Results
Lipopolysaccharide resulted inlearning and memory impairment in mice
The NORT is a widely used to evaluate learning and memory function in mice(W. Zhang et al., 2020; Zurek, Bridgwater, & Orser, 2012). In this study, no significant difference was found in time spent with objects between the Control and LPS mice (Figure 1A). In the testing stage, spent more time was spent on the novel object than on the familiar object in the Control mice (Figure 1B), but there was no statistic difference in LPS mice (Figure 1B). The result of discrimination ratio was similar to that in Figure 1B which shows that Control mice had greater discrimination ratio than LPS mice (Figure 1C). Then, we extracted serum to detect the concentrations of IL-6, IL-1β and TNF-α after LPS treatment. The result indicates that levels of IL-6, IL-1β and TNF-α were increased in the LPS group (Figure 1D). These findings demonstrate that the administration of LPS resulted in systemic inflammation that induced learning and memory impairment in mice.
Lipopolysaccharide disturbed the BDNF-TrkB signaling pathway and its downstream cascades in the hippocampus
After the novel object recognition test, we segregated the hippocampus to detect protein levels of BDNF-TrkB signaling using western blot. The results show that, compared with the Control group, the protein level of BDNF in the hippocampus was reduced in LPS mice (Figure 2A-B). No statistical difference was found in TrkB protein levels between the Control and LPS mice (Figure 2A, C). However, the protein level of the p-TrkB was reduced in LPS mice (Figure 2A, D). Then, we detected levels of apoptosis-related proteins. Compared with the Control mice, the level of pro-apoptotic protein Bax was distinctly increased, while the level of anti-apoptotic protein Bcl-2 was decreased in LPS mice (Figure 2 E-G). In addition, phosphorylation protein levels of TrkB downstream cascades were tested. The protein levels of p-ERK1/2, p-CaMK2, p-CREB and p-GluR1were obviously reduced in LPS mice (Figure 2H, J-K, M-N, P-Q, S). However, no difference was found in levels of ERK1/2, CaMK2, CREB and GluR1 proteins (Figure 2H-I, K-L, N-O, Q-R). These results illustrate that the BDNF-TrkB signaling pathway and its downstream cascades were regulated by inflammation and may be involved in learning and memory processes.
Lipopolysaccharide disturbed the B DNF-TrkB signaling pathway and its downstream cascades in the mPFC
Then, we tested the protein levels of the BDNF-TrkB signaling pathway and its downstream cascades in the mPFC. The western blot results revealed that the levels of BDNF and p-TrkB were reduced after administration of LPS (Figure 3A-B, D), and no difference was found in the protein level of TrkB (Figure 3A, C). Compared to the Control group, the expression of Bax was up-regulated in LPS mice (Figure 3E-F). At the same time, reduction of Bcl-2 expression was found in the LPS group (Figure 3E, G). Furthermore, the expression of p-ERK1/2, p-CaMK2, p-CREB and p-GluR1 proteins was decreased markedly in LPS mice (Figure 3H, J-K, M-N, P-Q, S). However, there were no significant differences in the expression of ERK1/2, CaMK2, CREB and GluR1 proteins (Figure 3H-I, K-L, N-O, Q-R). These results demonstrate that the BDNF-TrkB signaling pathway and its downstream cascades in the mPFC play a vital role in learning and memory processes.
Lipopolysaccharide disturbed the BDNF-TrkB signaling pathway and its downstream cascades in the EC
Next, the protein levels of the BDNF-TrkB signaling pathway and its downstream cascades in the EC were also examined using western blot. The results show that a qualitative reduction in the expression of BDNF and p-TrkB was observed in LPS mice (Figure 4A-B, D). There was no difference in the levels of TrkB protein between Control and LPS groups (Figure 4A, C). In addition, the protein level of Bax was increased compared to Control mice (Figure 4E-F), and the expression of Bcl-2 was markedly decreased in LPS mice (Figure 4E, G). No statistical difference was observed in protein levels of ERK1/2, p-ERK1/2, GluR1 and p-GluR1 between Control and LPS mice (Figure 4H-J, Q-S). The expression of p-CaMK2 and p-CREB were obviously reduced after using LPS (Figure 4K, M-N, P). No difference was shown in levels of CaMK2 and CREB (Figure 4K-L, N-O). These results suggest that, in the EC, the BDNF-TrkB signaling pathway, p-CaMK2 and p-CREB cascades participate in process of learning and memory.
7,8-DHF ameliorated lipopolysaccharide induced learning and memory deficits in mice
7,8-DHF was used to detect the role of the BDNF-TrkB signaling pathway in learning and memory(Bollen et al., 2013). ANA12 is a small-molecule TrkB antagonist(Cazorla et al., 2011), which was used to identify whether 7,8-DHF could reverse learning and memory dysfunction. The results of the novel object recognition test shows that there were no statistical changes in the time spent with identical objects during the training stage (Figure 5A). In the testing stage, administration of 7,8-DHF increased the time spent on the novel object in LPS mice, but ANA12 reversed the role of 7,8-DHF in LPS mice (Figure 5B). Further analysis of the discrimination ratio was in line with the result of time spent with an object. 7,8-DHF up-regulated the discrimination ratio in LPS+7,8-DHF mice, however, the discrimination ratio was reduced in LPS+7,8-DHF+ANA12 mice (Figure 5C). These results demonstrate that the preventive use of 7,8-DHF could effectively alleviate the dysfunction of learning and memory caused by LPS, while ANA12 reversed 7,8-DHF therapeutical effects.
7,8-DHF ameliorated lipopolysaccharide induced the BDNF-TrkB signaling pathway and its downstream cascade disorders in the hippocampus
The hippocampus was observed to test changes in protein levels after administration of 7,8-HDF. Western blot results show that 7,8-DHF up-regulated the expression of p-TrkB, Bcl-2, p-ERK1/2, p-CaMK2, p-CREB, p-GluR1, and decreased the protein level of Bax in LPS mice (Figure 6A, D-H, J-K, M-N, P-Q, S). But ANA12 reversed these changes in 7.8-DHF of LPS+7,8-DHF+ANA12 mice. There was no significant increase in expression of BDNF after using 7,8-DHF or reduction in the LPS+7,8-DHF+ANA12 group (Figure 6A-B). In addition, there were no significant differences in the expression of TrkB, ERK1/2, CaMK2, CREB and GluR1(Figure 6A, C, H-I, K-L, N-O, Q-R). These results demonstrate that 7,8-DHF effectively alleviated disorders of the BDNF-TrkB signaling pathway and its downstream cascades in the hippocampus of LPS mice, but ANA12 completely reversed the therapeutic effects of 7,8-DHF.
7,8-DHF ameliorated the lipopolysaccharide induced BDNF-TrkB signaling pathway and its downstream cascade disorders in the mPFC
Furthermore, we tested protein levels of the BDNF-TrkB signaling pathway and its downstream cascades in the mPFC after administration of 7,8-DHF using western blot.
The results are consistent with hippocampal data. The protein levels of p-TrkB, Bcl-2, p-ERK1/2, p-CaMK2, p-CREB, p-GluR1 were increased, and the expression of Bax was reduced in the LPS+7,8-DHF group (Figure 7A, D-H, J-K, M-N, P-Q, S). But the effects of 7,8-DHF were reversed by ANA12 in LPS+7,8-DHF+ANA12 mice. There were no changes in the level of BDNF after using 7,8-DHF or 7,8-DHF+ANA12 in LPS mice (Figure 7A-B). Furthermore, no statistical difference was observed in the expression of TrkB, ERK1/2, CaMK2, CREB and GluR1(Figure 7A, C, H-I, K-L, N-O, Q-R). These results confirm that the disorders of the BDNF-TrkB signaling pathway and its downstream cascades in the mPFC are alleviated using 7,8-DHF in LPS mice, but ANA12 antagonized the protective effects of 7,8-DHF.
7,8-DHF ameliorated lipopolysaccharide induced the BDNF-TrkB signaling pathway and its downstream cascade disorders in the EC
Finally, the expression changes in related proteins of the EC were detected by western blot. The results show that a qualitative increase in p-TrkB, Bcl-2, p-CaMK2, p-CREB expression and reduction in Bax expression was observed in LPS+7,8-DHF mice (Figure 8A, D-G, KC, M-N, P). However, the effects of 7,8-DHF were reversed by ANA12 in LPS+7,8-DHF+ANA12 mice. Consistent with previous results, 7,8-DHF and ANA12 had no effect on the expression of BDNF protein (Figure 8A-B). In addition, there were no significant differences in the expression of TrkB, ERK1/2, p-ERK1/2, CaMK2, CREB, GluR1 and p-GluR1 proteins (Figure 8A, C, H-L, N-O, Q-S). These results signify that 7,8-DHF can effectively alleviate disorders of the BDNF-TrkB signaling pathway, p-CaMK2 and p-CREB cascades in the EC of LPS mice, but ANA12 can antagonize the therapeutical effects of 7,8-DHF.