Catalpol ameliorates CFA-induced inflammatory pain by targeting spinal cord and peripheral inflammation

Abstract

Chronic, inflammatory pain is an international health concern that severely diminishes individuals' quality of life. Catalpol is an iridoid glycoside derived from the roots of Rehmannia glutinosa that possesses anti-inflammatory, antioxidant, and neuroprotective properties for the treating multiple kinds of disorders. Nevertheless, catalpol's impacts on inflammatory pain and its potential methods of action are still unclear. The purpose of this investigation is to determine the mechanism of catalpol to reduce the inflammatory pain behaviors in a rat model with complete Freund's adjuvant (CFA). Catwalk, Von-Frey, and open field testing were performed for behavioral assessment. Western blot analysis and real-time quantitative PCR (RT-PCR) were employed to identify variations in molecular expression, while immunofluorescence was utilized to identify cellular localization. Catalpol effectively reduced CFA-induced mechanical allodynia and thermal hyperalgesia when injected intrathecally. Moreover, catalpol can regulate the HDAC4/PPAR-γ-signaling pathway in CFA rat spinal cord neurons. Meanwhile catalpol significantly decreased the expression of the NF-κB/NLRP3 inflammatory axis in the spinal cord of CFA rats. In addition, both in vivo and in vitro research revealed that catalpol treatment inhibited astrocyte activation and increase inflammatory factor expression. Interestingly, we also found that catalpol could alleviate peripheral pain by inhibiting tissue inflammation. Taken together, the findings declared that catalpol may inhibit inflammatory pain in CFA rats by targeting spinal cord and peripheral inflammation.

Keywords: HDAC4/PPAR-γ -signaling pathway; NF-κB/NLRP3 inflammatory axis; astrocyte activation; catalpol; inflammatory pain; peripheral pain.

FIGURE 1

Catalpol could alleviate CFA-induced inflammatory pain in rats. (A). Timeline for single treatment of catalpol. (B,C). Effect of signal treatment of catalpol (50, 100, 200 μg, i t.) on PWT in response to Von Frey filament stimulation and PWL in response to thermal stimulation. PWT: Paw withdrawal threshold; PWL: Paw withdrawal latency. (D). Timeline for repeated injections of catalpol. (E,F). Effect of repeated injections of catalpol (50, 100, 200 μg, i. t.) on PWT and PWL. (G). Timeline for pre-administered treatment of catalpol. (H,I). Effect of pre-administered treatment of catalpol (50, 100, 200 μg, i. t.) on PWT and PWL. (*p < 0.05, **p < 0.01, and ***p < 0.001 vs. the CFA group; n = 6, two-way repeated measures ANOVA). (J–L). Representative traces of locomotor activity in the Open field. (M). Distance traveled in the central area in the Open field. (N). Time spent in the central area in the Open field. (∗∗∗p < 0.001 vs. CFA group; n = 8, one-way ANOVA).

FIGURE 2

Effects of catalpol on Cat Walk gait parameters in CFA rats. (O). Representative CATWALK gait analysis results, including Print view, Timing view and Print intensity. Changes of gait parameters including Print Area (P), Mean intensity (Q), Swing (R), and Swing Speed (S) in Sham, CFA, and CFA + CAT group. (∗p < 0.05, **p < 0.01 and ∗∗∗p < 0.001 vs. the CFA group; n = 8, one-way ANOVA). All data were calculated as left hind paw/right hind paw (LH/RH) formula to eliminate the effect of confounding factors. LH: left hind paw; RH: right hind paw; N.S. not statistically significant.

FIGURE 3

The endogenous expression and cellular localization of p-HDAC4 and PPAR-γ in the spinal cord of CFA rats. (A–C). The protein expressions of p-HDAC4 and PPAR-γ in the spinal cord of rats were detected by Western blotting. (D,E). The colocalization of p-HDAC4 and PPAR-γ with NeuN, iba1, and GFAP were detected by a double-label immunofluorescence assay in CFA rats. The white arrows indicate colocalization of p-HDAC4 and PPAR-γ with NeuN. Scale bar = 50 μm.

FIGURE 4

Effect of catalpol on HDAC4/PPAR-γ-signaling pathway in spinal cord of CFA rats. (F,G). Effect of intrathecal injection of HDAC4 siRNA (5 μg) on PWT and PWL. (**p < 0.01, and ***p < 0.001 vs. CFA group; n = 6, two-way repeated measures ANOVA). (H). The relative protein expression of p-HDAC4. (∗p < 0.05 vs. the CFA group; n = 4, one-way ANOVA, N.S: not statistically significant). (I,J). Effect of GW9662 administration on PWT and PWL. (*p < 0.05, **p < 0.01, and ***p < 0.001 vs. the CFA + si-HDAC4 group; n = 6, two-way repeated measures ANOVA). (K). The relative protein expression of PPAR-γ. (**p < 0.01, vs. the CFA + si-HDAC4 group; n = 4, one-way ANOVA). (L). The co-expression of p-HDAC4 and PPAR-γ in the spinal cord of rats was detected by a double-label immunofluorescence assay. The white arrows indicate colocalization of p-HDAC4 with PPAR-γ. Scale bar = 50 μm. (M-O). The relative protein expression of p-HDAC4 and PPAR-γ. (**p < 0.01, vs. the CFA group; n = 4, one-way ANOVA). (P-R). The fluorescence intensity of p-HDAC4 and PPAR-γ. (*p < 0.05, **p < 0.01, vs. the CFA group; n = 4, one-way ANOVA). Scale bar = 50 μm.

FIGURE 5

Effect of Catalpol on expression of NF-κB/NLRP3-inflammatory axis in the spinal cord of CFA rats. (A–C). The protein expressions of p-NF-κB and NLRP3 in the spinal cord of rats were detected by Western blotting. (*p < 0.05, **p < 0.01, vs. the CFA group; n = 4, one-way ANOVA). (D,E). The fluorescence intensity of NLRP3. (F,G). The fluorescence intensity of p-NF-κB. (*p < 0.05, **p < 0.01, vs. the CFA group; n = 4, one-way ANOVA). Scale bar = 50 μm.

FIGURE 6

Catalpol treatment inhibits the activation of astrocytes and the release of inflammatory factors in vivo and in vitro. (A,B). The protein expression of GFAP in the spinal cord of rats were detected by Western blotting. (C,D). The fluorescence intensity of GFAP in the spinal cord. (*p < 0.05 vs. the CFA group; n = 4, one-way ANOVA). Scale bar = 50 μm. The relative protein expression of iNOS (E), IL-1β (F), and TNF-a (G) in the spinal cord. (*p < 0.05, **p < 0.01, ***p < 0.001, vs. the CFA group; n = 4, one-way ANOVA). (H). Cell viability was measured using CCK8 assay. (***p < 0.001 vs. control; one-way ANOVA). (I). The protein expression of GFAP in primary astrocytes. (***p < 0.001, vs. LPS (1 μg/ml) group; one-way ANOVA). (J). Immunofluorescence results of GFAP in primary astrocytes. Scale bar = 50 μm. (K). The protein expression of iNOS in primary astrocytes. (L,M). The mRNA expression of IL-1β and TNF-α in primary astrocytes. (*p < 0.05, **p < 0.01, ***p < 0.001, vs. LPS (1 μg/ml) group; one-way ANOVA).

FIGURE 7

Catalpol reduces peripheral pain by inhibiting tissue inflammation. (A). Timeline for single subcutaneous injections of catalpol. (B,C). Effect of single subcutaneous injections of Catalpol (2.5 mg/kg and 10 mg/kg) on PWT and PWL. (***p < 0.001 vs. the CFA group; n = 6, two-way repeated measures ANOVA). (D,E). H&E staining of paw skin in different groups. (∗∗∗p < 0.001 vs. the CFA group; n = 4, one-way ANOVA). Scale bar = 50 μm. (F–H). The protein expression of COX-2, IL-1β and TNF-a in the paw skin of rats were detected by Western blotting. (**p < 0.01 and ∗∗∗p < 0.001 vs. the CFA group; n = 4, one-way ANOVA).