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8-O-Acetyl Shanzhiside Methylester From Lamiophlomis Rotata Reduces Neuropathic Pain by Inhibiting the ERK/TNF-α Pathway in Spinal Astrocytes

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8-O-Acetyl Shanzhiside Methylester From Lamiophlomis Rotata Reduces Neuropathic Pain by Inhibiting the ERK/TNF-α Pathway in Spinal Astrocytes

Wei Zhang et al. Front Cell Neurosci.

Abstract

Lamiophlomis rotata (L. rotata; Benth.) Kudo is an effective traditional herb in the clinical treatment of chronic pain syndromes in China. 8-O-acetyl shanzhiside methylester (8-OaS), a chief component in L. rotata, possesses potent immunosuppressive activities and favorable analgesic effects. This study was proposed to compare the analgesic effects of 8-OaS with those of lidocaine and ketamine in a spinal nerve ligation (SNL) model by behavioral tests, and then investigated its effects upon the expression of spinal glial fibrillary acidic protein (GFAP), phosphorylated extracellular regulated protein kinases (pERK) and tumor necrosis factor-alpha (TNF-α) via immunofluorescence staining and western blot analyses. The data showed consecutive intrathecal injection of 8-OaS for 2 weeks brought about remarkable palliation of neuropathic pain (NP), possessing similar anti-allodynia effects with those of lidocaine and ketamine. Two weeks after surgery, pERK within the spinal dorsal horn was mainly expressed in astrocytes more than neurons and microglia, and 8-OaS inhibited spinal astrocytic activation and TNF-α expression. Finally, co-treatment of 8-OaS and PD98059 (an Extracellular signal-regulated kinase, ERK inhibitor) did not lead to remarkable increase in pain relief or TNF-α expression comparing to rats treated with 8-OaS or PD98059 alone. In conclusion, the anti-nociceptive effects of 8-OaS in the condition of NP relied on the inhibition of SNL-induced astrocyte activation, probably via the down-regulation of the ERK/TNF-α pathway.

Keywords: 8-O-acetyl shanzhiside methylester; ERK; SNL; astrocytes; neuropathic pain; spinal dorsal horn.

Figures

Figure 1
Figure 1
Effects of consecutive intrathecal administration of 8-O-acetyl shanzhiside methylester (8-OaS) on spinal nerve ligation (SNL)-induced mechanical hypersensitivity. The time course of analgesia by different doses of 8-OaS (A). The area under the time-course curves (AUCs) of the analgesic effects for different doses of 8-OaS (B). The dose effect or log (dose) effect curves for the analgesic effects of 8-OaS are shown in (C,D), respectively. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with the SNL-saline group. n = 8 in each group.
Figure 2
Figure 2
The analgesic effects of lidocaine, ketamine and 8-OaS in the SNL model. Treatments with lidocaine, ketamine and 8-OaS for consecutive 7 days decreased SNL-induced mechanical hypersensitivity, **P < 0.01 and ***P < 0.001, SNL-lidocaine vs. SNL-saline group; &P < 0.05, &&P < 0.01 and &&&P < 0.001, SNL-ketamine vs. SNL-saline group; ##P < 0.01 and ###P < 0.001, SNL-8-OaS vs. SNL-saline group. n = 6 in each group.
Figure 3
Figure 3
The up-regulation of phosphorylated extracellular regulated protein kinases 1/2 (pERK1/2) within the spinal dorsal horn after SNL. SNL induced long-lasting ipsilateral mechanical hypersensitivity instead of contralateral hypersensitivity (A). ***P < 0.001 compared with sham group. n = 6 in each group. Western blot analyses showed that SNL enhanced the level of spinal pERK1/2 from post-operation days (POD) 3 to POD 15 (B,D). **P < 0.01 compared with sham group. The pERK1/2 in the SNL model was primarily expressed in the ipsilateral spinal dorsal horn instead of the contralateral part on POD 15 (C,E). **P < 0.01 compared with the contralateral side. Duplication was introduced in the western blot analyses for accuracy judgment. n = 4 in each group.
Figure 4
Figure 4
pERK was primarily expressed within spinal astrocytes in the SNL model on POD 15. Microphotographs indicating double-immunofluorescence histochemistry for pERK (red) and glial fibrillary acidic protein (GFAP; A1–A6) or NeuN (B1–B6) or Iba-1 (C1–C6; green) immunoreactivities within ipsilateral spinal dorsal horn in the SNL model on POD15. The framed areas in images (A1–A3,B1–B3,C1–C3) were magnified in images (A4–A6, B4–B6, C4–C6), respectively. White arrows showed double-labeled neurons in each set pictures. Bars = 100 μm in images (A1–A3,B1–B3,C1–C3) and 40 μm in images (A4–A6,B4–B6,C4–C6).
Figure 5
Figure 5
The effects of 8-OaS administration on SNL-induced spinal GFAP and pERK upregulation. Western blot analysis showed that GFAP (A,C) and pERK (B,D) expression increased in the ipsilateral spinal dorsal horn of SNL rats at POD 15. 8-OaS reduced the expression of GFAP in the SNL model (A,C). 8-OaS also suppressed SNL-induced pERK1 but not pERK2 (B,D) expression. *P < 0.05 compared with saline-SNL group. n = 4 for each group.
Figure 6
Figure 6
8-OaS inhibited SNL-induced spinal astrocytic ERK activation. SNL induced a marked increase in astrocytic activation and ERK activation in the ipsilateral spinal dorsal horn on POD 15, as indicated by the up-regulation of GFAP staining (A,G) and pERK staining (B,H) respectively. The GFAP/pERK double-labeled neurons (C,I) were also increased after SNL. 8-OaS decreased the density of GFAP (G,J) and pERK (H,K) staining as well as the number of GFAP and pERK double-labeled neurons (I,L) in the ipsilateral spinal dorsal horn after SNL. The framed area in images (A–L) was magnified in images (A′–L′), respectively. White arrows showed double-labeled neurons in each set of pictures. Bars = 100 μm in images (A1–L1) and 40 μm in images (A2–L2).
Figure 7
Figure 7
8-OaS suppressed SNL-induced spinal tumor necrosis factor-alpha (TNF-α) expression via inhibiting ERK activation. Both administration of 8-OaS and PD98059 increased mechanical paw withdrawal threshold (PWT) of the SNL model. But co-treatment of 8-OaS and PD98059 did not result in further significant increases in PWT compared to the 8-OaS group and the PD98059 group (A). *P < 0.05, **P < 0.01, SNL-PD98095 group compared with SNL-saline group. #P < 0.05, ##P < 0.01, SNL-8-OaS group compared with SNL-saline group. &P < 0.05, &&P < 0.01, SNL-8-OaS+PD98095 group compared with SNL-saline group. SNL surgery increased the expression of TNF-α within the ipsilateral spinal dorsal horn from POD 3 to 15 (B,D). *P < 0.05, **P < 0.01, compared with the sham group. n = 4 in each group. The expression of TNF-α were inhibited in 8-OaS and PD98059 treated groups but co-treatment of 8-OaS and PD98059 did not lead to further inhibition of TNF-α (C,E). *P < 0.05, compared with the SNL-saline group. n = 4 in each group. Duplication in the western blot analyses was introduced for accuracy judgment.

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