STUDY THE EFFECT OF HEMOXYGENASE-1 INDUCTION AND SUPPRESSION ON LIPOPOLYSACCHARIDE- BRAIN INJURY IN MALE RATS

Ahmed Fouad Hussein Hashad, Mahmoud Abd Elhameed Elghareeb, Ghada Mahmoud Ismail and Nermin Mohammed Madi Department of Physiology, Faculty of Medicine, Tanta University, Tanta, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 18 March 2020 Final Accepted: 20 April 2020 Published: May 2020


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Cognitive tests: At the end of the experimental protocol, all animals were tested for cognitive tests (two way active avoidance test)in a shuttle box apparatus, using conditioned stimuli (in the form of light) on the background of unconditioned stimuli (electrical shock), through pre-training, acquisition phase ad retention test described by Bures et al., 1976 [18] .

Tissue sampling:
Then, the animals were sacrificed by decapitation. Brains of experimental groups were harvested, snap-frozen in liquid nitrogen and subsequently used for measurement of oxidative stress, inflammatory markers and neurotransmitters.
Pieces of the dissected brains(after perfusion with a PBS solution, PH 7.4) are homogenized in 5-10 ml of ice cold buffer per gram tissue, and then centrifuged(at 10000 x g for 15 minutes at 4°C). The supernatant was removed for assay and store on ice.
The sacrificed rats were packed in a special package according to safety precautions and infection control measures and sent with hospital biohazards.

Statistical analysis:
Collected data were statistically analyzed by GraphpadInstat software, version 3.10, using one-way ANOVA, followed by Tukey-Kramer'stest. Statistical significance was considered at p-value ≤ 0.05, for all statistical tests.

Results:-
Two way active avoidance test: A significant decrease in avoidance number in a 10 block trials and a significant increase the highest avoidance latencies in LPS-treated group. These results were deteriorated with addition of Hemin. Addition of NM revealed a significant increase in avoidance number in a 10 block trials and a significant decrease in the highest avoidance latenciescompared to the LPS-treated group (table 1). Brain HO-1, NO, BDNF and Glu: Brain HO-1 and NO were significantly increased while brain BDNF and Glu were significantly decreased in LPStreatedgroup.These results were deteriorated with addition of Hemin. Addition of NM revealed a significant decrease in brain HO-1 and NO levels, together with a significant increase in brain BDNF and Glu levels compared to the LPS-treated group (table 2). Brain oxidative stressmarkers: Fig. 1(A,B) showed a significant increase of MDA levels and decrease of CAT activityin the brain tissues following LPS treatment. These results were deteriorated with addition of Hemin. With NM addition a significant decrease in brain MDA and a significant increase in brain CAT activity were detected in LPS + NM group versus LPS-treated group.

Discussion:-
The results of the current study revealed that, LPS-treated male rats showed high brain concentrations of HO-1 and NO. These findings could be attributed to the LPS-induced OS.These results were significantly deteriorated after treatment with Hemin in addition to LPS. However, treatment with NM significantly decreased HO-1 and NO brain levelsin the LPS + NM treated group.
The mechanism of brain damage induced by LPS may be due to generation of free radicals, which may produce lipid peroxidation and release of ROS.
The increase in HO-1can be explained by increased OS due to LPS injection.LPS increased HO activity significantly, especially in substantianigra and hippocampus, which was associated with increased both NOS activity and expression, showing brain OS indicated by regional distribution of lipid peroxides, that may lead to permanent dysfunction, this is according toNúñez and Hidalgo, 2019 [25] .
The significant increase in NO in LPS induced brain injury and in hemin group, may be secondary to direct activation of release of NO from glial cells, and may be indirectly due to increased HO-1 activity which increases both NOS expression and activity.Wegiel et al., 2014 [28] , recorded that the stress inducible HO-1 generates CO, which is included within the regulation of the cellular NO signal pathways.
The significant decrease of NO by NM may be due to decreased NO production from glial cells, NM may be suggested to suppress the HO-1 expression in apoptosis caused byaluminum neurotoxicity in mice, possibly via 1003 maintaining Fe homeostasis   [29] . Also, NM has shown inhibition of NO production and prostaglandin E2 secretion from LPS-stimulated microglia and reduced degeneration of DA neurons (Espinosa-Parrilla et al., 2015) [30] .
The significant decrease in BDNF levels in LPS induced brain injury and in hemin group may be secondary to increased inflammatory cytokines especially IL-1β,This effect was significantly decreased after NM administration that inhibits the release of inflammatory cytokines and subsequently stimulating the production of BDNF. This is supported by Kranjac et al., 2012 [31] ,who has reported that administration of LPS suppresses of BDNF secretion from glial cells, and diminished neuroplasticity resulting in impaired learning and memory.
Also, BDNF levels were significantly decreased in the rat hippocampus and in several cortical regions after injection of LPS. Also,neurotrophins as nerve growth factor and neurotrophin-3 levels were decreased even with different peaks (Boschen and Klintsova1, 2017) [32] . Tong et al., 2018 [33] , reported that NM may inhibit apoptosis of hippocampal neurons after brain radiotherapy and upregulate BDNF expression which is associated with the survival of neurons enhancing cognitive function.
Thepresent study shows significant decrease in Glu in LPS induced brain injury and in hemin group, may result from exaggerated release of inflammatory mediators and neuronal loss, which can be modulated by NO.This effect which was significantly abolished after NM administration which can decrease the release of both inflammatory cytokines and NO.
Choi et al., 2011 [38] , stated that NM can decrease neurotoxic Glu release, as it is a L-type Ca +2 channel blocker at both presynaptic and postsynaptic neuronal membranes, and may be also an intracellular Ca +2 antagonist, with a probable protective effect on surgical ischemic conditions of brain from Glu-induced neuronal damage.
Thepresent study shows significant increase in pro-inflammatory MDA brain tissue levels and the significant decrease in redox CAT activity, in LPS induced brain injury and in hemin group, which were reversed by NM. can be explained by increased ROS leading to exhaustion of antioxidant enzymes. Also, NM can effectively increase antioxidant capacity and inhibit OS expression.
Kaminska et al., 2016 [39] , reported that LPS administration resulted in the increased mRNA expression of proinflammatory cytokines as IL-1β, IL-6 and TNF-α due to increased binding of nuclear factor-κB to these cytokines gene promoters in microglia.Also, Sharma and Nehru, 2015 [40] ., reported significant increase in MDA levels in the brain (compared to control animals) after single systemic injection of LPS.
Wang et al., 2017 [41] , showed MDA elevation and reduction of CAT levels in early brain injury after subarachnoid hemorrhage in rats, in addition to increased tissue levels of TNF-α and IL-6. These increased cytokines and decreased antioxidant enzymes are due to activation of several signal cascades, such as NF-κB and nuclear factor erythroid 2-related factor/ heme oxygenase-1 (2Nrf2 /HO-1).This is supported byKajimura et al., 2010 [42] , There is a strong relationship between the activity of NOS, SOD, and other redox-active enzymes, and HOs reactions. Also, NO has other heme protein targets as CAT, cytochrome-c, hemoglobin and peroxidase.
NM can inhibit Ca +2 influx and reduce the apoptosis of cells. NM combined with edaravone (a potent free radical scavenger and antioxidant) can effectively increase antioxidant capacity and inhibit OS expression within brain cells. Thus, MDA levels were significantly lower in treated venous blood samples (Xie et al., 2016) [43] .

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In the present study, there were observed memory and behavioral impairments as evidenced via active avoidance test in LPS and hemin groups. This may be explained by the long term inflammatory conditions and accumulation of cytokines. This neurotoxicity leads to learning deficits.
Sharma et al., 2017 [44] , observed memory and behavioral impairments via active avoidance test in LPS treated rats, which escaped maximum number of trails in active avoidance test after prenatal LPS exposure as compared to the control rats.Also, many individuals have been observed for cognitive impairment in long term inflammatory conditions or treatment with cytokine based therapies.
According to Koh and Liang, 2017 [45] , neurotoxicity leading to learning deficits (decreased step through latency time and increased escape latency time in behavioral tests), was shown to be under several mechanisms including increased ROS formation, through enzymes involving as Nrf2, HO-1, and NADPH oxidase-4 enzyme (NOX4). Inhibited ROS formation is related to down-regulated NOX4, Nrf2, and HO-1.
In the present study, NM improves the two ways avoidance test. This is evidenced by usage of NM as antidepressant and can be explained by its action as Ca +2 channel blocker.
Koskimäki, 2015 [46] , reported that the NM used as antidepressant, as in its subacute treatment in helpless rodents which failed to avoid the resistant stress (e.g. mild electrical shock), which is a freezing behavior, leads to improvement of the learning behavior of these rodents. Also, Dominguez, 2011 [47] ., reported that the Ca +2 hypothesis of brain aging and AD can be proved by the role of Ca +2 antagonists in treatment of age related cognitive disorders and also symptoms of depressive mood.

Conclusion:-
Chronic HO-1 has a strong effect in a LPS brain injury rat model, through chronic inflammation, oxidative stress and modulation in NO signals, which can be exaggerated by Hemin and attenuated by Nimodipine, providing a new therapeutic intervention for neurodegenerative diseases, as assessed by biochemical findings.