Fig. 8
Expression feature of TRPC6 and the influence of LA on the protein. a Western blot analysis showed that nerve injury increased the level of TRPC6 protein in the ipsilateral SDH, which was dose dependently downregulated by LA at 10 and 30 μM. Upper panel, typical blotting band. Lower panel, summarized analysis of TRPC6 in five groups. *p < 0.05 vs. sham + veh group; # p <0.05 vs. SNI + veh group; n = 5. b Typical picture of TRPC6 immunostaining in the SDH of vehicle treated SNI rat. Upper panel, TRPC6 IR exhibited cloudy like even distribution in the neuropil, with preference in laminae I and II. The intensity of TRPC6 in the superficial dorsal horn of the ipsilateral side appeared stronger than that in the contralateral side, mainly within the medial section (denoted by the white rectangle). Lower panel, control picture of TRPC6 staining after the antibody was pre-absorbed by the antigen. CC central canal, Ipsi ipsilateral side, Contra contralateral side, Ab antibody, Ag antigen. Scale bar, 200 μm. c Triple-labeled staining of TRPC6, Iba-1, and DAPI in cultured microglia. First panel, normal staining showing that TRPC6 was mainly distributed in the membrane, cytosol and the processes of Iba-1-labeled microglia. Second panel, normal cells with pre-absorbed TRPC6 antibody staining. Third panel, triple staining after the cells were challenged by LPS at 500 ng/mL for 24 h. Microglia showed the typical M1 phenotype with hypertrophied cell body and short but thick processes. TRPC6 and Iba-1 IR were greatly elevated in the cells. Last panel, co-incubation of the cell with 10 μM of LA and LPS markedly decreased TRPC6 and Iba-1 IR with the regression of the cell shape. Scale bar, 30 μm