Tetrahydropalmatine exerts analgesic effects by promoting apoptosis and inhibiting the activation of glial cells in rats with inflammatory pain

Abstract

Background: Pain is an unpleasant sensory experience that usually plays a protective role. Inflammatory pain is often severe and stubborn, which has a great impact on the quality of life of patients. However, there has been no breakthrough in the treatment strategy and mechanism of inflammatory pain.

Methods: This study investigated the analgesic effect of tetrahydropalmatine (THP) in rats injected with complete Freund's adjuvant (CFA)-induced inflammatory pain. Allodynia and gait analysis of rats were used to evaluate the analgesic effect at different time points before and after operation. THP (2.5, 5, and 10 mg/kg) was administered intraperitoneally once daily for 7 days post Day 3. The expression levels of TNF-α and IL-1β in the spinal cord were measured by enzyme-linked immunosorbent assay. The activation of astrocytes and microglial cells in the spinal cord was tested by western blot before and after THP treatment. The apoptosis of glial cells was tested by flow cytometry after treatment with THP in the primary cultured glial cell model.

Results: CFA treatment induced significant allodynia and caused abnormal gait in rats. Administration of THP at 10 mg/kg significantly alleviated CFA-induced inflammatory pain behaviors. Moreover, CFA-induced activation of glial cells and the increased levels of TNF-α and IL-1β were inhibited by THP administration. In addition, THP promotes apoptosis in primary cultured glial cells. This study suggests the possible clinical utility of THP in the treatment of inflammatory pain.

Conclusion: THP plays an analgesic role by inhibiting the activation of glial cells and promoting apoptosis.

Keywords: Tetrahydropalmatine; apoptosis; glial cell; inflammatory pain.

Figure 1.

THP reduces CFA induced mechanical allodynia and heat hyperalgesia. Rats were treated with THP or vehicle (i. p.) once daily for 7 days post Day 3. (a) and(b) Intraperitoneal injection of THP dose dependently alleviated the mechanical allodynia and heat hyperalgesia in CFA induced inflammatory pain rats and this effect was observed at 5/10 mg/kg doses. (c) and (d) Intraperitoneal injection of THP time-dependently alleviated the mechanical allodynia and heat hyperalgesia in CFA induced inflammatory pain rats and this effect was observed at 2 h after THP injection. The results were exhibited as mean ± SD. (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Con + Veh group. #P < 0.05, ## P < 0.01 vs. CFA + Veh group.

Figure 2.

Effect of THP on functional gait-changes in CFA induced inflammatory pain rats. Rats were treated with THP or vehicle (i. p.) once daily for 7 days post CFA injected surgery. (a) Images of rats walking on the CatWalk were showed as above. Comparing these demonstrates increased duration of contact with the surface for the uninjured limb, compared with the injured, while the print representations above (RH, RF, LH, LF) shows the print-area in contact with the plate. Notice that the injured hind paw (LH: Left Hind, arrowhead) has a much smaller print-area, primarily with the heel of the paw. (b) Maximum contact area (cm²); the maximum surface area of a paw that comes into contact with the glass plate. Presented as a ratio between surgery and unsurgery hind paws. (c) Print length (cm); the print length of a paw. Presented as a ratio between surgery and unsurgery hind paws. The results were exhibited as mean ± SD. (n = 5). **P < 0.01, ***P < 0.001 vs. Con group. ###P < 0.001 vs. CFA group.

Figure 3.

THP promotes primary glial cells apoptosis by LPS treatment. Following 6 h treatment with LPS (1 µg/ml)) or THP (100 µM), primary astrocytes and microglia were determined by flow cytometry. (a) and (b) The purity of primary cultured astrocytes and microglia cells prepared from neonatal rat glia by the shaking was approximate 90% (n = 3) by GFAP/Iba-1 and DAPI immunofluorescence staining identification. Scale bar 50 µm. (c) and (d) The percentage of astrocytes and microglia apoptosis significantly increased after THP treatment by flow cytometry. The results were exhibited as mean ± SD. (n = 4). ***P < 0.001 vs. LPS group.

Figure 4.

THP inhibits astrocytes and microglia activation. (a) and (b) Repetitive administration of THP at 10 mg/kg (once daily for 7 days post Day 3) significantly suppressed astrocytes and microglial cells activation in spinal cord tissue of rats. (c) and (d) Treatment of THP at 100 µM significantly suppressed astrocytes and microglial cells activation in primary glial cells. The results were exhibited as mean ± SD. (n = 4). *P < 0.05, **P < 0.01 vs. Con group. #P < 0.05 vs. CFA group.

Figure 5.

THP suppresses CFA induced spinal cord increased levels of cytokine and NF‐κB activation. Seven days after intraplantar injection of CFA (100 μl per paw), the spinal cord was dissected for determination of TNF‐α (a), IL‐1β (b) and NF‐κB activation (c) and (d) by ELISA. NF‐κB activation was observed as a reduction of phosphorylated NF‐κB/NF‐κB ratio. The results were exhibited as mean ± SD. (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001 vs. Con group. #P < 0.05 vs. CFA group.