MiR-124 Promotes Microglial M2 Polarization Through TLR4/MyD88/NF-κB p65/NLRP3 Signaling In Palmitic Acid Treated-BV2 Cells

Aim Neuroin�ammation is an explanation why obesity or high-fat diet induce central nervous system disorders. MiR-124, as a highly expressed microRNA in brain, might alleviate neuroin�ammation through regulating microglial M1/M2 polarization, but its mechanism is unclear. The aim of the study was to explore whether miR-124 exerted its effect mentioned above through TLR4/MyD88/NF-κB p65/NLRP3 signaling in palmitic acid treated-microglia. Methods


Introduction
An increasing number of evidence suggest that obesity or high-fat diet cause cognitive impairment, and neuroin ammation is involved in such pathogenic process [1][2][3][4].However, regulatory mechanism of neuroin ammation in high-fat model has not been fully clari ed.But, academics agree that microglia play an irreplaceable role in it [5,6].Microglia are one kind of nerve cells which are distributed in central nervous system, and the main function is to regulate immune in ammatory response and to protect brain tissue from diseases and infections [7,8].Normally, the cells are dormant, but can be activated by in ammatory response.Activated microglia polarize to one functional state: M1 or M2 phenotype.The former promotes the in ammation, while the latter exerts an anti-in ammatory effect [9].So, promoting M2 polarization is a theoretically feasible strategy against neuroin ammation under high-fat diet condition.
In addition to microglia, NLRP3 in ammasome is also related to regulation of neuroin ammation induced by obesity and high-fat diet [10,11].In ammasomes are a class of multi-component protein complexes which play a crucial role in natural immune response, and NLRP3 in ammasome is one important member of them [12].Brie y, in ammasome is able to identify endogenous and exogenous risk molecules, and leads to release of in ammatory cytokines (i.e.IL-1β and IL-18) and development of pyroptosis [12].The process promotes strong neuroin ammation, which protects brain tissue against the risk molecules in initial stage and begins to play a harmful role when the in ammatory response gets out of control [12].Interestingly, Cui et al. suggest that inhibition of NLRP3 signaling promotes microglial M2 polarization in Alzheimer's disease [13].The ndings might shed light on functional relationship between microglia and NLRP3 in ammasome, but further research is needed to draw a nal conclusion, especially in high-fat diet model.
MicroRNA-124 (miR-124) is an important member of microRNA family, and is highly expressed in central nervous system [14].Previous studies reveal that miR-124 might promote anti-in amed microglial M2 polarization and inhibit NLRP3 in ammasome expression in other acute and chronic brain events [15,16].And, TLR4 is one kind of conserved natural pattern recognition receptor, and is involved in regulation of M2 polarization and NLRP3 in ammasome activation in microglia [17,18].More importantly, miR-124 has been proved to regulate the activity of TLR4, and the latter seems to be a bridge between the former and microglia [17,19].Therefore, miR-124/TLR4 signaling pathway may be a potential regulatory mechanism of neuroin ammation in high-fat diet condition, which should be fully verify.
Therefore, we conducted an in vitro experiment using a high-fat treated-BV2 cell line.The aim of the study was to reveal the potential effect and regulatory mechanism of miR-124/TL4/NLRP3 signaling pathway on microglial M2 polarization, and to provide the therapeutic strategy against neuroin ammation and cognitive impairment induced by obesity and high-fat diet.

Cell culture
A mouse BV2 microglial cell line was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).All reagents and materials in the process were obtained from Life Technologies (CA, USA).The cells were cultured in minimum Eagle's medium (MEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37℃ in a humidi ed environment with 5% carbon dioxide (CO 2 ).
The medium was renewed every 48 hours.In addition, the cells were regularly treated with 0.25% trypsin to transfer and place them in more culture bottles until enough cells were obtained.Finally, monolayers of the BV2 microglial cells were cultured on 24-well plates (1×10 5 cells/well) for further experiments.

Reagents and preliminary experiments
Palmitic acid (PA, one type of saturated fatty acids) was obtained from Sigma-Aldrich (USA), and was dissolved using 0.1 mmol/l sodium hydroxide at 70℃ for 5 minutes.Then, the solution is mixed using 10% bovine serum albumin at 55℃ for another 10 minutes, and was adjusted to a series of concentrations (0, 400, 800, 1200, 1600 µmol/l) using speci c neuronal medium (NM, Cat.#1521).
After that, as shown in Fig. 1, the prepared cells were separately treated with different concentrations of PA and TAK-242 for 12 hours.Cell activity was determined using CCK8 assay to assess the safety of the reagents.Expression of miR-124 and TLR4 was measured separately using qPCR and western blotting to evaluate the effectiveness of the reagents.As a result, 800 µmol/l of PA and 200 nmol/l of TAK-242 were determined to be the optimal concentrations for further experiments.

Grouping and cell transfection
The BV2 cells were divided into control group, PA group, PA + mir_mimic group, PA + mir_inhibitor group, PA + mir_ctl group, PA + TAK group, PA + nlrp_siR group and PA + nlrp_ctl group.Each group contained ten wells.
MiR-124 mimic, miR-124 inhibitor and negative control were mixed with Lipofectamine 2000 transfection reagent (RiboBio, Guangzhou, China), and were adopted for cell transfection separately in the PA + mir_mimic, PA + mir_inhibitor and PA + mir_ctl groups [20].Speci c experimental steps referred to the speci cation of the commercial kit.Cell transfection was performed in in 37℃, 5% CO2 incubator for 48 hours.

Modeling and intervention
After cell transfection, all the BV2 cells except those in the control group were treated with 800 µmol/l PA for 12 hours to construct an in vitro model of high-fat diet [22].Meanwhile, the BV2 cells in the PA + TAK group were treated with 200 nmol/l TAK-242 for 12 hours to inhibit the expression of TLR4.

Cell viability assay
After the intervention, cell viability was measured using a commercial CCK8 assay kit (#ab228554, Abcam, UK).Brie y, all of the wells to be tested were incubated with CCK-8 solution (10 µl) at 37℃ for 2 hours.Then, the absorbance of the specimens at 450 nm was determined using a Thermo Scienti c microplate reader (Waltham, MA, US).

Flow cytometry
Pyroptosis rate was determined by a FAM-FLICA in vitro caspase-1 detection kit (ImmunoChemistry, USA) [23].First, the cells were mixed with trypsin.Second, the cells were washed several times using phosphate buffer saline.Third, the cells were stained with 10 µl FAM-FLICA and 5 µl PI for 20 minutes in a dark environment at room temperature.Fourth, uorescence intensity was measured using a Coulter Epics XL ow cytometer.Pyroptosis rate was calculated using a formula: number of double-positive cells / number of total cells × 100%.

Statistical analysis
Continuous variables in the study were expressed in the form of mean ± standard deviation.Difference of two continuous variables was measured using independent sample t test, and difference of more than two continuous variables was measured using ANOVA with LSD test.If P value was less than 0.05, the difference was statistically signi cant.

Determination of concentrations for PA and TAK-242
In Figure 1, prepared BV2 cells were treated with different concentrations of PA or TAK-242.As a results, the study did not nd any abnormality in cell activity when the cells were treated with 800 μmol/l of PA or 200 nmol/l of TAK-242 (P = 0.344, P = 0.237, respectively).However, when the concentration of PA or TAK-242 exceed the boundary, the cell activity began to decline (P = 0.023, P = 0.005, respectively).The study also revealed that 800 μmol/l of PA and 200 nmol/l of TAK-242 can separately affect the expression of miR-21 and TLR4 (P 0.001, P 0.001, respectively), which proved that the reagents at these concentrations can play an effective biological role.Therefore, 800 μmol/l of PA and 200 nmol/l of TAK-242 were adopted to conduct following experiments.

Effect of miR-124 on NLRP3 in ammasome and pyroptosis in BV2 cells
In Figure 3, compared with the control group, PA treatment in the PA group elevated the expression of NLRP3 and IL-1β (P = 0.001, P 0.001, respectively), and also increased the rate of pyroptosis (P = 0.003).Meanwhile, up-regulation and down-regulation of miR-124 separately inhibited and re-elevated the expression of the proteins and the rate of pyroptosis (up-regulation: P = 0.011, P 0.001, P = 0.015, respectively; down-regulation: P 0.001, P 0.001, P = 0.011, respectively).These ndings indicated that miR-124 inhibited the activity of NLRP3 in ammasome and rate of pyroptosis in the PA-treated model.

Effect of miR-124 on microglial M2 polarization in BV2 cells
In Figure 4, PA treatment decreased the expression of CD206 and Arg-1 (P = 0.022, P = 0.020, respectively), and increased the expression of CD86 and iNOS (P 0.001, P 0.001, respectively).More importantly, up-regulation of miR-124 reversed the effect of PA on the expression of CD206, Arg-1, CD86 and iNOS (P 0.001, P 0.001, P 0.001, P = 0.001, respectively).And, down-regulation of miR-124 aggravated the effect of PA on such proteins (P 0.001, P = 0.003, P = 0.003, P 0.001, respectively).These ndings indicated that miR-124 inhibited the microglial M1 polarization and promoted the microglial M2 polarization in the PA-treated model.

Effect of TLR4 on microglial M2 polarization in BV2 cells
In Figure 6, TAK-242 was able to increase the expression of CD206 and Arg-1 (P 0.05, P 0.05, respectively), and decrease the expression of CD86 and iNOS in the PA-treated cells (P 0.05, P 0.05, respectively).These ndings indicated that down-regulation of TLR4 expression inhibited the microglial M1 polarization and promoted the microglial M2 polarization in PA-treated BV2 cells.

Relationship between NLRP3 in ammasome activation and microglial M2 polarization in BV2 cells
In Figure 7, down-regulation of NLRP3 elevated the expression of CD206 and Arg-1 (P 0.001, P 0.001, respectively), and inhibited the expression of CD86 and iNOS (P = 0.001, P 0.001, respectively).These ndings indicated that down-regulation of NLRP3 expression inhibited the microglial M1 polarization and promoted the microglial M2 polarization.

Discussion
High-fat diet and obesity contributed to cognitive impairment, which was related to in ammation in central nervous system.PA was one type of saturated fatty acids.High level of PA in peripheral circulation not only triggered in ammation in brain, but also enhanced susceptibility to develop a neurodegenerative disease [24].So, in the present study, the BV2 cells were treated with the reagent to establish an in vitro model of high-fat diet.In the process, we rst con rmed a optimal concentration of PA (800 µmol/l) for 12-hour treatment, which was largely consistent with a previous study [25].Second, in the main part of the experiment, we discovered that PA treatment signi cantly increased the activity of NLPR3 in ammasome and M1 polarization in microglia, and both promoted the development of neuroin ammation.Therefore, we believed that such in vitro model can simulate the state of microglia after high-fat diet.
Previous study revealed that miR-124 was an important regulator of triglycerides metabolism in liver tissue [26].Its expression can be up-regulated by high-fat diet, and its known function was to promote cellular triglyceride accumulation through inhibition of triglyceride and fatty acid catabolism, in parallel with decreased in ammatory factors [26].And, in vascular endothelial tissue, over-expression of miR-124 inhibited macrophage (one common in ammatory cell) proliferation and promoted macrophage apoptosis, which attenuated vascular local in ammation and atherosclerosis [27].As mentioned above, miR-124 was highly expressed in brain tissue, and was involved in regulation of neuroin ammation in brain trauma and Parkinson's disease [15,16].Actually, high-fat diet or obesity also triggered neuroin ammation in brain tissue and caused diseases.However, regulative effect of miR-124 on in ammation in such condition has not been clari ed.So, the present study focused this topic, and rst revealed that PA treatment was able to increase the expression of miR-124 in microglia, and up-regulated miR-124 in turn exerted an anti-in ammatory neuroprotective effect via promoting microglial M2 polarization and inhibiting NLRP3 in ammasome activity.
TLR4 played an special role in both acquired and innate immunity.More importantly, TLR4 was highly expressed in microglia, and the TLR4/MyD88/NF-κB p65 signaling pathway had been proved that it can regulate the microglial polarization in other models [28,29].The present study also revealed that PA treatment activated the signaling pathway and inhibited microglial M2 polarization, which can be reversed by up-regulation of miR-124.In addition, the study con rmed the regulatory effect of the TLR4/MyD88/NF-κB p65 signaling pathway on NLRP3 in ammasome.All of these ndings may partly explained the anti-in ammatory mechanism of miR-124 in high-fat treated BV2 cells.
Both microglial polarization and NLRP3 in ammasome were regarded as crucial mechanisms for regulating neuroin ammation.However, potential relationship between them were unclear.Only one study from Cui et al. reported that inhibition of TLR4 might induce microglial M2 polarization and exert neuroprotection through NLRP3 signaling in Alzheimer's disease [13].The present study further explored such relationship in high-fat model, and provided some evidences that NLRP3 may indeed be an upstream signal molecule for microglial polarization.And, detailed mechanism of NLRP3 on regulating microglial polarization should be fully explored in the future.
In conclusion, the study revealed that PA treatment induced signi cant neuroin ammation partly through inhibiting microglial M2 polarization.The study also reported that up-regulation of miR-124 may reverse the harmful effect of PA mentioned above through regulation of TLR4/MyD88/NF-κB p65/NLRP3 signaling pathway.highest concentrations of the reagents, which did not affect the cell activity.Meanwhile, the reagents of these concentrations were separately able to affect the expression of miR-124 and TLR4.So, 800 μmol/l of PA and 200 nmol/l of TAK-242 were adopted to conduct following experiments.Difference of two variables was measured using independent sample t test, and difference of more than two variables was measured using ANOVA with LSD test.P 0.05 indicated statistically signi cant.

Declarations Funding
Effect of miR-124 on TLR4/MyD88/NF-κB p65 signaling pathway in BV2 cells Prepared BV-2 cells were separately transfected with miR-124 mimic and inhibitor to up-regulate and down-regulate the expression of the nucleic acid.Then, PA was adopted to establish an in vitro model of high-fat.After that, expression of TLR4/MyD88/NF-κB p65 proteins was determined using western blotting.Difference of more than two variables was measured using ANOVA with LSD test.P 0.05 indicated statistically signi cant." †" indicated P 0.05 compared to control group; " ‡" indicated P 0.05 compared to PA group.
Effect of miR-124 on NLRP3 in ammasome and pyroptosis in BV2 cells Prepared BV-2 cells were separately transfected with miR-124 mimic and inhibitor to up-regulate and down-regulate the expression of the nucleic acid.Then, PA was adopted to establish an in vitro model of high-fat.After that, expression of NLRP3 and IL-1β proteins was determined using western blotting.Pyroptosis rate was determined using ow cytometry.Difference of more than two variables was measured using ANOVA with LSD test.P 0.05 indicated statistically signi cant." †" indicated P 0.05 compared to control group; " ‡" indicated P 0.05 compared to PA group.
Effect of miR-124 on microglial M2 polarization in BV2 cells Prepared BV-2 cells were separately transfected with miR-124 mimic and inhibitor to up-regulate and down-regulate the expression of the nucleic acid.Then, PA was adopted to establish an in vitro model of high-fat.After that, expression of CD206, Arg-1, CD86 and iNOS proteins was determined using western blotting.Difference of more than two variables was measured using ANOVA with LSD test.P 0.05 indicated statistically signi cant." †" indicated P 0.05 compared to control group; " ‡" indicated P 0.05 compared to PA group.
Effect of TLR4 on TLR4/MyD88/NF-κB p65 signaling pathway and pyroptosis in BV2 cells PA was adopted to establish an in vitro model of high-fat.Meanwhile, the BV-2 cells were treated with TAK-242 to regulate the expression of TLR4.After that, expression of TLR4, MyD88, NF-κB, NLRP3 and IL-1β proteins was determined using western blotting.Pyroptosis rate was determined using ow cytometry.Difference of more than two variables was measured using ANOVA with LSD test." †" indicated P 0.05 compared to control group; " ‡" indicated P 0.05 compared to PA group." †" indicated P 0.05 compared to control group; " ‡" indicated P 0.05 compared to PA group.
Effect of TLR4 on microglial M2 polarization in BV2 cells PA was adopted to establish an in vitro model of high-fat.Meanwhile, the BV-2 cells were treated with TAK-242 to regulate the expression of TLR4.After that, expression of CD206, Arg-1, CD86 and iNOS proteins was determined using western blotting.
Pyroptosis rate was determined using ow cytometry.Difference of more than two variables was measured using ANOVA with LSD test." †" indicated P 0.05 compared to control group; " ‡" indicated P 0.05 compared to PA group." †" indicated P 0.05 compared to control group; " ‡" indicated P 0.05 compared to PA group.
Relationship between NLRP3 in ammasome activation and microglial M2 polarization in BV2 cells Prepared BV-2 cells were transfected with NLRP3 siRNA to down-regulate the expression of NLRP3.Then, PA was adopted to establish an in vitro model of high-fat.After that, expression of NLRP3, CD206, Arg-1, CD86 and iNOS proteins was determined using western blotting.Difference of more than two variables was measured using ANOVA with LSD test.P 0.05 indicated statistically signi cant." †" indicated P 0.05 compared to control group; " ‡" indicated P 0.05 compared to PA group.