Journal of Traditional Chinese Medicine ›› 2023, Vol. 43 ›› Issue (5): 887-896.DOI: 10.19852/j.cnki.jtcm.20230802.003
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Zhenxin Anshen formula (镇心安神方) ameliorates atopic der-matitis-like skin dysfunction in mice and in vitro via regulation of transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 in Neural pathways
ZHAO Yiding1, YAN Xiaoning1, JIANG Shanshan1, LIU Yong1, DONG Chun1, CHI Huiyan2(
), MAO Chaoyi3()
- 1 Department of Dermatology, Shaanxi Provincial Hospital of traditional Chinese Medicine, Xi’an 710003, China
2 Department of Dermatology, Xiyuan Hospital China Academy of Chinese Medical Sciences, Beijing 100193, China
3 Department of Education Management, China Academy of Chinese Medical Sciences, Beijing 100700, China
-
Received:2022-02-20Accepted:2022-06-15Online:2023-10-15Published:2023-08-02 -
Contact:Dr. CHI Huiyan, Department of Dermatology, Xiyuan Hospital China Academy of Chinese Medical Sciences, Beijing 100193, China.chihuiyan@163.com ; Dr. MAO Chaoyi, Department of Education Management, China Academy of Chinese Medical Sciences, Beijing 100700, China.maochaoyi1204@126.com .Telephone: +86-10-62835136; +86-10-64089751 -
Supported by:National Natural Science Foundation of China: Study on the Mechanism of Zhenxin Anshen Formula Regulating GRPR/MrgprA3/TRPs Signaling Pathway for Atop-ic Dermatitis based on the Theory of All Kinds of Diseases with Pain, Itching, and Sores are Exclusively Related to the Heart(81704087);Regulation of Zhenxin Anshen Formula on Neuroimmune Function in Atopic Dermatitis from STIM1-ORAI1 Mediated Store Oper-ated Calcium Entry(82274537);Scientific and Technological Innovation Project of China Academy of Chinese Medical Sciences: Based on PAR2/TRPV1 Pathway to Regulate Nerve-Epidermal Pruritus Information Transmission to Explore the Mechanism of Longmu Decoction on “Stress-Type Atopic Dermatitis” Mice(CI2021A02315);Belt and Road Initiative Cooperation Project of Traditional Chinese Medicine of the Chinese Academy of Traditional Chinese Medicine: Evaluation of the Effect of Chinese Medicine on Allergic Diseases and Material Basis Research(GH201910)
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ZHAO Yiding, YAN Xiaoning, JIANG Shanshan, LIU Yong, DONG Chun, CHI Huiyan, MAO Chaoyi. Zhenxin Anshen formula (镇心安神方) ameliorates atopic der-matitis-like skin dysfunction in mice and in vitro via regulation of transient receptor potential vanilloid 1 and transient receptor potential ankyrin 1 in Neural pathways[J]. Journal of Traditional Chinese Medicine, 2023, 43(5): 887-896.
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Figure 1 Efficacy of ZXAS in the AD-like mice A: representative images of dorsal skin in each group on day 28. A1: NC; A2: MC; A3: CET; A4: LDZ; A5: MDZ; A6: HDZ; B: dermatitis scores were assigned on days 1, 4, 14, and 28; C: scratching frequency was recorded on days 1, 4, 14, and 28 for 30 min; D: skin sections from each group was stained with HE for morphological changes (× 400). D1: NC; D2: MC; D3: CET; D4: LDZ; D5: MDZ; D6: HDZ; E: skin thickness in each group. E1: epidermal thickness was measured in HE-stained sections using the microscope (μm, n = 9). E2: dermis thickness was measured in HE-stained sections using the microscope (μm, n = 9). Scale bar = 20 μm. Measurements were taken from day 0 until day 28. ZXAS: Zhenxin Anshen formula; AD: atopic dermatitis; NC: normal control; MC: model control; CET: cetirizine group treated with oral doses of 1.3 mg·kg-1·d-1; LDZ: low-dose ZXAS group treated with oral doses of 9.9 g·kg-1·d-1; MDZ: medium-dose ZXAS group treated with oral doses of 18.18 g·kg-1·d-1; HDZ: high-dose ZXAS treated with oral doses of 36.36 g·kg-1·d-1. Data are expressed as mean ± standard deviation (n = 9), aP < 0.01 vs the NC group; bP < 0.01 vs the MC group.
Figure 1 Efficacy of ZXAS in the AD-like mice A: representative images of dorsal skin in each group on day 28. A1: NC; A2: MC; A3: CET; A4: LDZ; A5: MDZ; A6: HDZ; B: dermatitis scores were assigned on days 1, 4, 14, and 28; C: scratching frequency was recorded on days 1, 4, 14, and 28 for 30 min; D: skin sections from each group was stained with HE for morphological changes (× 400). D1: NC; D2: MC; D3: CET; D4: LDZ; D5: MDZ; D6: HDZ; E: skin thickness in each group. E1: epidermal thickness was measured in HE-stained sections using the microscope (μm, n = 9). E2: dermis thickness was measured in HE-stained sections using the microscope (μm, n = 9). Scale bar = 20 μm. Measurements were taken from day 0 until day 28. ZXAS: Zhenxin Anshen formula; AD: atopic dermatitis; NC: normal control; MC: model control; CET: cetirizine group treated with oral doses of 1.3 mg·kg-1·d-1; LDZ: low-dose ZXAS group treated with oral doses of 9.9 g·kg-1·d-1; MDZ: medium-dose ZXAS group treated with oral doses of 18.18 g·kg-1·d-1; HDZ: high-dose ZXAS treated with oral doses of 36.36 g·kg-1·d-1. Data are expressed as mean ± standard deviation (n = 9), aP < 0.01 vs the NC group; bP < 0.01 vs the MC group.
Table 1 Primer sequences
| Gene | Primer | Sequence | Product size (bp) |
|---|---|---|---|
| β-actin | Forward | 5?-CACGATGGAGGGGCCGGACTCATC-3? | 240 |
| Reverse | 5?-TAAAGACCTCTATGCCAACACAGT-3? | ||
| Mus GRPR | Forward | 5?-CTGTGTGAACCCCTTTGCTC-3? | 157 |
| Reverse | 5?-CGAGGGGTTAGTGCTCTTGA-3? | ||
| Mus TRPV1 | Forward | 5?-TCATCACCGTCAGCTCTGTT-3? | 235 |
| Reverse | 5?-TCTGGGTCTTTGAACTCGCT-3? | ||
| Mus TRPA1 | Forward | 5?-CTGAGATCGACCGGAGTGTT-3? | 220 |
| Reverse | 5?-AAGGTCAGGACTGGGTATGC-3? |
Table 1 Primer sequences
| Gene | Primer | Sequence | Product size (bp) |
|---|---|---|---|
| β-actin | Forward | 5?-CACGATGGAGGGGCCGGACTCATC-3? | 240 |
| Reverse | 5?-TAAAGACCTCTATGCCAACACAGT-3? | ||
| Mus GRPR | Forward | 5?-CTGTGTGAACCCCTTTGCTC-3? | 157 |
| Reverse | 5?-CGAGGGGTTAGTGCTCTTGA-3? | ||
| Mus TRPV1 | Forward | 5?-TCATCACCGTCAGCTCTGTT-3? | 235 |
| Reverse | 5?-TCTGGGTCTTTGAACTCGCT-3? | ||
| Mus TRPA1 | Forward | 5?-CTGAGATCGACCGGAGTGTT-3? | 220 |
| Reverse | 5?-AAGGTCAGGACTGGGTATGC-3? |
Figure 2 Effect of ZXAS on type 2 inflammatory cytokines and IgE A-E: ELISA assay was performed to analyse the serum levels of (A) IL-4, (B) IL-5, (C) IL-13, (D) TSLP, and (E) IgE. NC: normal control; MC: model control; CET: cetirizine group treated with oral doses of 1.3 mg·kg-1·d-1; LDZ: low-dose ZXAS group treated with oral doses of 9.9 g·kg-1·d-1; MDZ: medium-dose ZXAS group treated with oral doses of 18.18 g·kg-1·d-1; HDZ: high-dose ZXAS treated with oral doses of 36.36 g·kg-1·d-1. ZXAS: Zhenxin Anshen formula; ELISA: enzyme-linked immunosorbent assay; IL: interleukin; TSLP: thymic stromal lymphopoietin; IgE: Immunoglobulin E. Data are expressed as mean ± standard deviation (n = 9). aP < 0.01 vs the NC group; bP < 0.01, cP < 0.05 vs the MC group.
Figure 2 Effect of ZXAS on type 2 inflammatory cytokines and IgE A-E: ELISA assay was performed to analyse the serum levels of (A) IL-4, (B) IL-5, (C) IL-13, (D) TSLP, and (E) IgE. NC: normal control; MC: model control; CET: cetirizine group treated with oral doses of 1.3 mg·kg-1·d-1; LDZ: low-dose ZXAS group treated with oral doses of 9.9 g·kg-1·d-1; MDZ: medium-dose ZXAS group treated with oral doses of 18.18 g·kg-1·d-1; HDZ: high-dose ZXAS treated with oral doses of 36.36 g·kg-1·d-1. ZXAS: Zhenxin Anshen formula; ELISA: enzyme-linked immunosorbent assay; IL: interleukin; TSLP: thymic stromal lymphopoietin; IgE: Immunoglobulin E. Data are expressed as mean ± standard deviation (n = 9). aP < 0.01 vs the NC group; bP < 0.01, cP < 0.05 vs the MC group.
Figure 3 Inhibitory effects of ZXAS on GRPR, TRPV1, and TRPA1 expression in the spinal cord A-C: Western blot and qRT-PCR were used to analyze the protein and mRNA expression levels of (A) GRPR, (B) TRPV1, and (C) TRPA1 in the spinal cord (n = 6). D-F: immunohistochemistry and optical density analyses was used to detect (D) GRPR-, (E) TRPV1-, and (F) TRPA1-positive area and positive cells (×100 magnification). High magnification was shown as an insert within the black frame (×400) (n = 3). D1, E1, F1: NC; D2, E2, F2: MC; D3, E3, F3: CET; D4, E4, F4: LDZ; D5, E5, F5: MDZ; D6, E6, F6: HDZ. ZXAS: Zhenxin Anshen formula; NC: normal control; MC: model control; CET: cetirizine group treated with oral doses of 1.3 mg·kg-1·d-1; LDZ: low-dose ZXAS group treated with oral doses of 9.9 g·kg-1·d-1; MDZ: medium-dose ZXAS group treated with oral doses of 18.18 g·kg-1·d-1; HDZ: high-dose ZXAS treated with oral doses of 36.36 g·kg-1·d-1. GRPR: gastrin-releasing peptide receptor; TRPV1: transient receptor potential vanilloid 1; TRPA1: transient receptor potential ankyrin 1; qRT-PCR: quantitative real-time polymerase chain reaction. Data are expressed as mean ± standard deviation. aP < 0.01 vs the NC group; bP < 0.05, cP < 0.01 vs the MC group.
Figure 3 Inhibitory effects of ZXAS on GRPR, TRPV1, and TRPA1 expression in the spinal cord A-C: Western blot and qRT-PCR were used to analyze the protein and mRNA expression levels of (A) GRPR, (B) TRPV1, and (C) TRPA1 in the spinal cord (n = 6). D-F: immunohistochemistry and optical density analyses was used to detect (D) GRPR-, (E) TRPV1-, and (F) TRPA1-positive area and positive cells (×100 magnification). High magnification was shown as an insert within the black frame (×400) (n = 3). D1, E1, F1: NC; D2, E2, F2: MC; D3, E3, F3: CET; D4, E4, F4: LDZ; D5, E5, F5: MDZ; D6, E6, F6: HDZ. ZXAS: Zhenxin Anshen formula; NC: normal control; MC: model control; CET: cetirizine group treated with oral doses of 1.3 mg·kg-1·d-1; LDZ: low-dose ZXAS group treated with oral doses of 9.9 g·kg-1·d-1; MDZ: medium-dose ZXAS group treated with oral doses of 18.18 g·kg-1·d-1; HDZ: high-dose ZXAS treated with oral doses of 36.36 g·kg-1·d-1. GRPR: gastrin-releasing peptide receptor; TRPV1: transient receptor potential vanilloid 1; TRPA1: transient receptor potential ankyrin 1; qRT-PCR: quantitative real-time polymerase chain reaction. Data are expressed as mean ± standard deviation. aP < 0.01 vs the NC group; bP < 0.05, cP < 0.01 vs the MC group.
Figure 4 Primary culture of DRG neuron cells and inhibitory effects of ZXAS-medicated serum on TRPV1, TRPA1, PLC expression and concentration of intracytoplasmic Ca2+ in DRG neurons A: the morphology and fluorescence immunocytochemistry of DRG neuron cells. A1: DRG neuron cells were stained with fluorescent markers (×40 magnification). High magnification was shown as an insert within the black frame (×100). A2: β-tubulin was expressed in DRG neuron cell bodies and axon fibers (×400). A3: Nuclei of neurons were stained with DAPI (×400). A4: merged picture from A2 and A3 (×400). B: proliferative capacity of DRG neuron cells in each group was measured using a 450 nm wavelength microplate reader with CCK-8. The relative expression levels of (C) TRPV1 and (D) TRPA1 in DRG neuron cells induced by capsaicin. Their protein levels were examined by Western blotting (E). Detection of (F) PLC and (G) Ca2+ using ELISA and (H) fluo-3AM fluorescence probe. Normal group: DRG neuron cells were pre-treated with 20% blank serum. Capsaicin group: DRG neuron cells were pre-treated with 20% blank serum and stimulated with 1 μM capsaicin. ZXAS group: DRG neuron cells were pre-treated with 10% blank serum and co-treated with 10% ZXAS-medicated serum and 1 μM capsaicin. Capsazepine group: DRG neuron cells were pre-treated with 20% blank serum and co-treated with 1 μM capsazepine and 1 μM capsaicin. ZXAS: Zhenxin Anshen formula; DRG: dorsal root ganglia; DAPI: 4,6-diamino-2-phenyl indole; CCK-8: cell counting kit-8; TRPV1: transient receptor potential vanilloid 1; TRPA1: transient receptor potential ankyrin 1; PLC: phospholipase C; ELISA: enzyme-linked immunosorbent assay. Data are expressed as mean ± standard deviation. aP < 0.05, cP < 0.01 vs the normal group; bP < 0.05, dP < 0.01 vs the capsaicin group.
Figure 4 Primary culture of DRG neuron cells and inhibitory effects of ZXAS-medicated serum on TRPV1, TRPA1, PLC expression and concentration of intracytoplasmic Ca2+ in DRG neurons A: the morphology and fluorescence immunocytochemistry of DRG neuron cells. A1: DRG neuron cells were stained with fluorescent markers (×40 magnification). High magnification was shown as an insert within the black frame (×100). A2: β-tubulin was expressed in DRG neuron cell bodies and axon fibers (×400). A3: Nuclei of neurons were stained with DAPI (×400). A4: merged picture from A2 and A3 (×400). B: proliferative capacity of DRG neuron cells in each group was measured using a 450 nm wavelength microplate reader with CCK-8. The relative expression levels of (C) TRPV1 and (D) TRPA1 in DRG neuron cells induced by capsaicin. Their protein levels were examined by Western blotting (E). Detection of (F) PLC and (G) Ca2+ using ELISA and (H) fluo-3AM fluorescence probe. Normal group: DRG neuron cells were pre-treated with 20% blank serum. Capsaicin group: DRG neuron cells were pre-treated with 20% blank serum and stimulated with 1 μM capsaicin. ZXAS group: DRG neuron cells were pre-treated with 10% blank serum and co-treated with 10% ZXAS-medicated serum and 1 μM capsaicin. Capsazepine group: DRG neuron cells were pre-treated with 20% blank serum and co-treated with 1 μM capsazepine and 1 μM capsaicin. ZXAS: Zhenxin Anshen formula; DRG: dorsal root ganglia; DAPI: 4,6-diamino-2-phenyl indole; CCK-8: cell counting kit-8; TRPV1: transient receptor potential vanilloid 1; TRPA1: transient receptor potential ankyrin 1; PLC: phospholipase C; ELISA: enzyme-linked immunosorbent assay. Data are expressed as mean ± standard deviation. aP < 0.05, cP < 0.01 vs the normal group; bP < 0.05, dP < 0.01 vs the capsaicin group.
| 1. |
Weidinger S, Novak N. Atopic dermatitis. Lancet 2016; 387: 1109-22.
DOI PMID |
| 2. | Drucker AM, Wang AR, Li WQ, Sevetson E, Block JK, Qureshi AA. The burden of atopic dermatitis: summary of a report for the National Eczema Association. J Invest Dermatol 2017; 137: 26-30. |
| 3. |
Nutten S. Atopic dermatitis: global epidemiology and risk factors. Ann Nutr Metab 2015; 66 Suppl 1: 8-16.
DOI URL |
| 4. |
Rønnstad ATM, Halling Overgaard AS, Hamann CR, Skov L, Egeberg A, Thyssen JP. Association of atopic dermatitis with depression, anxiety, and suicidal ideation in children and adults: a systematic review and Meta-analysis. J Am Acad Dermatol 2018; 79: 448-56.
DOI PMID |
| 5. |
Yosipovitch G, Berger T, Fassett MS. Neuroimmune interactions in chronic itch of atopic dermatitis. J Eur Acad Dermatol Venereol 2020; 34: 239-50.
DOI PMID |
| 6. |
Yang TB, Kim BS. Pruritus in allergy and immunology. J Allergy Clin Immunol 2019; 144: 353-60.
DOI URL |
| 7. |
Mollanazar NK, Smith PK, Yosipovitch G. Mediators of chronic pruritus in atopic dermatitis: getting the itch out? Clin Rev Allergy Immunol 2016; 51: 263-92.
DOI URL |
| 8. |
Gouin O, L'Herondelle K, Lebonvallet N, et al. TRPV1 and TRPA1 in cutaneous neurogenic and chronic inflammation: pro-inflammatory response induced by their activation and their sensitization. Protein Cell 2017; 8: 644-61.
DOI PMID |
| 9. | Tang L, Gao J, Cao X, Chen L, Wang H, Ding H. TRPV1 mediates itch-associated scratching and skin barrier dysfunction in DNFB-induced atopic dermatitis mice. Exp Dermatol 2021; 4: 14464. |
| 10. |
Cevikbas F, Wang X, Akiyama T, et al. A sensory neuron-expressed IL-31 receptor mediates T helper cell-dependent itch: Involvement of TRPV1 and TRPA1. J Allergy Clin Immunol 2014; 133: 448-60.
DOI PMID |
| 11. | Højland CR, Andersen HH, Poulsen JN, Arendt-Nielsen L, Gazerani P. A human surrogate model of itch utilizing the TRPA1 agonist trans-cinnamaldehyde. Acta Derm Venereol 2015; 95: 798-803. |
| 12. | Zhao YD, Yao CH, She YY, et al. The impact of tranquilizing method on clinical symptoms of youths and adults with atopic dermatitis. Zhong Guo Pi Fu Xing Bing Xue Za Zhi 2010; 24: 1151-53. |
| 13. |
Yu K, Wang Y, Wan T, et al. Tacrolimus nanoparticles based on chitosan combined with nicotinamide: enhancing percutaneous delivery and treatment efficacy for atopic dermatitis and reducing dose. Int J Nanomedicine 2017; 13: 129-42.
DOI URL |
| 14. | Xu SY. Pharmacological experimental techniques, 3rd ed. Beijing: People's Medical Publishing House, 2002: 179-81. |
| 15. | European Task Force on Atopic Dermatitis. Severity scoring of atopic dermatitis: the SCORAD index. Consensus report of the European task force on atopic dermatitis. Dermatology 1993; 186: 23-31. |
| 16. |
Shimada SG, LaMotte RH. Behavioral differentiation between itch and pain in mouse. Pain 2008; 139: 681-87.
DOI PMID |
| 17. |
Nam JH, Kim WK. The role of TRP channels in allergic inflammation and its clinical relevance. Curr Med Chem 2020; 27: 1446-68.
DOI PMID |
| 18. |
Lucaciu OC, Connell GP. Itch sensation through transient receptor potential channels: a systematic review and relevance to manual therapy. J Manipulative Physiol Ther 2013; 36: 385-93.
DOI URL |
| 19. |
Tóth BI, Szallasi A, Bíró T. Transient receptor potential channels and itch: how deep should we scratch? Handb Exp Pharmacol 2015; 226: 89-133.
DOI PMID |
| 20. |
Chen XJ, Sun YG. Central circuit mechanisms of itch. Nat Commun 2020; 11: 3052.
DOI |
| 21. |
Elmariah SB, Lerner EA. The missing link between itch and inflammation in atopic dermatitis. Cell 2013; 155: 267-69.
DOI PMID |
| 22. |
Indra AK. Epidermal TSLP: a trigger factor for pathogenesis of atopic dermatitis. Expert Rev Proteomics 2013; 10: 309-11.
DOI URL |
| 23. |
Yang TB, Kim BS. Pruritus in allergy and immunology. J Allergy Clin Immunol 2019; 144: 353-60.
DOI URL |
| 24. | Garcovich S, Maurelli M, Gisondi P, Peris K, Yosipovitch G, Girolomoni G. Pruritus as a distinctive feature of type 2 inflammation. Vaccines (Basel) 2021; 9: 303. |
| 25. |
Mack MR, Kim BS. The itch-scratch cycle: a neuroimmune perspective. Trends Immunol 2018; 39: 980-91.
DOI PMID |
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