Desensitization of TRPV1 Involved in the Antipruritic Effect of Osthole on Histamine-Induced Scratching Behavior in Mice

Abstract

Osthole has been isolated from the fruits of Cnidium monnieri (L.) Cusson, which has been used in Chinese traditional medicine to treat pruritic disorders for a long time. However, the antipruritic mechanism of osthole is not fully understood. In the present study, using calcium imaging, molecular docking, and animal scratching behavior, we analyzed the pharmacological effects of osthole on transient receptor potential vanilloid 1 (TRPV1). The results showed that osthole significantly induced calcium influx in a dose-dependent manner in dorsal root ganglion (DRG) neurons. Osthole-induced calcium influx was inhibited by AMG9810, an antagonist of TRPV1. Osthole and the TRPV1 agonist capsaicin-induced calcium influx were desensitized by pretreatment with osthole. Furthermore, molecular docking results showed that osthole could bind to TRPV1 with a hydrogen bond by anchoring to the amino acid residue ARG557 in the binding pocket of TRPV1. In addition, TRPV1 is a downstream ion channel for the histamine H1 and H4 receptors to transmit itch signals. Osthole attenuated scratching behavior induced by histamine, HTMT (histamine H1 receptor agonist), and VUF8430 (histamine H4 receptor agonist) in mice. These results suggest that osthole inhibition of histamine-dependent itch may be due to the activation and subsequent desensitization of TRPV1 in DRG neurons.

Figure 1

Chemical structure of osthole. (a) Two-dimensional structure of osthole. (b) Three-dimensional structure of osthole is displayed by the stick model.

Figure 2

Osthole increased intracellular calcium in DRG neurons. (a) Representative fluorescence images of osthole-induced changes in the intracellular calcium concentration. control (A) and osthole (B) (scale bar: 20 μm). (b) The representative trace showed that osthole repeatedly induced calcium influx in the same DRG neuron. (c) Fluorescence intensity of osthole induced is 0.34 ± 0.03 (first treated), 0.22 ± 0.05 (second treated), and 0.15 ± 0.05 (third treated) (cells = 114 (positive)/1202 (total), n = 3 animals). The mean fluorescence intensity was desensitization by the third application of osthole in the same neurons (^∗∗P < 0.01, one-way ANOVA followed by Dunnett's test versus Ost 1st). Ost, osthole.

Figure 3

Osthole activated DRG neurons in dose-dependent manner. (a) Representative fluorescence traces of 10 μM osthole-induced changes in DRG neurons. (b) Representative traces of DRG neurons responded to 30 μM osthole. (c) Representative traces of DRG neurons responded to 100 μM osthole. (d) The mean fluorescence intensity of osthole at different doses induced significant intracellular calcium increase. Fluorescence intensity of osthole at 10 μM is 0.29 ± 0.03 (cells = 161 (positive)/1785 (total), n = 3 animals), at 30 μM is 0.43 ± 0.04 (cells = 389 (positive)/2169 (total), n = 3 animals), and at 100 μM is 0.62 ± 0.09 (cells = 451 (positive)/1540 (total), n = 3 animals) (^∗P < 0.05, t-test, 10 μM osthole versus 30 μM osthole; 30 μM osthole versus 100 μM osthole).

Figure 4

Molecular docking of osthole combined with TRPV1. (a) The docking site based on the structure of TRPV1 protein active site. The green band represents TRPV1, and the yellow stick means the key residue of the active site. The overlapping display of 48 docking conformations of osthole on the activated site of TRPV1 is shown. The blue represents the docking conformation. (b) Combination model of osthole with TRPV1. Left: the surface of TRPV1, and the yellow stick represents osthole. Right: the gray band represents TRPV1, and the yellow stick represents osthole. (c–e) Chemical bond of osthole combined with TRPV1. (c) The hydrophobic interaction of osthole and TRPV1. Orange represents highly hydrophobic area, blue represents highly hydrophilic area, and the yellow ball-stick represents osthole. (d) The two-dimension schematic representation of the interaction between osthole and residues of TRPV1 protein's activated site. (e) The three-dimensional interaction structure of osthole and TRPV1.

Figure 5

Osthole induced calcium influx through TRPV1 in DRG neurons. (a) Representative images in cultured DRG neurons at normal (aA) and after treatment with osthole (30 μM) (aB), pretreatment with AMG9810 (1 μM) (AMG9810 + osthole) (aC). Kcl (aD) was used as the positive control (scale bar: 20 μm). (b) Representative traces showed the neurons activated by osthole were inhibited by pretreatment of TRPV1 antagonist AMG9810. (c) The mean fluorescence intensity of osthole positive neurons were restrained by pretreatment of AMG9810 (0.45 ± 0.06 (n = 262) vs 0.10 ± 0.04 (n = 146), total cells = 1654, ^∗∗P < 0.01, n = 3 animals). (d) The percentage of osthole positive neurons was inhibited by pretreatment of AMG9810 (17.36 ± 3.04% vs 9.49 ± 3.61%, ^∗P < 0.05, total cells = 1654, n = 3 animals).

Figure 6

Osthole desensitized capsaicin-induced calcium influx. (a, b) Representative traces showed the effect of pretreatment with 10 μM and 30 μM osthole on capsaicin-induced calcium influx. (c) Representative traces showed the effect of pretreatment capsaicin on secondary capsaicin-induced calcium influx. (d) The mean amplitudes of capsaicin-induced calcium influx were significantly different with pretreatment of osthole or capsaicin. Fluorescence intensity of capsaicin treated alone is 0.72 ± 0.04 and treated secondly is 0.26 ± 0.06 (cells = 161 (positive)/1785 (total), n = 3 animals). Fluorescence intensities of capsaicin after treatment with 10 μM and 30 μM osthole are 0.69 ± 0.05 (cells = 106 (positive)/1650 (total), n = 3 animals) and 0.57 ± 0.03 (cells = 102 (positive)/1360 (total), n = 3 animals). Cap, capsaicin (^∗P < 0.05, ^∗∗∗P < 0.001, one-way ANOVA followed by Dunnett's test versus capsaicin treated alone).

Figure 7

Osthole attenuated the scratching behavior induced by histamine, HTMT, and VUF8430. Histamine, HTMT, and VUF8430 induced scratch behavioral with or without pretreatment with osthole. Time course of itch-related behaviors during 30 minutes (a, c, e) and total scratch bouts (b, d, f) is shown. Osthole significantly inhibited the scratching behavior induced by histamine in mice in 30 minutes (n = 8) (a, b). Similar to histamine, HTMT-induced obvious scratching behaviors were significantly attenuated by osthole (n = 7) (c, d). Osthole also inhibited the VUF8430-induced scratching behavior in mice (n = 6) (e, f) (^∗P < 0.05, pair t-test).