BV2 microglial cells were purchased from Procell Life Science & Technology (Wuhan, Hubei, China) and were grown in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Anaheim, CA, USA) supplemented with 10% Fetal Bovine Serum (FBS) and 1% streptomycin/penicillin. PC12 cells were grown in dulbecco’s modified eagle medium (DMEM)-high glucose medium (Gibco) containing 10% FBS, 5% horse serum, and 1% streptomycin/penicillin. Both BV2 and PC12 cells were incubated at 37 °C in a 5% CO${}_2$ incubator. A quantitative polymerase chain reaction mycoplasma detection kit was used for mycoplasma testing (#4460623; Thermo Fisher, Waltham, MA, USA), and no mycoplasma infection was found in either cell line. cell line has been validated by short tandem repeat authentication.

BV2 cells were pretreated with the GRM8 agonist AZ12216052 (1 µM; 1290628-31-7; MedChemExpress, Monmouth Junction, NJ, USA) or saline vehicle for 2 h. Afterwards, the cells were treated with saline control or LPS (1 µg/mL) for 12 h.

For the establishment of PC12 and BV2 cell co-culture conditions, PC12 was first incubated in a 24-well plate for 72 h. Following this, BV2 cells were transferred to a 0.4 µm pore-sized Transwell insert. PC12 and BV2 cells (BV2:PC12 = 1:2) were subsequently co-cultured for 48 h.

Cells were seeded on a coverslip and subsequently treated with 0.5% Triton X-100 for 20 min. After washing, cells were incubated overnight at 4 °C with primary antibody. The primary antibodies used were rabbit anti-GRM8 (1:100; PA5-33830; Invitrogen, Waltham, MA, USA) and rat anti-CD11b (5 µg/mL; ab8878; Abcam, Cambridge, UK). Cells were subsequently incubated with secondary antibodies in a dark room. The secondary antibodies used in this experiment were fluoresceine isothiocyanate (FITC)-conjugated goat anti-rabbit immunoglobulin G (IgG) H&L (1:800; ab6717; Abcam) and Cy5-conjugated goat anti-rat IgG Heavy&Light (H&L) (1:800; ab6565; Abcam). Nuclei were stained with 4${}^{\prime }$,6-diamidino-2-phenylindole (DAPI) and cells were visualized using a laser scanning confocal microscope (Sp8, Leica, Heidelberg, Germany).

PC12 apoptosis in the co-culture system was detected using flow cytometry by staining PC12 cells with Annexin and propidium iodide (PI) (AP101; Multi Sciences, Hangzhou, Zhejiang, China) according to the manufacturer’s instructions. Preparations were analyzed on a CytoFLEX flow cytometer (Beckman, Pasadena, CA, USA).

Cell viability was assessed using a Cell Counting Kit-8 (CCK-8; C0039; Beyotime, Shanghai, China). Briefly, PC12 cells were inoculated into a 96-well plate and 10 µL of CCK-8 stock solution was added to each well and incubated for 4 h. Absorbance at 450 nm was measured using a Varioskan LUX microplate reader (Varioskan LUX, Thermo Fisher, Moorhead, MN, USA). Cell viability was calculated using the equation: [ODexperiemnt group – ODblank]/[ODControl group – ODblank] $\times$ 100%.

TRIZOL reagent (Sigma-Aldrich, St. Louis, MO, USA) was used to extract total RNA from BV2 cells according to the manufacturer’s instructions. RNA was subsequently reverse transcribed into cDNA using a PrimeScript${}^{\text{TM}}$ RT reagent kit (Takara, Kusatsu, Japan). Relative mRNA levels were measured using SYBR Premix EX Taq I (Takara) on a 7300 Plus Real-Time PCR System (Thermo Fisher). Each 20 µL reaction mixture contained 10 µL of 2$\times$ PCR master mix, 1 µL cDNA template, 5 pmol primer, and water. Thermocycling conditions were: 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s and 60 °C for 60 s. The primers are shown in Table 1. mRNA expression was determined relative to GAPDH mRNA levels.

Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure cAMP level (Abcam) and pro-inflammatory cytokine levels for interleukin (IL)-1$\beta$, tumor necrosis factor-alpha (TNF-$\alpha$), and IL-6 (Beyotime) in BV2 cells according to the manufacturer’s instructions.

Inositol-1,4,5-triphosphate receptor (IP3R) is expressed at the surface of the ER and when bound by IP3, calcium is released from the ER into the cytoplasm. Mag-Fluo-AM (GMS10189; GENMED, Shanghai, China) is a fluorescent probe with low affinity for calcium that can be specifically captured by the ER. This property is exploited for the detection of alterations in the concentration of ionized calcium within the ER. In this way, Mag-Fluo-AM can be used as a surrogate to examine the transfer activity of IP3R. Data were obtained 48 h after LPS treatment.

An IP3R functional fluorescence detection kit (GMS10189; GENMED, Shanghai, China) was used to evaluate IP3R activity. All procedures strictly followed the manufacturer’s instructions. In brief, Relative Fluorescence Units (RFUs) were detected using a fluorescence microplate reader (Varioskan LUX, Thermo Fisher, Moorhead, MN, USA). The induced calcium release rate was calculated using the following equation: [(RFU${}_{\text{ER Ca}}$${}^{2+}$ – RFU${}_{\text{induced Ca}}$${}^{2+}$ release)]/[(RFU${}_{\text{ER Ca}}$${}^{2+}$ – RFU${}_{\text{complete Ca}}$${}^{2+}$ release)] $\times$ 100%. In this assay system, the higher the induced calcium release rate, the greater the transport activity of IP3R.

The ER Ca${}^{2+}$ concentration was measured using an ER calcium concentration assay kit (GMS10267.1; GENMED) and by measuring the fluorescence intensity obtained using Mag-Fluo-AM and combining this with ER Ca${}^{2+}$ using a fluorescence microplate reader (Varioskan LUX, Thermo Fisher). The ER Ca${}^{2+}$ concentration (µM/100 µg) was calculated using the following equation: [RFU${}_{\text{sample}}$ – RFU${}_{\text{blank control}}$]/[RFU${}_{\text{max control}}$ – RFU${}_{\text{sample}}$] $\times$ 22.

A commercially available kit (MAK159; Sigma-Aldrich) was used to measure mitochondrial membrane potential (MMP) according to the manufacturer’s instructions. Cells were seeded into a 96-well plate and 25 µL of buffer A containing JC-10 was then added to each well and incubated for 45 min. The cells were then incubated with 25 µL of buffer B. Fluorescence measurements were made at an excitation wavelength of 540 nm and an emission wavelength of 490 nm in a microplate reader (Varioskan LUX, Thermo Fisher). The 540 nm/490 nm fluorescence intensity ratio indicates the MMP.

A luminescent ATP detection assay kit (ab113849; Abcam) was used to measure the ATP levels in BV2 cells. Briefly, detergent solution was added to cell cultures for cell lysis and ATP stabilization. Substrate solution was then added to each well and preparations were analyzed on a luminescence plate reader (Varioskan LUX, Thermo Fisher,) after incubating for 10 min in the dark.

Cells were seeded into 96-well plates and stained with 2${}^{\prime }$,7${}^{\prime }$-dichlorofluorescein diacetate (ab113851; Abcam) to measure intracellular ROS levels and with MitoSox (MAK145; Sigma-Aldrich) to measure mitochondrial ROS levels. Fluorescence intensity was measured using a microplate reader (Varioskan LUX, Thermo Fisher).

Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer supplemented with Phenylmethanesulfonyl fluoride (PMSF). The extracted proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently electrotransferred onto polyvinylidene fluoride (PVDF) membranes. These were blocked for 1 h and then incubated separately with primary antibody against ATF6 (1:1000; #DF6009; Affinity, Cincinnati, OH, USA), IRE1$\alpha$ (1:1000; #3294; CST, Danvers, MA, USA), p-ERK (1:1000; AF5304; Affinity), phospho-p65 (1:800; #3033; CST), $\beta$-actin (1:1000; ET1701-80; HuaBio, Hangzhou, Zhejiang, China), or TATA-binding protein (TBP) (1:1000; HA500518; HuaBio). After several washes, membranes were incubated with HRP-conjugated anti-rabbit secondary antibody (1:1000; A0208; Beyotime). Protein bands were visualized using chemiluminescence and the Tanon-4200 gel imaging analysis system (Tanon, Shanghai, China).

All values are expressed as the mean $\pm$ standard error of measurement (SEM). Data were analyzed using SPSS software (Version 22, IBM Corp., Armonk, NY, USA). Experiments with three or more groups were compared by one-way analysis of variance (ANOVA), followed by the least significant difference (LSD) t-test. The significance level was defined as p 0.05.

Immunofluorescence microscopy was used to examine the co-localization of GRM8 and CD11b, a marker for microglia. As shown in Fig. 1, GRM8 co-localized with CD11b, indicating that GRM8 is expressed in microglia.

Fig. 1.

Metabotropic glutamate receptor 8 (GRM8) and CD11b co-localize in BV2 cells. Red fluorescence indicates CD11b-positive cells, while green fluorescence indicates GRM8-positive cells. Nuclei were counter stained with 4${}^{\prime }$,6-diamidino-2-phenylindole (DAPI). Scale bar = 20 µm.

Quantitative RT-PCR was used to measure relative mRNA levels of microglial M1/M2 markers. Compared with the controls, LPS markedly upregulated the expression of pro-inflammatory M1 phenotypic markers, including inducible nitric oxide synthase (iNOS) and TNF-$\alpha$ (iNOS, p 0.001; TNF-$\alpha$, p 0.001; Fig. 2A). In contrast, microglial GRM8 activation significantly reduced the expression of M1 markers (iNOS, p = 0.036; TNF-$\alpha$, p = 0.003; Fig. 2A) when the LPS and LPS+GRM8 groups were compared. LPS also significantly reduced the expression of anti-inflammatory M2 phenotype markers relative to controls, including arginase 1 (Arg-1) and cluster of differentiation 206 (CD206) (Arg-1, p = 0.0012; CD206, p 0.001, Fig. 2B). Combined treatment of GRM8 agonist and LPS was found to increase the expression of M2 markers relative to LPS-only group (Arg-1, p = 0.03; CD206, p = 0.51; Fig. 2B).

Fig. 2.

GRM8 activation shifts M1/M2 polarization and inhibits the release of pro-inflammatory cytokines. (A) Relative mRNA expression of the pro-inflammatory M1 phenotype markers iNOS and TNF-$\alpha$. n = 4 per group. (B) Relative mRNA expression of the anti-inflammatory M2 phenotype markers Arg-1 and CD206. n = 4 per group. (C) Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of pro-inflammatory cytokines in BV2 cell extracts. n = 5 per group. **p 0.01, ***p 0.001 compared with the control (Con) group. #p 0.05, ###p 0.001 compared with the lipopolysaccharide (LPS) group.

We used ELISA to further evaluate pro-inflammatory cytokine levels in BV2 cells. GRM8 activation significantly inhibited the release of LPS-induced pro-inflammatory cytokines (TNF-$\alpha$, p = 0.014; IL-1$\beta$, p = 0.03; IL-6, p 0.001; Fig. 2C). These results suggest that microglial GRM8 activation shifts the M1/M2 polarization state and improves the inflammatory microenvironment in microglia challenged with LPS.

After incubating with LPS, GRM8, or LPS combined with a GRM8 agonist, BV2 cells were transferred to a co-culture system with PC12 cells and grown for 48 h (Fig. 3A). PC12 cells were then analyzed using flow cytometry (Fig. 3B) and CCK-8 (Fig. 3D) assays. The flow cytometry results showed that BV2 cells challenged with LPS significantly increased the apoptosis of PC12 cells in the co-culture model system relative to controls (p 0.001, Fig. 3C). However, activated GRM8 markedly attenuated LPS-induced apoptosis (p = 0.002, Fig. 3C).

Fig. 3.

Microglial GRM8 activation promotes neuronal survival in a co-culture model system. (A) Simplified schematic of the co-culture model. (B) Representative flow cytometry analyses showing apoptotic cell populations. (C) Bar graphs indicating mean numbers of apoptotic cells measured in the indicated experimental groups. n = 4 per group; dots show the individual assay values. (D) Cell viability examined by CCK-8 assays. n = 5 per group. ***p 0.001 compared with the control (Con) group. #p 0.05, ##p 0.01 compared with the LPS group. CCK-8, Cell Counting Kit-8; PI, propidium iodide.

In CCK-8 viability assays, LPS treatment markedly reduced the viability of PC12 cells relative to untreated controls (p 0.001, Fig. 3D), while microglial GRM8 activation markedly increased cell viability following LPS challenge (p = 0.022, Fig. 3D). Taken together, these results indicate that microglial GRM8 activation can protect neurons from inflammation-induced death.

To explore the mechanism controlling the anti-inflammatory response to GRM8 in microglia, the effects of GRM8 on IP3R-dependent calcium release were assessed. As shown in Fig. 4A, LPS treatment significantly increased calcium release from the ER to the cytoplasm relative to controls (p 0.001, Fig. 4A). This subsequently depleted calcium storage levels within the ER (p 0.001, Fig. 4B). Notably, GRM8 activation inhibited calcium release (p = 0.004, Fig. 4A) and maintained ER Ca${}^{2+}$ concentrations (p = 0.011, Fig. 4B) in BV2 cells challenged with LPS.

Fig. 4.

Microglial GRM8 restores calcium homeostasis in the endoplasmic reticulum (ER) and reduces cAMP levels in BV2 cells. (A) Induced calcium release from the ER to the cytoplasm in BV2 cells. n = 5 per group. (B) ER Ca${}^{2+}$ concentration in BV2 cells. n = 5 per group. (C) cAMP levels in BV2 cells measured by ELISA. n = 5 per group. ***p 0.001 compared with the control (Con) group. #p 0.05, ##p 0.01 compared with the LPS group.

cAMP is a common second messenger that may facilitate calcium release by sensitizing IP3R. Therefore, we next measured cellular cAMP levels using ELISA. cAMP levels increased significantly following LPS stimulation (p 0.001, Fig. 4C). However, GRM8 activation reduced cAMP levels in BV2 cells treated with LPS (p = 0.002, Fig. 4C). These results suggest that GRM8 modulates IP3R-dependent calcium homeostasis by regulating the cellular level of cAMP.

Since calcium overload can trigger mitochondrial dysfunction and promote excessive ROS production, ROS could also be responsible for increasing ER stress. We observed a marked decrease in MMP in BV2 cells treated with LPS relative to controls (p 0.001, Fig. 5A), as well as lower ATP levels (p 0.001, Fig. 5B). In contrast, GRM8 activation significantly increased both MMP (p = 0.009, Fig. 5A) and ATP levels (p = 0.038, Fig. 4B). LPS caused ROS accumulation in BV2 cells compared with untreated controls (intracellular ROS, p 0.001; mitochondrional ROS, p 0.001; Fig. 5C,D, respectively), whereas GRM8 activation reduced ROS accumulation (intracellular ROS, p = 0.016; mitochondrional ROS, p 0.001; Fig. 5C,D, respectively). These findings indicate that microglial GRM8 activation restores toxin-induced mitochondrial dysfunction and inhibits the accumulation of ROS.

Fig. 5.

Effects of GRM8 on mitochondrial function in BV2 cells challenged with LPS. (A) Relative mitochondrial membrane potential (MMP). (B) Relative ATP levels. (C) Intracellular reactive oxygen species (ROS) levels. (D) Mitochondrial ROS levels. n = 5 per group. ***p 0.001 compared with the control (Con) group. #p 0.05, #p 0.01, ###p 0.001 compared with the LPS group.

Increased ER stress can activate inflammatory signaling pathways such as the canonical nuclear factor kappa B (NF-$\kappa$B) pathway. Elevated expression of ER stress sensing proteins (ATF6, p 0.001; IRE1$\alpha$, p 0.001; p-ERK, p 0.001; Fig. 6A), and phosphorylation of the NF-$\kappa$B subunit p65 (p 0.001, Fig. 6B) were observed in BV2 cells treated with LPS. However, GRM8 activation markedly inhibited these markers of ER stress compared with treatment with LPS alone (ATF6, p 0.001; IRE1$\alpha$, p 0.001; p-ERK, p = 0.03; Fig. 6A), as well as reducing p65 phosphorylation (p = 0.01, Fig. 6B). These results indicate that ER stress and NF-$\kappa$B signaling are attenuated by the anti-inflammatory action of GRM8.

Fig. 6.

GRM8 activation deactivates ER stress and NF-$\kappa$B signaling. (A) Expression of ER stress sensing protein detected by western blotting. n = 3 per group. (B) Western blot analysis of NF-$\kappa$B p65 subunit phosphorylation. n = 3 per group. ***p 0.001 compared with the control (Con) group. #p 0.05, ###p 0.001 compared with the LPS group. TBP, TATA binding protein.