Necroptosis of hippocampal neurons in paclitaxel chemotherapy-induced cognitive impairment mediates microglial activation via TLR4/MyD88 signaling pathway

Abstract

Background: Paclitaxel (PTX) chemotherapy frequently induces cognitive impairment, which is closely associated with two key pathological processes: necroptosis of hippocampal neurons and microglial polarization. Necroptotic neurons release damage-associated molecular patterns, triggering inflammatory responses. As the primary immune cells in the central nervous system, microglia can exhibit either pro-inflammatory or anti-inflammatory activity depending on their polarization state. However, the relationship between PTX-induced neuronal necroptosis and microglial activation remains unclear.

Methods: In this study, both in vivo and in vitro experiments were conducted. In vivo, an adult male C57BL/6N mouse model of PTX-induced cognitive impairment was established and divided into three groups: Veh (vehicle control), PTX (paclitaxel only), and P + N (paclitaxel with Nec-1 treatment). Necrostatin-1 (Nec-1), a specific inhibitor of RIPK1, was used to inhibit necroptosis. In vitro, HT22 cells were used to prepare necroptosis-conditioned medium, and BV-2 cells were treated with this medium. TAK-242, a TLR4 inhibitor, was used to explore the role of the TLR4/MyD88 signaling pathway. Immunofluorescence staining, western blot, and ELISA were employed to detect relevant markers and cytokines.

Results: The results demonstrated that PTX-induced necroptosis of hippocampal neurons activated microglia. Nec-1 effectively suppressed neuronal necroptosis and reduced M1 polarization of microglia. The TLR4/MyD88 signaling pathway was involved in microglial polarization induced by the necroptotic-conditioned medium of PTX-treated HT22 cells. TAK-242 significantly blocked the regulatory effect of PTX-induced neuronal necroptosis on BV-2 microglial polarization.

Conclusion: This study reveals that hippocampal neuron necroptosis activates microglia through the TLR4/MyD88 signaling pathway in PTX-induced cognitive impairment, promoting M1 polarization and neuroinflammation. Inhibiting necroptosis promotes M2 polarization and neuroprotection. These findings uncover a novel mechanism of PTX-induced cognitive impairment and suggest potential therapeutic targets.

Keywords: DAMPs; TLR4/MyD88 signaling pathway; microglia polarization; necroptosis; paclitaxel.

Figure 1

Workflow chart of PTX-induced cognitive impairment mice model construction and group treatment.

Figure 2

Necroptosis of hippocampal neurons induces microglial polarization toward M1 and inhibits its polarization toward M2. (a) Microphotographs of fluorescent immunohistochemical staining showing labeling of Iba-1 (red). Bar = 50 µm, n = 3 mice/group. (b) Number of microglial cells. Results were expressed as the Mean ± SEM, n = 3 mice/group, ***P < 0.001, ****P < 0.0001. Abbreviation: Veh, vehicle; PTX, paclitaxel; P + N, paclitaxel + necrostatin-1. (c) Microphotographs of fluorescent immunohistochemical staining showing double labeling of iNOS (green) and Iba-1 (red). Bar = 50 µm. (d) Number of microglial cells and iNOS co-labeled cells as a percentage of the number of microglial cells. Results were expressed as the Mean ± SEM, n = 3 mice/group. (e) Microphotographs of fluorescent immunohistochemical staining showing double labeling of Arg-1 (green) and Iba-1 (red). Bar = 50 µm. (f) Number of microglial cells and Arg-1 co-labeled cells as a percentage of the number of microglial cells. Results were expressed as the Mean ± SEM, n = 3 mice/group. (g)–(i) Western blot expression of iNOS and Arg-1 in the hippocampus of mice in different groups. (j) and (k) Effects on the levels of inflammatory molecules TNF-α and IL-1β secreted by M1 microglia in the hippocampus of mice in different groups. All data are shown as the Mean ± SEM, n = 3 for each group. (l) and (m) Effects on the levels of anti-inflammatory molecules IL-4 and IL-10 secreted by M2 microglia in the hippocampus of mice in different groups. All data are shown as the Mean ± SEM, n = 3 for each group.

Figure 3

Necroptosis of hippocampal neurons leads to decreased BDNF release from microglia and activation of TLR4/MyD88 signaling pathway. (a) Microphotographs of fluorescent immunohistochemical staining showing double labeling of BDNF (green) and Iba-1 (red). Bar = 50 µm. (b) Number of microglial cells and BDNF co-labeled cells as a percentage of the number of microglial cells. Results were expressed as the Mean ± SEM, n = 3 mice/group, **P < 0.01, ****P < 0.0001. Abbreviation: Veh, vehicle; PTX, paclitaxel; P + N, paclitaxel + necrostatin-1. (c) Microphotographs of fluorescent immunohistochemical staining showing double labeling of TLR4 (green) and Iba-1 (red). Bar = 50 µm. (d) Number of microglial cells and TLR4 co-labeled cells as a percentage of the number of microglial cells. Results were expressed as the Mean ± SEM, n = 3 mice/group. (e) Microphotographs of fluorescent immunohistochemical staining showing double labeling of MyD88 (green) and Iba-1 (red). Bar = 50 µm. (f) Number of microglial cells and MyD88 co-labeled cells as a percentage of the number of microglial cells. Results were expressed as the Mean ± SEM, n = 3 mice/group. (g)–(j) Western blot expression of BDNF, TLR4, and MyD88 in the hippocampus of mice in different groups. All data are shown as the Mean ± SEM, n = 3 for each group.

Figure 4

Preparation of DAMPs in the conditioned medium for PTX-induced necroptosis of HT22 cells and the conditioned medium for PTX-induced necroptosis of HT22 cells can induce BV-2 cells to polarize toward M1. (a) Preparation of PTX-induced necroptosis-conditioned culture medium of HT22 cells. (b) and (c) Effects of necroptosis-conditioned culture medium of different groups of HT22 cells on iNOS expression in BV-2 cells. (d) Effects of necroptosis-conditioned culture medium of different groups of HT22 cells on TNF-α release of BV-2 cells. (e) Effects of necroptosis-conditioned culture medium of different groups of HT22 cells on IL-1β release of BV-2 cells. All data are shown as the Mean ± SEM, n = 3 mice/group, **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviation: Veh, vehicle; PTX, paclitaxel; P + N, paclitaxel + necrostatin-1.

Figure 5

Polarization of microglia induced by conditioned medium of HT22 cells necroptosis is mediated by TLR4/MyD88 signaling pathway. (a) and (b) Effect of TAK-242 on MyD88 protein expression in BV-2 cells caused by necroptosis of HT22 cells in conditioned culture medium. All data are shown as the Mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Abbreviation: Veh, vehicle; PTX, paclitaxel; P + N, paclitaxel + necrostatin-1. (c) Morphological changes of BV-2 cells in different treatment groups. Bar = 100 µm. (d)–(f) Western blot expression of iNOS and Arg-1 in the BV-2 cells in different groups. (g)–(h) Effects on the levels of inflammatory molecules TNF-α and IL-1β secreted by M1 microglia in the BV-2 cells in different groups. (i) and (j) Effects on the levels of anti-inflammatory molecules IL-4 and IL-10 secreted by M2 microglia in the BV-2 cells in different groups. All data are shown as the Mean ± SEM, n = 3 for each group.