Activation of alpha-4 beta-2 nicotinic receptor against cardiopulmonary bypass surgery induced brain injury by regulating NLRP3 pathway

Present report evaluates the role of alpha-4 beta-2 nicotinic receptor (α4β2 nAChRs) in the development of cardiopulmonary bypass (CPB) surgery induced brain injury. Brain injury was induced by CPB and animals were treated with α4β2 nAChRs agonist (DHβE 9 mg/kg, s.c.) and α4β2 nAChRs antagonist (MLA 10 mg/kg, i.p.) 3 hr before the induction of CPB in the separate groups. Effect of α4β2 nAChRs agonist was determined on the neurological function in CPB induced brain injured rats. Level of cytokines, ROS and expression of NLRP3, ZO-1 and Occluding proteins were estimated in CPB induced brain injured rats. Effect of α4β2 nAChRs agonist was determined on the neuronal apoptosis and histopathological changes in the brain tissue. Result of the study suggest that neurological score was reversed in α4β2 nAChRs agonist treated group than CPB group. Level of cytokines and ROS was reduced in α4β2 nAChRs agonist treated group than CPB group. Neuronal apoptosis in α4β2 nAChRs agonist treated group was found to be reduced compared to CPB group of rats. Moreover Activation of α4β2 nAChRs ameliorates the altered expression of NLRP3, ZO-1 and Occluding protein in the brain tissue of CPB induced brain injured rats. In conclusion, Data of report suggest that treatment with α4β2 nAChRs agonist protects the brain injury in cardiopulmonary bypass surgery induced brain injury by regulating NLRP3 pathway.


Introduction
Open heart surgery is performed by using cardiopulmonary bypass (CPB) technology, which has reduces the rate mortality occurs due to postoperative fatal arrhythmia, heart failure, myocardial infarction and other cardiac issues (Hausenloy et al, 2012).However patients with cardiac surgery have been appearing to be cognitive dysfunction which causes mental and neuropsychiatric disorders.Literature suggests that CPB induced neuropsychiatric and neurological disorders leads to enhance the mortality rate up to 19.6% (Salameh et al, 2016).Management of several heart disorders by surgery using CPB induces hypoxia and lower perfusion in the brain tissues, which causes occurrence of oxygen debt (Lee et al, 2008).These alterations in the oxygen supply causes activation of in ammatory cascade and thereby enhances the in ammatory response.Systemic in ammatory response syndrome occurs due to cerebral embolism in CPB (Sarkar and Prabhu, 2017).There is a need of development of novel therapy for the management of it.
Nicotinic acetylcholine receptors (nAChRs) is activated by exerting nicotine and homomeric α7 and heteromeric α4β2containing receptors were the identi ed subunit of nAChR (Albuquerque et al, 2009).nAChRs is widely spread and abundantly present in the brain tissue and activation of it regulates the production of cytokines.Literature reveals that nAChR ameliorates immune response and in ammation by regulating the activity of NF-B and JAK/STAT pathway (Ibrahim et al, 2018) and also reported to posses' neuroprotective effect due to its anti in ammatory property (Hone and McIntosh, 2018).α4β2 nAChRs agonist reported to be bene cial effect in cessation of smoking (Singh and Budhiraja, 2008).Moreover α4β2 nAChRs also reduces the NF-B activity which is induced by LPS by regulating JAK/STAT3 pathway (Zdanowski et al, 2015).Thus present report determines the role of α4β2 nAChRs in cardiopulmonary bypass surgery induced brain injury.

Animal
Male Sprague-Dawley rats weighing 250-300 g were kept under a 12-h light/dark cycle at 60 ± 5% humidity and 24 ± 3 °C.Protocols used in the animals were approved by Institutional Animal Ethical Committee of First Hospital of Hebei Medical University, China (IAEC/FH-HMU/2018/05).

Experimental
Cardiopulmonary bypass surgery induced brain injury was performed as per earlier reported study (Grogan et al, 2008).22G and 24G trocar was inserted in the left femoral artery and right femoral vein respectively after anesthetizing all the animals with iso urane.Further extracorporeal venous out ow ends by inserting 18G puncture needle through right internal jugular vein to the right atrium.CPB model was established by connecting the connecting pipe, lter, arterial infusion tube, blood reservoir and drainage tube to 1.6 mm internal diameter PVC tube.Mechanical ventilation was terminated at the beginning and ow rate was maintained by inserting the circulating solution (lactate Ringer's solution, hydroxyethyl starch, mannitol 20%, sodium bicarbonate 5% and heparin) at 80 ml/kg/min and lung rate was maintained at 800 ml/min by passing oxygenair mixture (1:4 ratio) through the membrane lung.
Diameter of venous ow was reduced to decrease the ow rate and later restarted the mechanical ventilation.Later incision was sutured after the removal of catheter and once the berating was resumed animals were fed in laboratory for observation.
Forty rats were separated into four different groups each group carries ten animals such as control group; CPB group; α4β2 nAChRs agonist group which receives DHβE 9 mg/kg, s.c. and α4β2 nAChRs antagonist treated group which receives MLA 10 mg/kg, i.p. 3 hr before the induction of CPB.

Neurological dysfunction
Neurological dysfunction was assessed by determining neurological scores in I/R-induced neuronal injury rats using Longa's method, as follows: 0, no de cit; 1, forelimb failed to extend entirely and weak; 2, circling to the contralateral side; 3, weight-bearing capacity reduced on the injured side; and 4, no spontaneous locomotor activity.

Estimation of cytokines
The levels of the in ammatory mediators interleukin (IL)-1β, IL-6, nuclear factor kappa B (NF-κB) and tumour necrosis factor (TNF)-α were determined in the brain tissue homogenates by ELISA using commercial kits in accordance with the manufacturer's protocols (R&D Systems).

Estimation of ROS
MitoSOX red mitochondrial superoxide indicator was used to estimate the levels of reactive oxygen species (ROS) in the brain tissues.Brie y, tissue homogenates were stained at 37 °C in the dark for 30 minutes with 5 µM MitoSOX red.A uorescent plate reader was used to estimate the intracellular ROS levels at excitation and emission wavelengths of 510 and 580 nm, respectively.

Western blotting
The protein expression levels of Bax, Bcl-2, caspase-3, pDrp1, Drp1, NLRP3, ZO-1 and occludin were assessed by Western blotting analysis of brain tissue homogenates.A BCA assay kit was used to quantify the protein from each tissue homogenate, and 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to separate the proteins, which were electroblotted onto nitrocellulose membranes.Subsequently, each membrane was blocked with 5% blocking solution (non-fat milk) and incubated in blocking buffer with primary antibodies overnight at 4 °C.Goat secondary antibodies conjugated with horseradish peroxidase were added to the blocking buffer, and a chemiluminescence kit was used to detect the proteins.

TUNEL assay
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay was used to determine apoptosis of myocardial cells.The apoptosis index was calculated as follows: no. of stained myocytes/total no. of myocytes × 100%.

Histopathology study
Hippocampus was isolated from each animal after the 6 hr of CPB and tissue was xed in formalin solution.Ethanol was used to dehydrate the brain tissue and seed it into liquid para n.Microtome was used to section the hippocampus tissue and stained it with H&E staining.

α4β2 nAChRs activation ameliorates the neurological functions
Neurological function was estimated in α4β2 nAChRs agonist and antagonist treated CPB induced brain injured rats by determining the neurological score.Neurological function score was enhanced up to 3.7 score in CPB group than control group (0.1 score).There was reduction in the neurological score to 1.06 in α4β2 nAChRs agonist treated group than CPB group.However α4β2 nAChRs antagonist treated group shows the enhancement of neurological score compared to α4β2 nAChRs agonist and control group of rats (Figure 1).

α4β2 nAChRs activation reduces cytokines
Effect of α4β2 nAChRs activation was observed on the level of cytokines in the serum of CPB induced brain injured rats as shown in Figure 2.There was increase in the level of cytokines such as IL-1β, IL-8, TNF-α and NF-kB in the serum of CPB group than control group of rats.Level of cytokines was found to be reduced in the serum of α4β2 nAChRs agonist treated group and enhances in the serum of α4β2 nAChRs antagonist treated group compared to CPB group.α4β2 nAChRs activation reduces the level of ROS Level of ROS was estimated in the brain tissue of α4β2 nAChRs agonist and antagonist treated CPB induced brain injured rats.There was increase in the level of mitochondrial ROS in the brain tissue of CPB group than control group of rats.Treatment with α4β2 nAChRs agonist reduces the level of ROS in the brain tissue of CPB induced brain injured rats.However level of ROS was enhanced in the brain tissue of α4β2 nAChRs antagonist treated group compared to control and α4β2 nAChRs agonist treated group of rats (Figure 3).

α4β2 nAChRs activation reduces the neuronal apoptosis
Western blot assay was performed to determine the effect of α4β2 nAChRs activation on the expression of proteins responsible for apoptosis in the neuronal tissue of CPB induced brain injured rats and apoptosis index by TUNEL assay (Figure 4 A&B).There was increase in the expression of caspase-3 and Bax protein and decrease in the expression of Bcl-2 protein in the brain tissue of CPB group than control group of rats.Treatment with α4β2 nAChRs agonist ameliorates the altered expression of caspase-3, Bax and Bcl-2 protein in the neuronal tissue of CPB induced brain injured rats (Figure 4A).Moreover apoptosis in the neuronal tissue was enhanced in CPB group than control group of rats.There was reduction in the apoptosis of neuronal tissue in α4β2 nAChRs agonist treated group compared to CPB group (Figure 4B).α4β2 nAChRs activation ameliorates the NLRP3 pathway Expression of NLRP3, ZO-1 and Occluding protein was determined in the brain tissue of α4β2 nAChRs agonist and antagonist treated CPB induced brain injured rats.Expression of ZO-1 and occluding was reduced signi cantly and enhances the expression of NLRP3 in the brain tissue of CPB group than control group of rats.There was increase in the expression of ZO-1 and occluding protein and decrease in the expression of NLRP3 in the α4β2 nAChRs agonist treated group than CPB group.However treatment with α4β2 nAChRs antagonist further enhances the expression of NLRP3 and reduces the expression of ZO-1 and occluding proteins in CPB induced brain injured rats (Figure 5).

α4β2 nAChRs activation ameliorates the pathological changes
Effect of α4β2 nAChRs activation was observed on the histopathological changes in the hippocampal tissue of CPB induced brain injured rats as shown in Figure 6.Hippocampus tissue was observed to be normal structured with clear boundary and regular.In CPB group hippocampal neurons were found to be damaged with change in regular structure and cell band.However treatment with α4β2 nAChRs agonist reverses the histopathological changes occurs due to CPB induced brain injury (Figure 6).

Discussion
Cardiopulmonary bypass (CPB) technology is used for the treatment of several types of cardiovascular disorder by surgery and reported to reduces the mortality occurs due to cardiovascular issues (Ranganath et al, 2019).However CPB reported to induces the injury to different organs of the body including brain (Kim et al, 2016).There is no speci c treatment is available for the management of it, moreover exact pathogenesis remain to establish.Thus present report evaluates the role of α4β2 nAChRs in the development of CPB induced brain injury.Effect of α4β2 nAChRs agonist was determine on the neurological function in CPB induced brain injured rats.Level of cytokines, ROS and expression of NLRP3, ZO-1 and Occluding proteins were estimated in CPB induced brain injured rats.Effect of α4β2 nAChRs agonist was determined on the neuronal apoptosis and histopathological changes in the brain tissue.
Postoperative cognitive dysfunction was found to be higher in the geriatric patients, in which loss of social integration, language comprehension, concentration and memory (Berger et al, 2015).All these leads to loss of neurological function (Rundshagen, 2014) and data of the report also suggest it, as CPB group shows enhancement in the neurological score compared to control group of rats.However activation of α4β2 nAChRs with α4β2 nAChRs agonist reverses the neurological score to normal.Literature suggest that Ach release enhances in several conditions such as embolism, injury or infection by the activation of vagus nerve and activation of nAChRs reported to inhibit the release of cytokines and also enhances the formation of anti in ammatory factor (de Jonge and Ulloa, 2007).All these lead to produces the anti in ammatory property and data of presented report suggest that activation of α4β2 nAChRs reduces the level of cytokines in the serum of CPB induced brain injured rats.
Literature suggest that NLRP3 has a proven role in the neuronal dysfunction by enhancing the apoptosis through the activation of caspase pathway and NLRP3 in ammasome pathway reported to be involved in CPB surgery induced brain injury (Eren and Özören, 2019).ROS also contributes in the development of neuronal injury by regulating NLRP3 pathway (Abais et al, 2015).Result of the study suggests that level of ROS was reduced in the brain tissue of α4β2 nAChRs agonist treated group compaired to CPB group rats.Further treatment with α4β2 nAChRs agonist ameliorates the altered level of NLRP3 pathway in the brain tissue of CPB induced brain injured rats.In conclusion, Data of report suggest that treatment with α4β2 nAChRs agonist protects the brain injury in cardiopulmonary bypass surgery induced brain injury by regulating NLRP3 pathway.
Effect of α4β2 nAChRs activation on the neurological function in CPB induced brain injured rats mean±SEM (n=10); **p<0.01compared to control group; ##p<0.01compared to CPB group; @@p<0.01compared to α4β2 nAChRs agonist Figure 2 Effect of α4β2 nAChRs activation on the cytokine level in the serum of CPB induced brain injured rats mean±SEM (n=10); **p<0.01compared to control group; ##p<0.01compared to CPB group; @@p<0.01compared to α4β2 nAChRs agonist Figure 3 Effect of α4β2 nAChRs activation on the level of ROS in the brain tissue of CPB induced brain injured rats mean±SEM (n=10); **p<0.01compared to control group; ##p<0.01compared to CPB group; @@p<0.01compared to α4β2 nAChRs agonist Figure 4 Effect of α4β2 nAChRs activation on the neuronal apoptosis in the brain tissue of CPB induced brain injured rats.A: Expression of Caspase-3, Bax and Bcl-2 protein in the brain tissue of CPB induced brain injured rats by western blot assay; B: Apoptosis index by TUNEL assay mean±SEM (n=10); **p<0.01compared to control group; ##p<0.01compared to CPB group; @@p<0.01compared to α4β2 nAChRs agonist Figure 5 Effect of α4β2 nAChRs activation on the expression of NLRP3, ZO-1 and Occluding protein in the brain tissue of CPB induced brain injured rats mean±SEM (n=10); **p<0.01compared to control group; ##p<0.01compared to CPB group; @@p<0.01compared to α4β2 nAChRs agonist Figure 6 Effect of α4β2 nAChRs activation on the histopathological changes in the brain tissue of CPB induced brain injured rats