Progesterone attenuate autism-like-phenotype through modulation of cerebral inflammation and oxidative stress

INTRODUCTION

Autism spectrum disorder (ASD) is a neurodevelopment disorder that characterises impairment in two major behaviours: Repetitive behaviour and deficit in social communication as well as interaction.^[1] Other comorbid conditions observed in an autistic individual include anxiety, aggressiveness, sleeplessness, gastrointestinal disturbances and motor impairments.^[2] Studies suggest that men are more susceptible to the ASD condition than women.

Propionic acid (PPA) is a naturally occurring saturated fatty acid that is produced through anaerobic fermentation of dietary substrates by gut microbiota. PPA modulates cellular processes and metabolism and exhibits immunosuppressive properties.^[3] PPA can permeate the blood-brain barrier and cause developmental delays as well as disruption in neurotransmitter release.^[3,4] PPA has been implicated in inflammation, oxidative stress, immune dysfunction and cognitive impairment, which is also observed in ASD.^[5-11]

PPA exposure induces behavioural abnormalities akin to ASD. Administration of PPA induces stereotypy and causes impairment in attention, social behaviour, locomotion and information processing abilities in rodents.^[5,6,12,13] Postnatal PPA exposure causes a decline in the levels of cerebral phosphorylated – cAMP response element binding protein and brain-derived neurotrophic factor (BDNF). Furthermore, PPA elevates the levels of oxidative (thiobarbituric acid reactive substance [TBARS]) and inflammation stress markers (interleukin 6 [IL-6] and tumour necrosis factor-alpha [TNF-a]) in rodents.^[14,15] Consequently, postnatal exposure to PPA serves as a reliable model for replicating behavioural and biochemical phenotypes that are associated with and observed in ASD.^{[5,6,11,13,16]}

Progesterone is an endogenous steroid hormone that is essential for pregnancy, embryogenesis and the menstrual cycle of humans and other species. The potential role of progesterone has been implicated in several neurological such as traumatic brain injury, hypoxia/ischemic brain injury, Parkinson’s disease and subarachnoid haemorrhage.^[17-20] Progesterone corrects behavioural abnormalities that are observed in ASD in various animal experimental models for diseases. This includes social behaviour, stereotypy, anxiety, depression and hyperactivity.^[21-25] In addition to this, progesterone promotes neuroprotection, neurotransmission, myelination and neurogenesis through inhibition of neuroinflammation, oxidative stress and neurodegeneration.^[26,27] However, the role of progesterone is yet to be elucidated in the experimental model of ASD. We have hypothesised that progesterone might play a key role in ameliorating behavioural as well as biochemical alterations that are observed in ASD. In this study, we have investigated the effects of progesterone against impaired behavioural (stereotypy, hyperlocomotion and exploration) and biochemical (cerebral inflammation and oxidative stress) parameters that are associated with ASD.

MATERIALS AND METHODS

RESULTS

Effect of progesterone on locomotion in postnatal PPA exposed rats

PPA-administered rats, compared to control groups, showed an increase in the number of square crossings that indicate hyper-locomotion. However, the administration of progesterone (4 mg/kg; 8 mg/kg; sc) to PPA-administered rats showed a significant reduction in the number of square crossings in open field apparatus in comparison to the PPA-administered group [Figure 1a].

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Figure 1:

Effect of progesterone administration on locomotion and repetitive behaviour. Results are expressed as mean ± S.D (n = 8; male); two-way ANOVA followed by Bonferroni’s post hoc test. (a) Number of square crossings: (F [1,42] = 1511.663), a: P < 0.001 versus control group; (F [2,42] = 152.296), b: P < 0.05 versus PPA group. (b) Grooming: (F [1, 42] = 3493.017), a: P < 0.001 versus control group; (F [2, 42] = 796.243), b: P < 0.05 versus PPA group. PBS: Phosphate buffer saline, P8: Progesterone (8 mg/kg), PPA: Propionic acid, P4: Progesterone (4 mg/kg), ANOVA: Analysis of variance, S.D: Standard deviation.

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Effect of progesterone on exploratory activity in postnatal PPA exposed rats

PPA rats showed an increase in the exploratory behaviour as observed through an increase in the latency of the first poke as well as a decrease in the number of hole poking and rearing in comparison to control rats. However, treatment with progesterone (4 mg/kg; 8 mg/kg; sc) significantly attenuated the PPA-induced increase in the exploratory activity in the PPA-administered rats [Figure 2a-c].

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Figure 2:

Effect of progesterone administration on exploratory activity. Results are expressed as mean ± S.D (n = 8; male); two-way ANOVA followed by Bonferroni’s post hoc test. (a) Latency to first poke: (F [1, 42] = 2898.639), a: P < 0.001 versus control group; (F [2, 42] = 272.418) and b: P < 0.05 versus PPA group. (b) Hole poking: (F [1, 42] = 1448.832), a: P < 0.001 versus control group; (F [2, 42] = 74.818), b: P < 0.05 versus PPA group. (c) Rearing: (F [1, 42] = 2108.065), a: P < 0.001 versus control group; (F [2, 42] = 86.364), b: P < 0.05 versus PPA group. PBS: Phosphate buffer saline, P8: Progesterone (8 mg/kg), PPA: Propionic acid, P4: Progesterone (4 mg/kg), ANOVA: Analysis of variance, S.D: Standard deviation.

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Effect of progesterone on brain oxidative stress in postnatal PPA-exposed rats

PPA-administered rats showed a significant increase in oxidative stress (increased TBARS and decreased GSH) in different brain regions compared to control animals; however, treatment with progesterone (4 mg/kg; 8 mg/kg; sc) significantly attenuated the PPA-induced increase in the brain TBARS levels and decrease in the brains’ GSH levels in the postnatal PPA-exposed rats [Figures 3a and b].

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Figure 3:

Effect of progesterone administration on cerebral oxidative stress and inflammatory markers Results are expressed as mean ± S.D (n = 8; male); two-way ANOVA followed by Bonferroni’s post hoc test. (3a) TBARS: Frontal cortex (F (1, 42) = 1130.157), a: p<0.001 versus control group; (F (2, 42) = 36.501), b: p<0.05 versus PPA group. Cerebellum (F (1, 42) = 443.856), c: p<0.001 versus control group; (F (2, 42) = 30.389), d: p<0.05 versus PPA group. Hippocampus (F (1, 42) = 513.7), e: p<0.001 versus control group; (F (2, 42) = 59.402), f: p<0.05 versus PPA group. (3b) GSH: Frontal cortex (F (1, 42) = 1606.387), a: p<0.001 versus control group; (F (2, 42) = 115.005), b: p<0.05 versus PPA group. Cerebellum (F (1, 42) = 919.987), c: p<0.001 versus control group; (F (2, 42) = 177.589), d: p<0.05 versus PPA group. Hippocampus (F (1, 42) = 1127.581), e: p<0.001 versus control group; (F (2, 42) = 129.703), f: p<0.05 versus PPA group. (3c) TNF-α: Frontal cortex (F (1, 42) = 946.783), a: p<0.001 versus control group; (F (2, 42) = 642.398), b: p<0.05 versus PPA group. Cerebellum (F (1, 42) = 5502.655), c p<0.001 versus control group; (F (2, 42) = 386.948), d: p<0.05 versus PPA group. Hippocampus (F (1, 42) = 6046.561), e: p<0.001 versus control group; (F (2, 42) = 126.985), f: p<0.05 versus PPA group. (3d) IL-6: Frontal cortex (F (1, 42) = 3724.988), a: p<0.001 versus control group; (F (2, 42) = 727.146), b: p<0.05 versus PPA group. Cerebellum (F (1, 42) = 15333.05), c: p<0.001 versus control group; (F (2, 42) = 5393.849), d: p<0.05 versus PPA group. Hippocampus (F (1, 42) = 6811.425), e: p<0.001 versus control group; (F (2, 42) = 1166.197), f: p<0.05 versus PPA group. PBS: Phosphate buffer saline, P8: Progesterone (8 mg/kg), PPA: Propionic acid, P4: Progesterone (4 mg/kg), ANOVA: Analysis of variance, S.D: Standard deviation, TNF-a: Tumour necrosis factor-alpha, IL-6: Interleukin, GSH: Glutathione, TBARS: Thiobarbituric acid reactive substance. [Note: For the frontal cortex, “a” is for the statistical comparison of the PPA group with the control group; “b” is for the statistical comparison of treatment groups with the PPA group for all the parameters, i.e., TBARS, GSH, TNF-α, and IL-6. For the cerebellum region, “c” is for the statistical comparison of the PPA group with the control group; “d” is for the statistical comparison of treatment groups with the PPA group for all the parameters, i.e., TBARS, GSH, TNF-α, and IL-6. For the Hippocampus region, “e” is for the statistical comparison of the PPA group with the control group; “f ” is for the statistical comparison of treatment groups with the PPA group for all the parameters, i.e., TBARS, GSH, TNF-α, and IL-6.]

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DISCUSSION

Autistic animals have shown hyperlocomotion during the experimental studies.^[3] In the present study, rats administered with PPA have shown an increase in the number of square crossings during an open field test, signifying an increase in locomotor activity as compared to the normal control animals. Evidence suggests hyperlocomotion in rodents with elevated levels of cerebral TNF-a in comparison to rodents with normal cerebral TNF-a levels.^[34] Furthermore, hyperlocomotion was also recorded in GSH-knockout mice during the experiment of the open field apparatus.^[35] In the present study, we have observed a significant elevation and reduction in the TNF-a and GSH levels, respectively, in the studied brain regions of the PPA-administered rats. Therefore, the observed hyper-locomotory effect in these PPA rats might have been due to an increase and decrease in brain TNF-a and GSH levels, respectively.

PPA-administered rats in the present study have shown an increase and decrease in repetitive and exploratory behaviour, respectively. Clinical observation suggests the association of restricted and repetitive behaviour with increased cerebral IL-6 levels in autistic children.^[36] Furthermore, oxidative stress in the frontal cortex and hippocampal region regions of the brain was observed to impact exploratory behaviour in the experimental animals.^[37] Consequently, a rise in the cerebral inflammatory status and oxidative stress markers in PPA-administered rats in the present study might have been responsible for an increase and decrease in the repetitive and exploratory behaviour, respectively. In addition, cerebral inflammation and oxidative stress are reported to cause behavioural impairment in the PPA-induced ASD animal model.^{[6-8,31-33,38]} PPA induces the excessive formation of reactive oxygen species in both the frontal cortex and hippocampus. Oxidative stress activates the nuclear factor kappa-light-chain-enhancer of activated B-cells, which further increases the secretion of inflammatory markers, forming a vicious circle of inflammatory markers secretion and free radicals formation.^[39,40] Furthermore, a decrease in the GSH levels was recorded in the postmortem brain of individuals suffering from autism, suggesting the crucial role of GSH depletion in autistic phenotype.^[41]

Progesterone potentiates BDNF mRNA and protein levels in the hippocampus and cerebral cortex regions of the brain^[42], which further initiates the activation of prosurvival cell signalling pathways.^[43] In ADHD mice, an increase in the expression of BDNF is reported to protect against hyperactivity and hyper-locomotion.^[44] Furthermore, administration of progesterone inhibits the release of proinflammatory cytokines such as IL-6, TNF-a and interferon-g that are known to cause hyperlocomotion in ketamine-induced cognitive dysfunction and hyperlocomotion in rats.^[33,45] Furthermore, another study has demonstrated that progesterone plays a beneficial role in synapse formation in the hippocampal region of the brain.^[46] These results are akin to the results obtained in the present study, where subcutaneous administration of progesterone (4 mg/kg and 8 mg/kg) in postnatal PPA-exposed experimental animals increases the expressions of antioxidant marker GSH as well as decreases the expression of brain inflammatory markers such as IL-6, TNF-a in the cerebellum, hippocampus and frontal cortex regions of the brain. Thus, in this study, the progesterone-mediated decrease in hyperlocomotion could have been due to an increase in the GSH and a decrease in the TBARS expression in the brain.

The present study has observed the protective effects of progesterone administration (4 mg/kg and 8 mg/kg) against increased repetitive behaviour as well as impaired exploratory activity in the PPA-treated rats. These results are in accordance with the previously published reports where the cocaine sensitisation rat model has demonstrated the beneficial effects of progesterone administration against stereotypic behaviour in animals.^[22] Furthermore, oxidative stress in the frontal cortex and hippocampal regions of the brain is observed to impair exploratory activities in rodents during pre-clinical studies.^[37] Progesterone (4 mg/kg and 8 mg/kg) administration to the PPA-treated rats in this study caused a marked decrease in the levels of oxidative stress markers (TBARS) in the frontal cortex as well as hippocampal regions of the brain. Therefore, progesterone-mediated beneficial effects against PPA-induced decrease in the exploratory behaviour as well as an increase in the repetitive behaviour might have been due to its ameliorating effects against elevated inflammation and oxidative stress markers in animals. Furthermore, the present study utilises sesame oil as a vehicle for progesterone administration. Sesame oil is a delivery vehicle for all fat-soluble compounds, such as steroidal hormones and toxins. The administration of sesame oil to male offspring from PND 24 to PND 50 has shown no significant effects against any of the parameters assessed, which clearly suggests that the beneficial effects observed in the present study were observed through the administration of progesterone and not its vehicle.

CONCLUSION

Therefore, we can conclude that the administration of progesterone (4 mg/kg and 8 mg/kg) has significantly enhanced the exploratory behaviour as well as reduced the hyperlocomotion and stereotypy behaviour in postnatal PPA-treated rats in the present study. This beneficial effect of progesterone was observed through the activation of anti-inflammatory as well as anti-oxidative stress markers and the inhibition of inflammatory as well as oxidative stress markers in the frontal cortex, hippocampus and cerebellum regions of the brain.