Fig 2: The expressed and electrophysiology properties of TRPV1 in primary cultured microglia. (A) Photomicrograph immunocytochemistry indicated different co-expression of TRPV1 (visualized in red Cy3), Iba1 (visualized in green FITC), and cell nuclei (visualized in dark blue DAPI) from control and hyperthermia groups microglia. Note the colocalization indicated by merge yellow fluorescence, the fluorescence intensity of TRPV1 and Iba1 along the indicated line were scanned by using ImageJ software, and their colocalization was determined by the PCC method (C,D; Scale bars: 50 µm). (B) The histograms represent the ratio of the mean PCC calculated from the colocalizating in three samples (n = 3 per groups, ***p < 0.001, unpaired, two-tailed Student’s t-test compared with control). (E,F) Representative Immunoblot bands and densitometric analysis of total protein (E) and membrane protein expression (F) of TRPV1 in cortical primary cultured microglia with or without pretreatment of CAP (10 µM), hyperthermia (43°C, 4*30 min) or LPS (1.0 µg/ml, 24 h). Data were presented as means ± SEM, n = 3 per groups, *p < 0.05, **p < 0.01, one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test with DMSO. Ctrl, Control; CAP, capsaicin; HT, hyperthermia. (G) Whole-cell patch clamp (I = 0 mode) recorded resting membrane potential (RMP) in microglia from wild-type (WT) and TRPV1-/- mice (n = 11 per groups, ns, not significant, unpaired, two-tailed Student’s t-test compared with WT). (H) TRPV1 currents amplitude (pA) is significantly increased in 1.0 µM capsaicin group (-99.05 ± 2.632, n = 4) than 0.5 µM capsaicin group (-60.73 ± 6.445, n = 3) in cortical activated microglia (**p < 0.01, unpaired, two-tailed Student’s t-test compared with 0.5 µM CAP). (I) Representative whole-cell voltage-clamp mode recorded current traces from individual primary microglia challenged by 0.5 µM, 1.0 µM Capsaicin and Capsazepine (10 µM) applications, the largest current is 104.9 pA in 1.0 µM capsaicin perfusion pretreatment with LPS (1.0 µg/ml, 24 h). (J) Representative traces of intracellular calcium ([Ca2+]i) responses by CAP (1.0 µM), (Ja,b) CAP (1.0 µM) with LPS (1.0 µg/ml) in microglia from WT mice. (Jc,d) Representative traces of CAP (1.0 µM) induced-([Ca2+]i) response in LPS (1 ng/ml, 24 h)-stimulated microglia and to Capsazepine (10 µM). (Je) Representative traces of CAP (1.0 µM) induced-([Ca2+]i) response in LPS (1.0 µg/ml, 24 h)-stimulated microglia in TRPV1-/- mice microglia.

Fig 3: TRPV1 activation enhances migration activity in cultured mice microglia. (A–F) Microglial cultures were scratched with tips, and immunocytochemistry was acquired with low magnification at 24 h with or without hyperthermia (43°C) and hyperthermia with CPZ (10 µM) in wild type microglia. (A) Control, (B) capsaicin (10 µM), (C) hyperthermia (4*30 min), (D) hyperthermia and capsazepine (CPZ, 10 µM), (E) hyperthermia and EGTA (2 mM), (F) hyperthermia and BAPTA-AM (10 µM). (G) Migration activity of microglia induced by capsaicin (10 µM) and hyperthermia (43°C) with or without Capsazepine (10 µM), EGTA (2 mM), BAPTA-AM (10 µM). Dotted lines show the initial location of the microglia. Notably, the cell number of the microglia invading the wound increases (Scale bar: 100 µm). Data are presented as mean ± SEM, n = 3 per groups, *p < 0.05, **p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparison test with control.

Fig 4: Relative gene expression ratios of Transient Receptor Potential Ankyrin 1 (TRPA1) and Vanilloid 1 (TRPV1) receptors normalized to the Importin 8 (IPO8) reference gene in the healthy control oral mucosa (n = 10), compared to oral squamous cell carcinoma (OSCC; n = 15). Columns represent the mean + SEM, *** p < 0.001, Mann–Whitney U test; (A). Expression of TRPA1 and TRPV1 mRNA in the PE/CA-PJ41 cell line and TRPA1 and TRPV1 expressing CHO cells (positive controls) (n = 4/group; (B)).

Fig 5: Schematic representation of TRPV1 in inhibiting the microglial TGF-ß1 signaling via TLR4 after hyperthermia-induction seizures. (1) TRPV1 is upregulated and trafficked to the membrane from microglia; (2) TRPV1 leads to excessive Ca2+ influx in microglia and promotes microglia polarization; (3) TRPV1 activation triggers marked proliferation, migration, morphology changes and inflammatory responses; and (4) increased microglial TRPV1 inhibited TGF-ß1 signaling and Arg1 and Ym1 via interaction with TLR4. Consistently, TRPV1-/- have the microglial M2 phenotype.