BBR promoted phagocytosis of Aβ1−42 by BV-2 cells
Western blot analysis revealed that BBR treatment could significantly increase the protein expression level of TREM2 compared with the control group in BV2 cells (Fig. 1a). To investigate the pathophysiological function of TREM2, siRNA interference was used to knock down TREM2. Western blot results showed that the expression of TREM2 in three different siRNA-targeted interferences was significantly lower than that in the blank control group (Fig. 1b). Based on this result, siTREM2-1 was selected for following knockdown experiments. Then, BV2 cells were stained with Hoechst 33258, a special fluorescent dye that can distinguish apoptotic cells from normal cells, under a fluorescence microscope, and the cell morphology was observed. As shown in Fig. 1c, after BBR treatment, the nuclear morphology did not change, showing diffuse homogeneous blue fluorescence as in normal nuclei. When treated with si-TREM2 or Aβ1−42, the cells show apoptotic cell-like nucleus fragmentation, chromosome condensation and strong blue fluorescence. However, the addition of BBR in combination with si-TREM2 or Aβ1−42 treatment can restore the nuclear state of apoptosis. The effect of TREM2 on BV2 cell viability was assessed using the CCK8 assay. As shown in Fig. 1d, the cell viability of BBR treatment group decreased slightly by 11.8%, whereas si-TREM2 and Aβ1−42 treatment significantly decreased by 53.8% and 62.4%. Compared with si-TREM2 group and Aβ1−42 group, BBR supplementation reversed cell viability by 58.1% and 94.3%. These results indicated that BBR has no significant effect on cell viability, and can rescue the decreased cell viability caused by si-TREM2 and Aβ1−42. ELISA assay was used to detect the effect of BBR on the Aβ phagocytosis of BV2 cells. In BV2 cell contents, Aβ1−42 treatment significantly increased Aβ1−42 content compared with the control group, while the addition of BBR significantly reversed this effect (Fig. 1e Left). Similarly, in the supernatant of BV2 cells, Aβ1−42 treatment significantly increased Aβ1−42 content, while BBR significantly reversed this result (Fig. 1e Right). These data suggest that BBR promotes Aβ phagocytosis in BV2 cells.
BBR promotes the phenotype changes of microglia in vitro.
Microglia cells have different phenotypes. Activated microglia cells include neuro-damaging M1, neuro-protective M2, and a new type of DAM that can significantly limit the progression of AD. Therefore, the role of microglia cells in AD is a double-edged sword. We should take advantage of its favorable side and inhibit its harmful side to achieve the effect of treating AD. In this study, we found that BBR inhibits the transition of resting BV2 cells to M1 and promotes the transition of resting BV2 cells to M2 by regulating TREM2 (Fig. 2a). As shown in Fig. 2b-c, compared with the control group, BBR significantly decreased the expression of M1 markers CD32 and IL-1β, and significantly increased the expression of M2 markers CD206 and IL-10, while si-TREM2 significantly promoted the expression of CD32 and IL-1β, and significantly inhibited the expression of CD206 and IL-10. However, compared with si-TREM2 group, BBR supplementation significantly decreased the expression of CD32 and IL-1β, and significantly increased CD206 and IL-10 expression. In addition, we evaluated the expression of CD32 (Fig. 2d) and CD206 (Fig. 2e) in microglia (IBA1) using immunofluorescence staining, which was consistent with Western blot results. In short, our results prove that BBR could inhibit the transformation of resting microglia to M1 and promote the transformation of resting microglia to M2. Then, immunofluorescence was used to detect BBR's effect on microglial transformation to DAM cells. As shown in Fig. 3a-b, BBR significantly promoted DAM marker CD11c expression and si-TREM2 significantly promoted CD11c expression compared with the control group in microglia (IBA1). However, BBR combined with si-TREM2 treatment did not restore CD11c expression compared with si-TREM2 group, suggesting that BBR may promote microglial conversion to DAM through TREM2. In conclusion, these data demonstrate that BBR promotes BV2 cells transformation to neuroprotective M2 and DAM and inhibit BV2 cells transformation to neurotoxic M1.
BBR promotes the phenotype changes of microglia in vivo.
To further confirm the effect of BBR on microglia phenotypic changes, wild-type (WT) mice, AD control mice (Saline treatment) and AD experimental mice (BBR treatment) were used as animal models. In vivo experiments indicated that BBR inhibits the transition of resting microglia to M1 and promotes the transition of resting microglia to M2 and DAM, which was in accordance with in vitro results (Fig. 4a). Western blot assay showed that CD32 and IL-1β expression in the Saline group was significantly higher than that in the WT group. Compared with the Saline group, the expression of CD32 and IL-1β in AD mice after BBR treatment was significantly decreased, while the expression of CD206 and IL-10 was significantly increased (Fig. 4b-c). Immunofluorescence results showed that the CD11c fluorescence intensity in microglia (IBA1) of AD mice treated with BBR was significantly increased compared with that of Saline group (Fig. 4d). These results suggest that BBR can not only inhibit the transformation of microglia into neurotoxic M1, but also promote the transformation of microglia into neuroprotective M2 and DAM in mice.
BBR promotes phenotypic altered microglia to surround Aβ in vivo.
The results of both in vivo and in vitro experiments showed that BBR could promote the transformation of microglia to M2 and DAM phenotypes. Next, we studied the effects of M2 and DAM phenotypic microglia on Aβ. Here we report that BBR promotes microglial transformation of M2 and DAM to encircle Aβ and inhibit Aβ expression through TREM2 (Fig. 5a). Immunofluorescence results showed that BBR promoted the increase of M2 (CD206) in microglia (IBA1), and the increased M2 tended to surround Aβ (Fig. 5b). Similarly, BBR promoted the significantly increased DAM (CD11c) in microglia (IBA1) to be in proximity to Aβ (Fig. 5c), which may have the phagocytic effect on Aβ. Both M2 with anti-inflammatory effect and DAM with phagocytic effect were close to Aβ, which may have a positive promoting effect on Aβ clearance. Immunohistochemical results showed that BBR significantly increased the expression of TREM2 and significantly reduced Aβ plaques compared with AD control group (Fig. 5d), suggesting that BBR may promote the transition of resting microglia to M2 and DAM by promoting the expression of TREM2, thereby leading Aβ clearance.
BBR regulated the spatial learning and memory of APP/PS1 mice.
The MWM test was performed to evaluate spatial learning and memory of APP/PS1 mice followed the mouse experiment process diagram (Fig. 6a). The escape latency of WT, APP/PS1 control (Saline), and APP/PS1 + BBR groups decreased in a time-dependent way during 5 consecutive days of pre-training. On day 5, APP/PS1 control group had significantly higher escape latency than WT group, while APP/PS1 + BBR had significantly lower escape latency than APP/PS1 control group (Fig. 6b). After pre-training, the platform was removed to measure the platform location time, the across platform times and the retention time in the target quadrants to test the long-term spatial memory ability of experimental mice. As shown in Fig. 6c-e, APP/PS1 control group showed worse spatial memory ability than WT group, while the platform location time, the across platform times and the retention time of APP/PS1 + BBR group were significantly improved compared with that of APP/PS1 control group. Typical trajectories of the mice clearly showed differences in the spatial memory abilities of the three groups (Fig. 6f). These results indicated that BBR could significantly improve spatial learning and memory of APP/PS1 mice.