Recombinant adiponectin peptide ameliorates cortical neuron damage induced by chronic 1 cerebral hypoperfusion by inhibiting NF-κB signaling and regulating microglial polarization

Background 27 Chronic cerebral hypoperfusion (CCH) is common in multiple central nervous system diseases that 28 are associated with neuronal death and cognitive impairment. Microglial activation-mediated 29 polarization changes may be involved in CCH-induced neuronal damage. Adiponectin (APN) is a 30 fat-derived plasma protein that affects neuroprotection. This study investigated whether a 31 recombinant APN peptide (APN-P) improved the cognitive function of CCH rats by regulating 32 microglial polarization in the cortex. 45 In the cortical microglia of 12-week-old CCH rats, the expression of APN protein was significantly 46 downregulated compared to the sham rats. CCH damages neurons and activates cortical microglial 47 polarization to an M1-type by upregulating inflammatory factors. APN-P supplementation 48 upregulated APN expression in cortical microglia, with neuronal survival as well as microglial 49 polarization from an M1 toward an M2 phenotype in CCH cortex. In vivo and in vitro experiments 50 revealed that APN-P promoted the expression of anti-inflammatory factors and neuronal survival 51 by inhibiting NF-κB signaling, thus improving the cognitive function in CCH rats. 52 53 Our study revealed a novel mechanism by which APN-P suppresses the NF-κB pathway and 54 promotes microglial polarization from M1 toward the M2-type to reduce neuron damage in the 55 cortex after CCH. M1-type APN-P treatment might be a novel therapeutic strategy for CCH, evidenced by the increased neuronal survival and improved neurological function observed in CCH mice treated with APN-P when compared to the CCH control group. APN-P treatment alleviated neuroinflammation and promoted microglia M2-type polarization CCH and LPS stimulation. positive


Nissl staining 222
Nissl staining was used to observe morphological changes in neurons within the cortex of CCH rats. 223 Rats were transcardially perfused (chest ribs were dissected to expose heart tissue for perfusion) 224 with physiological saline solution followed by 4% paraformaldehyde [25] and following the 225 manufacturer's instruction of the Nissl staining kit (E607316, Sangon Biotech, China). Neurons in 226 three different fields were counted. Intact neurons were shown as a rich cytoplasm, one or two large 227 round nuclei with large cell bodies, but damaged neurons were shown as dark cytoplasm, condensed 228 nuclei, empty vesicles or shrunken cell bodies. 229 230

qRT-PCR 264
Cortex tissue of rats and BV2 cells were collected from each treatment groups. Total RNA was 265 isolated using an RNA Extraction Kit (Thermo Fisher Scientific, USA) and quantified. Then, reverse 266 transcription using a reverse transcriptase kit (TaKaRa, Japan). Real-time PCR was performed 267 according to the manufacturer's manual for SYBR green (TaKaRa, Japan). The reaction was 268 performed at 95°C for 3 min followed by 40 cycles of 95°C for 10 s and 55°C for 30 s on the Bio-269 Rad PCR Detection System (Bio-Rad, USA). The primers (

APN-P improves learning and memory function in CCH rats 440
Using the Morris water maze test, we showed that APN-P treatment improved learning, which was 441 compared to the CCH group (Fig. b-c). Representative images of frequency of platform crossing in 447 different groups are displayed in Fig. 7d. These results indicated that APN-P treatment could 448 improve cognitive function in CCH rats.  Because the full-length APN has restricted blood-brain barrier permeability that reduces its potential 481 for future clinical applications, a recombinant APN-P was synthetized [16,23] and used in the 482 present study. We found that APN-P significantly improved the cognitive function, attenuated 483 microglial activation, and mediated neuroinflammation and neuronal survival in the rat 484 cortex/hippocampus. These findings highlight the promising neuroprotective effects of APN-P 485 treatment on CCH in human patients. 486 Previous study has suggested that neuroinflammation plays a crucial role in the cognitive 487 dysfunction induced by CCH [29]. Microglia are resident immune cells of the central nervous 488 system that maintain brain homeostasis. A previous study reported that neuroinflammation is mainly 489 manifested by microglial activation and the subsequent release of inflammatory factors, which lead 490 to brain damage ultimately [30]. In this study, we also found that microglia activation accompanied 491 CCH in the cortex. Neuron death is a key hallmark of CCH-related cognitive impairment [7], and it 492 is well established that inflammatory responses induced by CCH, including the activation of glial 493 cells and inflammatory cytokine production, could further result in neuron death and cognitive 494 deficits [30]. In addition, we found that microglia activation was accompanied by neuronal damage 495 in the cortex of CCH rats in this study. Therefore, inhibiting microglial activation to the pro-inflammatory phenotype could alleviate neuronal injury [29].
Activated microglia represent a variety of phenotypes after injury, including the broadly 498 classified M1 and M2 phenotypes that are useful for understanding the function and effect of 499 microglia in diversiform brain diseases [12]. The M2-to-M1 phenotypic changes during chronic 500 inflammation may be regulated by a common pathologic mechanism that underlies multiple types 501 of injuries in the central nervous system, including traumatic brain injury, white matter lesions, 502 spinal cord injuries, and strokes [31]. These dynamic phenotypic changes observed in brain diseases 503 suggest that manipulating microglial polarization might be a promising therapeutic strategy with 504 neuroprotective effects. However, research on the underlying mechanism and related potential 505 interventions of microglial polarization during CCH are currently limited. We used three methods 506 to evaluate how CCH affects the microglial polarization process. First, we detected increased iNOS 507 and decreased Arg-1 protein levels in the cortexes of CCH rats compared to sham controls. Second, 508 immunofluorescence was used to evaluate Iba-1, CD16, and CD206 expression as biomarkers for 509 microglial polarization. We found that the ratio of CD16/CD206 in Iba-1+ cells in the cortex of 510 CCH rats gradually increased between 4 and 12 weeks after BCCAO surgery. Third, microglial 511 polarization was further shown to significantly increase the mRNA levels of iNOS, IL-6, IL-18, IL-512 1β, and TNF-α in CCH groups compared to those in the sham control. These results demonstrated 513 that during CCH, the microglia switched towards an M1 phenotype, which is associated with 514 neuroinflammation. Conversely, APN-P treatment in CCH rats promoted the microglia towards the 515 detrimental M2 phenotype, with decreased iNOS and increased Arg-1 expression, an increased 516 CD206/CD16 ratio in Iba-1+ cells, and secretion of anti-inflammatory factors. 517 Research have focused on the role of the hippocampus in cognitive impairment, but spatial reference memory is thought to be related to the integrity of both the hippocampus and cortex [32]. 519 The onset of CCH is in the associative cortical areas; then, it spreads throughout the brain via the 520 neuronal network, affecting cognitive function [33]. Our previous studies also showed that CCH 521 resulted in hypoperfusion of cortical cerebral blood flow in rats, as well as white matter fiber injury 522 and neuron death in the cortex [7,9]. Considering cerebral white matter is comprised of nerve fibers 523 that interconnect neurons in the cortex or the deep structures [34], pathological changes in the 524 cerebral cortex caused by CCH could also be associated with cognitive impairment. Therefore, we 525 focused on the CCH effects on cortex in this study. We found APN-P not only regulated microglial 526 polarization changes in the cortex of CCH rats but also showed a protective effect on cortical and 527 hippocampal neurons in vivo. In vitro results also indicated a neuroprotective effect in the two 528 neuronal cell lines when co-cultured with activated BV2 microglia cells. Together, these results 529 indicated that APN-P treatment could reduce cortex injury through the mediation of pro-and anti-530 inflammatory responses that contribute to CCH tissue damage, further suggesting a therapeutic 531 potential for ANP-P treatment strategies for cortex injuries. 532 CCH causes a cascade of pathological processes during which diverse signaling pathways are 533 activated [30]. Our in vivo study found that CCH could activate the NF-κB signaling pathways, 534 which are closely related to neuroinflammation [35] and can induce the production of pro-535 inflammatory molecules that promote apoptosis in neural cells and cause secondary neurotoxicity 536 [36]. A previous study indicated that APN inhibits ROS-induced cardiac remodeling in rat 537 ventricular myocytes by inhibiting NF-κB activation and that APN mediates the suppression of NF-538 κB activation and pro-inflammatory cytokine expression, leading to a suppression of M1 539 macrophage proliferation and function [15]. Consistent with these previous findings, we observed that APN-P could efficiently inhibit NF-κB activation in the cortex of CCH rats, and our in vitro 541 results from LPS-activated BV2 microglial cells showed that an NF-κB inhibitor and and APN-P 542 treatment could mediate the suppression of M1 polarization and pro-inflammatory factor expression. 543 Consistent with the effect of the NF-κB inhibitor, APN-P mediated the promotion of M2 polarization 544 and anti-inflammatory factor expression in LPS-induced BV2 cells. 545 This study has several limitations. First, APN-P-mediated protection after CCH was studied 546 using a single dose concentration; future studies should assess concentration-dependent effects of 547 APN-P. Second, we focused on changes in microglial cells after CCH and APN-P treatment, but the 548 potential effects on microglia-neuron crosstalk deserve further exploration. Third, exploration of the 549 effects of APN-P on CCH in aging, diabetes, or obesity is warranted in follow-up studies due to the 550 vital role of APN in metabolic regulation. Finally, none of the experimental CCH models currently 551 in use can completely mimic the clinical CCH process, and before clinical application, the 552 translational challenges of this novel treatment must be fully considered.

Availability of data and materials 582
The datasets generated and/or analyzed in the current study are available from the corresponding 583 author on reasonable request. 584 Values are mean ± SD, n = 3 slices from 4 animals per group, *P < 0.05, **P < 0.01 vs sham group, 738 Student's t test and Pearson correlation analysis. ROI, 200μm 2 . 739 e. A heat map representation of the protein level analysis of antibody arrays that show that APN was 740 the most statistically significant among 10 proteins in the cortex of CCH 12w rats compared to the 741 sham (fold change = -4.23, P < 0.05; n = 6 animals per group). Values are mean ± SD, n = 3 slices from 3 animals per group, **P < 0.01 CCH vs sham groups, ##P 763 < 0.01 CCH+APN-P vs CCH groups, One way ANOVA test, Tukey tests. ROI, 100μm 2 .