced increases in pro-inflammatory cytokine levels in the hippocampus. (a) IL-1β levels are shown. One hour of IMM stress increased levels of IL-1β in the hippocampus. (b) IL-1β levels are shown. BHB pretreatment decreased levels of IL-1β in the hippocampus in IMM model rats. (c) Hippocampal TNF-α levels after 1 h IMM stress are shown. 1 h of IMM did not change TNF-α levels in the hippocampus. (d) Hippocampal TNF-α levels after 1 h of IMM with pretreatment by BHB are shown. BHB pretreatment decreased TNF-α levels in the hippocampus in IMM model rats. (e) Hippocampal IL-10 levels after 1 h IMM stress are shown. 1 h of IMM did not change IL-10 levels in the hippocampus. (f) Hippocampal IL-10 levels after 1 h of IMM with pretreatment by BHB are shown. BHB pretreatment did not change IL-10 levels in the hippocampus in IMM model rats. IMM: immobilization stress, PBS: phosphate buffered saline, BHB: beta-hydroxybutyrate, *p < 0.05, Error bars, standard error of the mean (SEM).

Figure 4

Chronic peripheral BHB treatment reduces levels of TNF-α, but not of IL-1β, in the hippocampus after CUS. (a) IL-1β levels in the hippocampus are shown. Chronic BHB treatment did not alter levels of IL-1β in the hippocampus in CUS model rats. (b) Hippocampal TNF-α levels in the hippocampus are shown. Chronic BHB treatment decreased levels of TNF-α in the hippocampus in CUS rats. (c) IL-10 levels in the hippocampus are shown. Chronic BHB treatment did not alter levels of IL-10 in the hippocampus in CUS model rats. CUS: chronic unpredictable stress, PBS: phosphate buffered saline, BHB: beta-hydroxybutyrate, *p < 0.05, Error bars, standard error of the mean (SEM).

Figure 5

Pretreatment with BHB attenuates stress induced-serum corticosterone levels in IMM stress rats. (a) Serum corticosterone levels at 1 h after IMM is shown. 1 h of IMM significantly increased serum corticosterone levels. (b) Serum corticosterone levels at 1 h after IMM stress, with pretreatment by BHB are shown. BHB pretreatment decreased serum corticosterone level in IMM model rats. IMM: immobilization stress, PBS: phosphate-buffered saline, BHB: beta hydroxybutyrate, *p < 0.05, ****p < 0.0001, Error bars, standard error of the mean (SEM).

Figure 6

Experimental procedures. (a) The procedure for measurement of hippocampal BHB concentration is shown. In each group, four rats were treated with 250 mg/kg BHB subcutaneously, and were then sacrificed for sample collection at 1, 2, and 3 h after BHB injection. (b) Schematic representation of the CUS experimental procedure and behavioral tests. Three groups were studied: non-CUS + PBS, CUS + PBS, and CUS + BHB, n = 16 per group. Rats were exposed to two variable sequences of unpredictable stressors per day in the daytime and night. All rats were administered PBS or BHB (250 mg/kg) subcutaneously, twice a day. Behavioral tests were conducted after 4 weeks of CUS. (c) The experimental paradigm for IMM stress is shown. Rats were sacrificed after exposure to 1 h of stress, and the brain and blood were collected. (d) The experimental paradigm for pretreatment and IMM stress is shown. PBS or BHB was subcutaneously administered 1 h prior to IMM, and rats were sacrificed after 1 h of IMM stress, and the brain and blood were collected. CUS: chronic unpredictable stress, PBS: phosphate buffered saline, BHB: beta-hydroxybutyrate, IMM: immobilization stress, Sac: sacrifice, s.c.: subcutaneous injection, FST: forced swim test, SPT: sucrose preference test, NSFT: novelty suppressed feeding test, EPM: elevated plus maze test.