Protective effect of high n-3/n-6 PUFA ratio on acute hypoxic-ischemic brain damage in mfat-1 transgenic mice and possible inammation-related targets identied by transcriptome analysis

Background Acute hypoxic-ischemic brain damage (HIBD) occurs not only in newborns but also in adults. It is associated with series of cellular and biochemical pathways that lead to neuronal injury. N-3 polyunsaturated fatty acids (PUFAs) have been reported to improve neuron functions via G protein-coupled receptor 120 signal pathway in cells or with exogenous supplementation. Possible protective targets and underlying mechanisms of high proportion of n-3/n-6 PUFAs contained in the brains of mfat-1 transgenic mice on HIBD-induced adult brain damage needed to be further investigated. were to were after assay. and were by real-time quantitative PCR. Key factors related to were detected by immunouorescence and western blot.


Berderson score
Mice were held gently by the tail, suspended one meter above the oor, and observed for forelimb exion. The evaluation standards of Bederson method were divided into 4 grades, normal grade 0: no observable de cit; moderate grade 1: forelimb exion; severe grade 2: decreased resistance to lateral push (and forelimb exion) without circling; grade 3: same behavior as grade 2, with circling [35].

Rotarod test
For the rotating test, an accelerating rotarod (Giliang DigBehv-010, Shanghai, China) was used as described previously [36,37], which accelerated in speed of 20 rpm over a 5min period. Record the time for the mouse to fall for the rst time and the number of drops (mice were reloaded on rotarod as soon as possible after falling) within 300s. mice were acclimatized to the rotarod for three trials, with an intertrial interval of 30 min.

2,3,5-Triphenyte-trazoliumchloride (TTC) staining
After anesthesia, fresh brain tissue was taken immediately, washed with pre-cooled buffer solution (1X PBS) and directly put into quick-frozen at -20 ℃ for 20-30 minutes. The brain was cut into 2-3mm tissue blocks with a lycra blade in the brain tank. Then put it into the pre-preheated TTC incubation solution (Sigma-Aldrich, Missouri, USA), placed in the oven at 37 °C and incubated in the dark for 15min. The reaction between TTC reagent and dehydrogenase in normal tissues is red, while the dehydrogenase activity in ischemic tissues is reduced and cannot be reacted, thus presenting a pale color. The Image Pro Plus software was used to analysis the infarct volume. Infarct volume (%) = (right ischemic pale area / left brain area + right brain area) × 100.
Nissl staining and hematoxylin and eosin staining After perfusion and xation, drying, samples were dewaxed with xylene and hydrated with alcohol of class gradient concentration. Coronal brain sections (4um) were used to observe the change of the gross morphology by Hematoxylin-Eosin staining and Nissl staining, including brain hemisphere swelling, subcortical petechial hemorrhage, tissue necrosis, nerve cell loss, and in ammatory cell in ltration. The damaged neurons appear vacuolated and pale blue. Normal cells have relatively large, plump nidellae with a darker bluish tint.

Immunohistochemical analysis
Immuno uorescence staining was carried out to detect GPR120 expression in mice after HIBD. Para nembedded formaldehyde xed specimens were cut into 4um thick slices, dewaxed with xylene, and rehydrated with a series of graded alcohols. Microwave high temperature antigen repair in citric acid buffer (3% trisodium citrate and 0.3% sodium citrate). Brain serial coronal sections were washed with PBS before xed with 4% paraformaldehyde at room temperature for 30 min. Subsequently, they were incubated with a blocking solution (5% FBS) for 30 min at 37 °C. Then, they were incubated with anti-GPR120 antibody (Santa Cruz Biotechnology, CA, USA) overnight at 4 °C. The antibodies used in this section also illustrated in supplementary Table S1. On the following day, they were washed with PBS and incubated with secondary antibodies anti-IgG Conjugates (Invitrogen, Carlsbad, CA, USA) for 1h and 4',6diamidino-2-phenylindole (DAPI) for 1 min at room temperature in the dark. Images were obtained using a confocal microscope (CarlZeiss LSM710, Oberkochen, German).
Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay TUNEL staining was performed according to the manufacturer's instructions (KeyGEN BioTECH, Nanjing, China) for quanti cation of neuronal cell death [38].Para n sections (4μm) were dried for 1 h at 70 °C, and depara nized in xylene and graded ethanol solutions. Images were obtained using a confocal microscope (CarlZeiss LSM710, Oberkochen, German). The TUNEL-positive cells were counted in the cortex, hippocampus and striatum in three separate elds for each animal by an observer who was blind to the experimental condition. Data was expressed as ratio of TUNEL-positive neurons (%).

Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IL-1β, IL-6, and TNF-α in ischemic penumbra in brain of each group were quanti ed using an ELISA kit (Elabscience Biotechnology, Wuhan, China) according to the manufacturer's instruction. Absorbance at 450nm was recorded and the concentration of the target protein was calculated according to the standard curve and normalized against the protein of the samples. Result was expressed as pg/mg protein.

RNA-seq analysis
Total RNA extraction Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to manual instruction. Ischemic side brain tissues were ground into powder by liquid nitrogen, followed by being homogenized and rested horizontally. The mix was centrifuged, then the supernatant was transferred into a new EP tube with chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15s, and then centrifuged, the upper aqueous phase where RNA remained was transferred into a new tube with equal volume of supernatant of isopropyl alcohol, then centrifuged at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 75% ethanol, then the mix was centrifuged at 4°C to collect residual ethanol, followed by the pellet air dry in the biosafety cabinet. Finally, DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was quali ed and quanti ed using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scienti c, MA, USA).

mRNA Library Construction
Oligo(dT)-attached magnetic beads were used to puri ed mRNA. Puri ed mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were ampli ed by PCR, and products were puri ed by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the nal library. The single strand circle DNA (ssCir DNA) was formatted as the nal library. The nal library was ampli ed with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).

Bioinformatics analysis
Primary sequencing data produced by RNA-Seq (raw reads) were subjected to quality control (QC). The sequencing data was ltered with SOAPnuke (v1.5.2) by (1) Removing reads containing sequencing adapter; (2) Removing reads whose low-quality base ratio (base quality less than or equal to 5) is more than 20%; (3) Removing reads whose unknown base ('N' base) ratio is more than 5%, afterwards clean reads were obtained and stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2 (v2.0.4). Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set, then expression level of gene was calculated by RSEM (v1.2.12) . The heatmap was drawn by pheatmap (v1.0.8) according to the gene expression in different samples. Essentially, differential expression analysis was performed using the DESeq2(v1.4.5) with Q value ≤ 0.05. To take insight to the change of phenotype, GO (http://www.geneontology.org/) and KEGG (https://www.kegg.jp/) enrichment analysis of annotated different expressed gene was performed by Phyper (https://en.wikipedia.org/wiki/Hypergeometric_distribution) based on Hypergeometric test. The signi cant levels of terms and pathways were corrected by Q value with a rigorous threshold (Q value ≤ 0.05) by Bonferroni.

Western blotting
Western blotting was performed according to the manufacturer's speci cation. The ischemic side brain tissue samples were collected at 24h after HIBD. Proteins were extracted by homogenizing in RIPA buffer (Sigma-Aldrich, Missouri, USA) with phenylmethanesulfonyl uoride (PMSF) and phosphatase inhibitor (Bimake, TX, USA) and further centrifuged at 12,000 rpm at 4 °C for 10 min. The concentrations were determined with BCA protein assay kit (Thermo Fisher Scienti c, MA, USA). the samples were separated by 8-12% SDS-PAGE gel (Bio-Rad, CA, USA) and then transferred to polyvinylidene uoride (PVDF) (Bio-Rad, CA, USA) membranes. Subsequently, the membranes were blocked in 5% BSA (Sigma-Aldrich, Missouri, USA) for 1 h at room temperature following incubation with primary antibodies overnight at 4°C . Dilutions for primary and secondary antibodies were listed in supplementary Table S1. Membranes were washed three times in TBST and speci c binding was visualized by ECL reaction (Bio-Rad, CA, USA). The density of bands was detected using an imaging densitometer, and the gray value of the bands was quanti ed using ImageJ Software (version 1.41).

Real-time PCR
Total RNA was extracted from the ischemic cerebral cortices at 24 h after HIBD using Trizol reagent (Invitrogen, CA, USA) according to the manufacturer's protocol. The quantity of total RNA was measured with a UV spectrophotometer (Thermo Fisher Scienti c, MA, USA). Next, reverse transcription was performed using a cDNA synthesis kit HiScript II Q RT SuperMix (Vazyme Biotech, China). Quantitative PCR was performed with ChamQ SYBR qPCR Master Mix (Vazyme Biotech, China) at the following conditions (denaturing at 95 °C for 30s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s) and detected by StepOnePlus Real-Time PCR Systems (Applied Biosystems; MA, USA). The expression of target genes was measured in triplicate and normalized to β-actin as an internal control. The −ΔΔCt values of each group were analyzed, and mRNA expression levels were normalized to 2 −ΔΔCt . Primers are listed in Supplementary table S2.

Statistical analysis
GraphPad Prism 8.0.1 software (GraphPad Software Inc, La Jolla, CA) was used to analyze data and form the graphs in this work (including which tests were performed, exact P values, and sample sizes).
Simply, one-way ANOVA with a test for linear trend, Tukey test was used as appropriate to analyze parametric statistics. The statistics of neural score longa score and Berderson score data use rank sum test in nonparametric test. At least three independent experiments were applied to collect effective data. Bias was avoided by making sure that assessor was blinded to collecting and analyzing data. P < 0.05 was considered signi cant. Average values represent the mean ± SD. *: P<0.05; **: P<0.01; ***: P<0.001.

Results
Expression of the mfat-1 transgene elevated the ratio of n-3/n-6 PUFAs The genotypes of the mice were identi ed by PCR and showed in Supplementary Fig.S1. The whole brain tissues of mfat-1 transgenic and WT mice were analyzed for fatty acid composition using GC. As showed in Fig. 1A, the ratio of n-3/n-6 PUFAs was signi cantly higher than that in WT mice in the same litter (86.89±9.61 in mfat-1 vs. 62.11±3.38 in WT, p <0.05) due to decreased proportion of n-6 PUFAs, including AA and LA, increased expression of n-3 PUFAs, including EPA, DPA, ALA and DHA. Moreover, we performed RNA-seq analysis on the whole brains of WT and mfat-1 mice in the same litters on physiological conditions. A total of 46 differentially expressed genes (DEGs) was identi ed in supplementary table S3, including 43 up-regulated genes and 3 down-regulated genes. Fig. 1B showed the cluster heat map of all DEGs. GO and KEGG pathway analysis were used for functional annotation to determine the biological signi cance of the differential clustering of all DEGs mRNA and related enrichment pathways in Supplementary Fig. S2. Most of these signi cant pathways were closely related to the synthesis and metabolism of unsaturated fatty acids, neurogenesis and neuron development, and signal transduction. The above results showed that the brains of mfat-1 transgenic mice not only contain a high proportion of n-3/n-6 PUFAs, but also differs from WT mice in terms of gene expression levels related to polyunsaturated fatty acid metabolism and neurotrophic metabolism. This was in line with the expectation of using mfat-1 transgenic mice to study neuroprotection in this study. In sum, mfat-1 mice are ideal for addressing the effects of n-3/n-6 ratio in the brain tissues.
Neuroprotective effects of high ratio of n-3/n-6 PUFAs in mfat-1 mice on HIBD In order to investigate the protective effect of high ratio of n-3/n-6 PUFAs in mfat-1 transgenic mice on brain lesions induced by HIBD, brain tissue was carefully checked. In Fig. 2A, compared with mfat-1 transgenic mice brain, WT brain tissue showed that the central part of the ischemia is more swollen and more bleeding around. In addition, H/E staining found in Fig. 2B, in WT+HIBD group, small focal hemorrhage occurred in the cerebral cortex, and a large number of red blood cells over owed around the damaged vessel wall, however, this type of hemorrhage rarely appeared in the mfat-1+HIBD group (pointed by the arrow). Infarction is an important manifestation of brain injury, and its area can directly re ect the degree of injury [39]. In Fig. 2C, TTC staining exhibited that the infarct volume in the mfat-1 group was signi cantly reduced (39.32±6.34 in WT+HIBD vs. 15.42±6.06 in WT+HIBD, p <0.05). In Fig. 2D, H/E staining showed in the WT+HIBD group, much more hippocampus and cortical neurons were lost and more typical red neurons were observed which de ned by severely chromatin agglutination, shrank and deformed cell body, dark red cytoplasm, unclear outline and appearance of foam cells [40] (pointed by the arrow). While in mfat-1+HIBD groups, there was only slight condensation of chromatin in the hippocampus and cortical neurons, but no signi cant neuron loss and typical red neurons were observed. These results indicated that mfat-1 mice containing high ratio of endogenous n-3/n-6 PUFAs in mfat-1 transgenic mice have a neuroprotective effect on HIBD.
High ratio of n-3/n-6 PUFAs in mfat-1 mice on HIBD improved neurobehavioral performance Classical Longa score and Berderson score were used as indicators to evaluate the neurological defect and neurobehavioral performance at 24h after HIBD. As showed in Fig.3A, all mice in sham groups had a neurologic grade of 0, while in the HIBD group, mfat-1 mice obtained a lower Longa score compared with WT group and exhibited better neurobehavior. In addition, Berderson score in Fig. 3B showed similar results. Subsequently, we investigated motor coordination and balance using the rotarod test at 24 h after HIBD. In the sham operation groups, the mice had coordinated limbs on the rotating rod and had a strong balance ability, although they would rotate with the rotating rod due to fatigue in the later stage. In HIBD groups, WT mice got more signi cant motor function defects than mfat-1 mice exhibited by shorter latency to fall and increased times of drops within 300s compared with mfat-1 mice (Fig. 3C, 3D). These results re ected that mfat-1 mice possessed low neurological de cits and relatively better motor coordination and balance maintenance ability compared with WT mice at 24 h after HIBD.
Endogenous high ratio of n-3/n-6 PUFAs in mfat-1 mice protected neurons against HIBD-induced neuronal apoptosis Nissl staining was used to identify apoptosis and loss of neurons in the damaged area. Typical neuronal lesions in the hippocampus and cortex in the WT+HIBD group were showed in Fig. 4A: the central Nissl corpuscle was dissolved, mainly manifested as neuron swelling and rounding; nucleolar got larger, the cytoplasmic central Nissl body disintegrated, dissolved and the disappeared. Next, western blotting was performed to verify the expression level of apoptotic relative protein (Fig. 4B). 24 h after HIBD, cleaved caspase-3 showed higher expression in the ischemic hemisphere of WT group compared with that in mfat-1 group. In Fig. 4C, TUNEL staining further con rmed that apoptotic neurons in mfat-1 group were signi cantly reduce compared with WT group in the hippocampus as well as the cortex and striatum. The above results demonstrated that mfat-1 transgenic mice could prevent apoptosis damage caused by acute hypoxia-ischemia in the perioperative period.
The mfat-1 transgenic mice showed reduced in ammation on HIBD Mounting evidence suggests that in ammation is a key contributor to the severity of CNS hypoxiaischemia injury [41,42] After HIBD, their ipsilateral hemispheres may display an in ammatory response, we evaluated changes in the mRNA and protein levels of pro-in ammatory cytokines at 24 h after HIBD insult by q-PCR and ELISA. HIBD caused a signi cant increasement in the secretion of IL-1β, IL-6, and TNF-α compared to sham treatment as expected. However, mRNA expression level of IL-6 (137.03±11.33 in WT+HIBD vs. 56

RNA-seq transcriptome analysis of DEGs on HIBD
After acute hypoxia-ischemia, a series of pathological processes are triggered in the lesion area. RNA-seq transcriptome was used to identify the full gene expression pro le of WT and mfat-1 mice in the same litters at 24 hours after HIBD with ipsilateral hemispheres. After DEGs screening, 1936 down-regulated genes and 1315 up-regulated genes were obtained showing by volcano map (Fig. 6A). Next, we performed KEGG pathway analysis on differentially down-regulated genes and up-regulated genes respectively. TOP20 enrichment pathways in the down-regulated and up-regulated genes were showed in the form of bubble chart (Fig. 6B1 and 6C1). And then, we used Cytoscape software to perform enrichment analysis on the DEGs involved in the signi cant pathways (Fig. 6B2 and 6C2). Statistical analysis found that in the down-regulated 1,936 DEGs, 247 enrichment pathways were involved, mainly involving cytokine-cytokine receptor interaction, osteoclast differentiation, ribosome, TNF signaling pathway, NOD-like receptor signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, chemokine signaling pathway, neuroactive ligand-receptor interaction and apoptosis, etc. Most of the down-regulated pathways were closely related to in ammatory response, indicating that mfat-1 transgenic mice could inhibit the in ammatory response induced by HIBD. In contrast, among the 1,315 up-regulated DEGs, there were 247 enriched pathways involved, mainly involving phosphatidylinositol signaling system, glutamatergic synapse, aldosterone synthesis and secretion, neurotrophin signaling pathway, phospholipase D signaling pathway, axon guidance, long-term depression and calcium signaling pathway, etc. Compared with WT mice, most of these up-regulated pathways were closely related to nerve growth and differentiation, cholinergic synapse development, and nervous system signal transduction. Neurotrophic factors and growth factors could promote tissue repair and vascular remodeling [43]. They were critical for the recovery of ischemic brain. This detailed analysis showed for the rst time that the genetic regulatory network of neurotrophic factors, its receptors and the protein kinases that in uence metabolism might mediate brain damage induced by ischemia and hypoxia.

Veri cation of the DEGs on HIBD
In the enrichment network analysis, we found that IL-6, IL-1β, TNF-α, AP-1, Ifnar2, Mmp3, Tlr4, Cxcl1, Cxcl2, Cxcl3, Ccl2 and Ccl12 etc. were involved in the regulation of most of signi cant pathways in all down-regulated genes, and these genes all belong to in ammatory signaling pathway. The mRNA and protein expression levels of pro-in ammatory factors IL-6, IL1β, and TNF-α have been veri ed in Fig. 5.
which were consistent with RNA-Seq results. In addition, we also found that Mapk7, Pik3ca, Pik3r3, Itpr3, Foxo3, Itrp1, Mapk12, Grid2 and Map2k7 are involved in the regulation of most of signi cant pathways in all up-regulated genes, and these genes all belonged to neurotrophin signaling pathway. Then, String public database was used to perform interaction analysis on these selected genes ( Fig.7A and 7C). Next, qPCR was used to verify the expression of these screened differential genes, and the qPCR results were consistent with RNA-seq transcriptome analysis (Fig.7B, 7D).
Activation of GPR120, suppression phosphorylation of TAK1 and NF-КB involved in protection against HIBD in mfat-1 mice by alleviating in ammation As an important receptor for n-3 PUFAs, the expression of GPR120 in the mouse brain is mainly concentrated in the olfactory bulb, cerebral cortex, and a small amount of expression in the hippocampus (Allen brain map data). And recent study showed that the expression of GPR120 was remarkably increased in the microglia, neurons, astrocytes of penumbra of cortex after ischemic injury [44]. Indeed, in our case, the expression level of GPR120 was found higher in mfat-1 mice compared with WT mice with or without HIBD, and its expression was veri ed not only from RNA-seq but also by immuno uorescence analysis and western blot (Fig. 7A ,7B, 7C). These ndings illustrated the existence and participation of GPR120 in HIBD itself and its further protective effects in mfat-1 mice. Previous research reported that through GPR120 signaling pathway, n-3 PUFAs pretreatment could inhibit the pro-in ammatory responses in downstream such as the phosphorylation level of TAK1 and NF-κB [45,46]. Therefore, we performed western blot on p-NF-κB P65 and p-TAK1. As expected, the phosphorylation level of NF-κB P65 and TAK1 protein in the brains signi cantly decreased in the mfat-1 mice compared to the WT mice on HIBD ( Fig.  7D and 7E). Mechanistically, we concluded that mfat-1 transgenic mice alleviated in ammation by activating the GPR120 receptor, reducing the phosphorylation of TAK1 and NF-κB P65 and then signi cantly inhibited the release of pro-in ammatory cytokines: IL-1β, TNF-α and IL-6.

Discussion
One of the signi cant risk factors that patients undergoing major cardiothoracic surgery is ischemic stroke [47]. In order to prevent subsequent occurrence of central nervous system diseases, related events must be either reduced in severity or prevented completely. Different methods had been tried including the use of pharmacological drugs and other non-pharmacological methods [48][49][50]. Previous studies have demonstrated that exogenous and endogenous n-3 PUFAs exerted protective effects in ischemic stroke after focal cerebral ischemia [16,20]. However, what is protective effect of endogenous high proportion of n-3/n-6 PUFAs on HIBD model and the underlying mechanism involved in this process still need to be thoroughly researched.
Well-controlled mfat-1 transgenic mice on a normal diet were used in current study, and the ratio of n-3/n-6 PUFAs (DHA+EPA+DPA+ALA/LA+AA) was statistically higher in the brain tissues of littermates in mfat-1 mice. Without interference from long-term dietary of other bioactive compounds, the transgenic mice proved to be a better model compared with exogenous supplementation. Consistent with our pervious report [26], the n-3 PUFAs in the brains of mfat-1 mice not only contain a higher proportion of DHA, but also contain EPA and DPA that are almost absent in the brain of WT mice, which leading to an increase in the overall n-3/n-6 PUFA ratio. The overall increase in n-3 PUFAs should be better than exogenous supplementation of speci c kind of n-3 PUFAs. Whole-transcriptome deep sequencing of the whole brain tissues of mfat-1 transgenic mice were performed on normal condition and on HIBD for the rst time, although transcriptome sequencing of the hippocampus of mfat-1 mice had been reported [51]. First, the transcription level of mfat-1 transgene was proved to be at a stable and high expression level in the brain tissues. And then, we found that many DEGs, such as SCD1, GPX3, DUSP1, Susd2, Ngfr, were involved in the synthesis, metabolism and transportation of fatty acids in the brain [52][53][54]. This further suggested that mfat-1 transgenic mice were suitable model to research protective effect of endogenous high n-3/n-6 PUFA ratio in the brain.
In this study, mfat-1 transgenic mice had protective effects on HIBD-induced brain damage by signi cantly reduced infarct range, greatly improved neurobehavioral defects, relatively lower level of neuronal necrosis, apoptosis and in ammation. Although many studies have revealed its pathological mechanism [55,56], the molecular details of the subsequent events after HIBD and the underlying protective mechanism from mfat-1 mice still needed to be further elucidated. RNA-seq technology was used to identify the full gene expression pro les of WT and mfat-1 mice at 24 h after acute ischemia and hypoxia. To our knowledge, this is so far the rst study to pro le the gene expression changes and key pathways on HIBD in mfat-1 mice. The results provided a multi-faceted research directions and targets for the subsequent study of mfat-1 transgenic mice in ischemic and hypoxic diseases. Among them, antiin ammatory signaling pathway was the most signi cant one. In ammation was one of the crucial factors of secondary neuronal injury after global HI neonatal perinatal period [57,58], however, the in ammatory related targets and pathways after HIBD in adults during perioperative period are still unclear. As an important receptor for n-3 PUFAs, GPR120 was found mainly expressed in the olfactory bulb, cerebral cortex and hippocampus on physiological condition (Allen brain atlas), and activated expression in this HIBD model. GPR120 activation has been shown to produce anti-in ammatory effects in previous studies [44,59]. GPR120 couples with b-arrestin2 to induce receptor endocytosis, which in turn inactivates phosphorylation of NF-κB, thereby providing a mechanism to inhibit in ammation signaling pathways [44]. Therefore, we speculated that the activation of GPR120 might protect brain injury in HIBD model by alleviating the in ammation in the ischemic area. As expected, we found that GPR120 expression in the penumbra of the affected side of WT and mfat-1 mice was signi cantly increased after acute hypoxia-ischemic injury. It was worth noting that in the sham operation group, the expression of GPR120 in mouse brain tissue of WT mice and mfat-1was different as well. Although the expression level of GPR120 in the penumbra of WT mice was signi cantly activated within 24 hours after acute ischemia and hypoxia, but the downstream TAK1 activated by the receptor after endocytosis was not weakened, neuronal necrosis and apoptosis caused by in ammation have not improved as a result. On the contrary, the expression of GPR120 in mfat-1 mice was at a high level under physiological conditions. 24 h after acute ischemic hypoxia, GPR120 is overactivated further, which signi cantly inhibits phosphorylation of TAK1 and NF-κB induced by acute ischemic stroke. Therefore, the TAK1-NFκB in ammatory pathway in the brain is signi cantly interfered. Of note, endogenous n-3 PUFAs might also exert anti-in ammatory effects through other mechanisms. For example, recent studies have shown that metabolites of n-3 PUFAs such as resolvins, protectins and maresin may play a role in improving in ammation [60].
Although the CT imaging of adult and neonatal individual brains was different for hypoxia-ischemia damage, the mechanisms of in ammation and apoptosis induced by hypoxia-ischemia injury were similar.

Conclusions
In summary, we concluded the endogenous high proportion of n-3/n-6 PUFAs in the brains of mfat-1 mice had a certain neuroprotective trend for acute hypoxia-ischemic stroke that occurs during the perioperative period. Among the multiple complicated pathological protection mechanisms, current improved neurological outcomes mainly from activation of GPR120 pathway in alleviation of in ammation.
Understanding of these insights can serve as the basis for broadening the scope of treatment of hypoxiaischemia-related encephalopathy during the perioperative period and nally bene t more patients. KEGG pathway enrichment result between mfat-1 and WT mice. According to the KEGG pathway annotation classi cation, use the phyper function in the R software to perform enrichment analysis, calculate the Pvalue, and then perform FDR correction on the Pvalue. Figure 1 Expression of the mfat-1 transgene elevated the ratio of n-3/n-6 PUFAs. A Brain tissues were collected from mfat-1 transgenic mice and WT control littermates. Compositions of n-3 or n-6 PUFAs were expressed using relative percentages-that is, the distribution areas of n-3 or n-6 PUFAs peaks divided by the total peak areas of all detectable saturated and unsaturated free fatty acids (from the same sample) resolved from the gas chromatography column. Data are expressed as mean ± SD; n = 3 per group. AA=arachidonic acid; ALA=α-lipoic acid; DPA=docosapentaenoic acid; DHA=docosahexaenoic acid; EPA=eicosapentaenoic acid; LA=linoleic acid. B The mapping data for this analysis was the centralized and standardized gene expression (FPKM), and the color gradient from blue to red indicates the change of gene expression from low to high. And make KEGG pathway term level2 annotations based on all differentially expressed genes. Horizontal: clustering between samples, re ecting the repeatability of samples. Longitudinal: Gene clustering, re ecting the similarity of different gene functions. Different square color blocks corresponded to speci c gene enrichment pathways.

Figure 2
High ratio of n-3/n-6 in mfat-1 transgenic mice showed neuroprotective effects after HIBD. A The picture showed that at 24 hours after HIBD, more severe diffuse hemorrhage and edema occurred in the WT group (Circled). B H/E staining showed cerebral cortex hemorrhage after HIBD. In the WT group, the edge of the cortex was damaged, the boundary was blurred, and there were scattered bleeding points (as shown by the arrow). Scale = 100um in left panel; Scale = 50um in the right panel. C TTC staining in the coronal section of typical cerebral infarction at 24 h after HIBD. The infarct was seen in white and the normal tissue in red. Statistics in the bar chart below showed quanti cation of infarct volume in mfat-1 groups and WT groups. The data were expressed as the mean ± SD; n = 5 per group. D H/E staining indicated neuronal morphology and pathological characteristics in ischemic penumbra at 24 h after Page 22/28 HIBD. The red arrows represented typical red neurons with shrunken bodies, highly condensed chromatin, and vacuolated cytoplasm.    High ratio of n-3/n-6 PUFAs in mfat-1 transgenic mice reduced in ammatory cytokine release after HIBD.
A, B, C Representation of the fold changes of the mRNA expression levels of IL-1β, IL-6 and TNF-α. mfat-1 group inhibited the upregulation of IL-1β, IL-6 and TNF-α compared to WT+HIBD groups. Average values represented the mean ± SD; n = 5 per group. D, E F Expression levels of pro-in ammatory factors, IL-1β, IL-6 and TNF-α in groups were measured by ELISA assay. Data were presented as the means ± SD; n = 5 per group. groups; The horizontal axis was log2 fold change (mfat-1-HIBD/WT-HIBD), the vertical axis was -log10 (Q value), and each point represented a gene. Blue dots represented down-regulated genes; red dots represented up-regulated genes. B1 KEGG pathway analysis of differential down-regulated genes between WT+HIBD and mfat-1+HIBD groups; B2 Enrichment of KEGG pathways. The network was visualized using Cytoscape 3.7.1; C1 KEGG pathway analysis of differential up-regulated between WT+HIBD and mfat-1+HIBD; C2 Enrichment of KEGG pathways. The network was visualized using Cytoscape 3.7.1. The phyper function in R software was used for enrichment analysis, P value was calculated, and P value was then FDR corrected. Generally, functions with Q value ≤ 0.05 ware regarded as signi cant enrichment.

Figure 7
Veri cation of the DEGs. A Protein-protein interaction network among differential down-regulated genes obtained from RNA sequencing. Colors of the inside nodes indicate that the genes came from different signi cant KEGG pathways above. Edges represented protein-protein associations. B Transcriptional level expression of down-regulated differential genes by qPCR in groups n = 3 per group. C Protein-protein interaction network among differential up-regulated genes obtained from RNA sequencing. Colors of the inside nodes indicated that the genes came from different signi cant KEGG pathways above. Edges represented protein-protein associations. D Transcriptional level expression of up-regulated differential genes by qPCR in groups, n = 3 per group.

Figure 8
Activation of GPR120, suppression of TAK1 and NF-КB signaling protected against HIBD in mfat-1 mice by alleviating in ammation. A Immuno uorescence analysis was performed to study the expression level of GPR120 in the cerebral cortex (red). Nuclei were uorescently labeled with DAPI (blue). Scale bar = 50um. B Quantitative analysis of GPR120 protein immuno uorescence experiment. The results were expressed as the number of GPR120+ neurons/DAPI (%); The data were expressed as the mean ± SD n = 5 per group. C Western blot analysis and quanti cation of GPR120 protein levels in brain tissues of different groups. The data were expressed as the mean± SD; n = 3 per group. D, E Western blot analysis and quanti cation of TAK1 and NF-κB P65 corresponding phosphorylation p-NF-κB P65 and p-TAK1 protein levels in brain tissues of different groups. The data were expressed as the mean ± SD; n = 3 per group.

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