1. Effects Of Notch Pathway Inhibitor Dapt On Paclitaxel-induced Neuropathic Pain.
The rats received nab-paclitaxel at days 1, 3, 5, and 7. The mechanical allodynia and heat hypersensitivity were tested at 0 day before nab-paclitaxel administration, 8 days (d8) and 15 days (d15) after nab-paclitaxel first administration, respectively. Compared to the control group, the PWT and TWL were significantly decreased in the PTX group at d8 and d15. Compared to the PTX group at d8 and d15, PWT and TWT were significantly increased in PTX + DAPT group (Fig. 2). The results revealed that nab-paclitaxel could efficiently induce the mechanical and heat hypersensitivity and notch pathway inhibitor DAPT could alleviate mechanical and heat hypersensitivity induced by nab-paclitaxel.
2. Dynamic Expression Of Proteins Related To The Notch Signalling Pathway After After Nab-paclitaxel Administration.
Western blotting analysis was performed to examine the expression of proteins related to the notch signalling pathway, including NICD, JAG1 and Hes1 at different time points. The results showed that compared to the control group, the expression of these proteins was significantly upregulated in the spinal cord on d8, d15, d21 after nab-paclitaxel first administration. Compared to the PTX d15 group, the level of these proteins was significantly lower than that in PTX d8 group and PTX d21 group (Fig. 3). Considering the tendency of change of the expression of proteins related to the notch signalling pathway, PTX d15 group (PTX group) was chosen to conduct the further studies.
3. Role of notch pathway in pathological changes and microtubule histomorphology in the rat spinal cord after nab-paclitaxel administration.
Histopathological features were examined to evaluate the pathological changes of rat spinal cord by H&E staining and the microtubule histomorphology by TEM. In the H&E staining sections, no abnormalities were observed in the control group. In the PTX group, H&E staining revealed neuronal pyknosis, torsion, and cell body deformation in the spinal cord of rats. Compared to the PTX group, the pathological changes including neuronal pyknosis, torsion, and cell body deformation were reduced in the PTX + DAPT group. The structure of microtubule was found to be consecutive, integrated, and compact in the control group. In the PTX group, the microtubules were fractured, discontinuous and displayed conspicuous free ends. Compared to the PTX group, the number, integrity, and compactness of microtubules were reserved (Fig. 4).
4. Inhibition Of Notch Pathway Relieved The Axonal Injury Induced By Nab-paclitaxel.
NF-L, NF-M, and NF-H were considered as markers of axonal injury. To investigate the role played by notch pathway in axonal injury after nab-paclitaxel administration, the expression of NF-L, NF-M and NF-H was assessed by immunostaining in rat spinal cord. NF-L, NF-M, and NF-H are rarely detected in the control group. In contrast, compared to expression in the control group, the expression of NF-L, NF-M and NF-H was increased in the PTX group. Compared to expression in the PTX group, DAPT decreased the expression of NF-L, NF-M and NF-H in PTX + DAPT group (Fig. 5).
5. Notch Pathway Inhibition Significantly Ameliorated Apoptosis After Nab-paclitaxel Administration.
Apoptosis was detected by TUNEL assay in the spinal cord of rats. Few TUNEL-positive cells were detected in the control group. Compared to the control group, TUNEL-positive cells were evident in the PTX group. Compared to the PTX group, the number of apoptotic cells was significantly decreased in the PTX + DAPT group (Fig. 6).
6. Role Of Notch Pathway In Glial Responses After Nab-paclitaxel Administration.
Compared to the control group, the IHS of GFAP and Iba-1, assessed by the number and staining intensity of Iba-1-positive microglial cells and GFAP-positive astrocytes, was significantly increased in the PTX group. Compared to the PTX group, staining and numbers of Iba-1-positive microglia and GFAP-positive astrocytes were significantly decreased in the PTX + DAPT group (Fig. 7).
7. Inhibition Of Notch Pathway Promoted Neural Regeneration After Nab-paclitaxel Administration.
Western blotting analysis was performed to measure the expression levels of GAP-43 and MAP-2, which are molecular indicators of axonal plasticity in spinal cord after nab-paclitaxel administration. The results showed that compared to the control group, the expression levels of GAP-43 and MAP-2 were slightly higher in the PTX group. Compared to the PTX group, the expression levels of GAP-43 and MAP-2 were significantly increased in the PTX + DAPT group (Fig. 8).
8. Dapt Significantly Downregulated The Expression Of Notch Signalling Pathway Related Molecules.
Western blotting analysis was performed to measure the expression of several Notch1 signaling components after nab-paclitaxel administration, including NICD, Hes1 (a downstream effector of Notch), and jagged1 (JAG1, a ligand of Notch). The results showed that compared to the PTX group, notch inhibition by DAPT significantly downregulated the expression of NICD, Hes1 and JAG1 (Fig. 9).
9. The Neuroprotective Effects Of Dapt Was Related To Hmgb1/tlr4 Pathway And Downstream Inflammatory Cytokines.
In order to determine the downstream targets of notch pathway after after nab-paclitaxel administration, the expression of analyses of HMGB1 and TLR4 immunostaining were performed in rat spinal cord. HMGB1 and TLR4-positive cells were rarely found in control group. Compared to control group, the expression of HMGB1 and TLR4 was significantly increased in the PTX group. Compared to the PTX group, the expression of HMGB1 and TLR4 was decreased in PTX + DAPT group (Fig. 10). Furthermore, the levels of proinflammatory factors, such as TNF-𝛼, IL-1𝛽 and anti-inflammatory factors, such as IL-4, IL-10 in the spinal cord of rats after nab-paclitaxel administration were determined by ELISA. The results showed that the levels of inflammatory factors (TNF-𝛼, IL-1𝛽, IL-4 and IL-10) were increased in the PTX group compared with control group. Compared to PTX group, the levels of proinflammatory factors were decreased and the levels of anti-inflammatory factors were further increased in the PTX + DAPT group (Fig. 10).