Journal of Traditional Chinese Medicine ›› 2022, Vol. 42 ›› Issue (5): 749-757.DOI: 10.19852/j.cnki.jtcm.2022.05.008
• Research Articles • Previous Articles Next Articles
Zebrafish facilitates drug screening: potential of 3-deoxy-andrographoside from Chuanxinlian (Herba Andrographitis Paniculatae) as an anti-inflammatory agent
HE Xuemei1,2, XIAO Junjie1,2, FAN Chunlin3, LU Zibin1,2, CAO Huihui1,2, YU Linzhong1,2, ZHENG Yuanru1,2,4(
), LIU Junshan1,2,5()
- 1 Third Level Research Laboratory of State Administration of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
2 Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics, Guangzhou 510515, China
3 Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, China
4 School of Pharmacy, Guangdong Pharmaceutical university, Guangzhou 510006, China
5 Department of Pharmacy, Zhujiang Hospital, Southern Medical University, Guangzhou 510515, China
-
Received:2021-05-23Accepted:2021-08-21Online:2022-10-15Published:2022-09-02 -
Contact:ZHENG Yuanru,LIU Junshan -
About author:Associate Prof. LIU Junshan, Third Level Research Laboratory of State Administration of Traditional Chinese Medicine, School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China. liujunshan@smu.edu.cn;
-
Supported by:National Science Foundation-funding Project: Study of the Attenuating Effects of 3-dehydroandrographolide on Acute Lung Injury through Modulating Alveolar Macrophage Polarization via Targeting α7nAChR(81973544);Guangzhou Education Bureau University Scientific Research Project: Construction of Traditional Chinese Medicine Discipline(201831845);Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme: Study on the Mechanisms of Anti-Tumor and Anti-Inflammatory Components from Traditional Chinese Medicine based on Detoxification Therapy(GDHVPS2018);Young Elite Scientists Sponsorship Program by CACM: Study on the Mechanisms of Antitumor Components from Traditional Chinese Medicine based on Detoxification Therapy(2019-QNRC2-C14)
Cite this article
HE Xuemei, XIAO Junjie, FAN Chunlin, LU Zibin, CAO Huihui, YU Linzhong, ZHENG Yuanru, LIU Junshan. Zebrafish facilitates drug screening: potential of 3-deoxy-andrographoside from Chuanxinlian (Herba Andrographitis Paniculatae) as an anti-inflammatory agent[J]. Journal of Traditional Chinese Medicine, 2022, 42(5): 749-757.
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Table 1 Sequences of the primers in qRT-PCR analysis
| Gene | Primer sequence |
|---|---|
| β-actin | Forward: ATGGATGAGGAAATCGCTG |
| Reverse: ATGCCAACCATCACTCCCTG | |
| IL-6 | Forward: AGACCGCTGCCTGTCTAAAA |
| Reverse: TTTGATGTCGTTCACCAGGA | |
| TNF-α | Forward: GCTGGATCTTCAAAGTCGGGTGTA |
| Reverse: TGTGAGTCTCAGCACACTTCCATC | |
| NF-κB | Forward: GAGCCCTTTGTGCAAGAGAC |
| Reverse: TGGGATACGTCCTCCTGTTC | |
| IκBα | Forward: GGTGGAAAGACTCCTGAAAGC |
| Reverse: TGTAGTTAGGGAAGGTAAGAATG | |
| MyD88 | Forward: GAGGATGGTGGTGGTCATCT |
| Reverse: CGACAGGGATTAGCCGTTTA | |
| STAT3 | Forward: CCCTGGGACTAACTCTGGCA |
| Reverse: AGAGGTCCT GGATTGGCCTC |
Table 1 Sequences of the primers in qRT-PCR analysis
| Gene | Primer sequence |
|---|---|
| β-actin | Forward: ATGGATGAGGAAATCGCTG |
| Reverse: ATGCCAACCATCACTCCCTG | |
| IL-6 | Forward: AGACCGCTGCCTGTCTAAAA |
| Reverse: TTTGATGTCGTTCACCAGGA | |
| TNF-α | Forward: GCTGGATCTTCAAAGTCGGGTGTA |
| Reverse: TGTGAGTCTCAGCACACTTCCATC | |
| NF-κB | Forward: GAGCCCTTTGTGCAAGAGAC |
| Reverse: TGGGATACGTCCTCCTGTTC | |
| IκBα | Forward: GGTGGAAAGACTCCTGAAAGC |
| Reverse: TGTAGTTAGGGAAGGTAAGAATG | |
| MyD88 | Forward: GAGGATGGTGGTGGTCATCT |
| Reverse: CGACAGGGATTAGCCGTTTA | |
| STAT3 | Forward: CCCTGGGACTAACTCTGGCA |
| Reverse: AGAGGTCCT GGATTGGCCTC |
Figure 1 Inhibitory effects of 17 diterpenoid lactones on LPS-stimulated inflammation in 3-dpf zebrafish larvae A: overview of the experimental timeline and location of LPS microinjection; B: representative images of LPS-induced zebrafish treated with different diterpenoid lactones; i: PBS group, ii: LPS group, iii: Dex group, iv-xx: AP-1-AP-17, respectively; C: neutrophils in the region of interest (red ovals) were counted. PBS: phosphate-buffered saline; Ctl: control, M: model (treated with LPS); Dex: dexamethasone; LPS: lipopolysaccharidei. aP < 0.001 vs the control group; bP < 0.001, cP < 0.01vs the LPS group.
Figure 1 Inhibitory effects of 17 diterpenoid lactones on LPS-stimulated inflammation in 3-dpf zebrafish larvae A: overview of the experimental timeline and location of LPS microinjection; B: representative images of LPS-induced zebrafish treated with different diterpenoid lactones; i: PBS group, ii: LPS group, iii: Dex group, iv-xx: AP-1-AP-17, respectively; C: neutrophils in the region of interest (red ovals) were counted. PBS: phosphate-buffered saline; Ctl: control, M: model (treated with LPS); Dex: dexamethasone; LPS: lipopolysaccharidei. aP < 0.001 vs the control group; bP < 0.001, cP < 0.01vs the LPS group.
Figure 2 Inhibitory effects of 17 diterpenoid lactones on CuSO4-induced inflammation in 3-dpf zebrafish larvae A: overview of the experimental timeline; B: representative images of CuSO4-induced zebrafish treated with different diterpenoid lactones; i: PBS group, ii: LPS group, iii: Dex group, iv-xx: AP-1-AP-17, respectively; C: neutrophils recruited to the lateral line (red regions) were counted. PBS: phosphate-buffered saline; LPS: lipopolysaccharidei; CuSO4: Copper sulfate; M: model (treated with LPS); Dex: dexamethasone. aP < 0.001 vs the control group; bP < 0.001, cP < 0.05, dP < 0.01 vs the CuSO4 group.
Figure 2 Inhibitory effects of 17 diterpenoid lactones on CuSO4-induced inflammation in 3-dpf zebrafish larvae A: overview of the experimental timeline; B: representative images of CuSO4-induced zebrafish treated with different diterpenoid lactones; i: PBS group, ii: LPS group, iii: Dex group, iv-xx: AP-1-AP-17, respectively; C: neutrophils recruited to the lateral line (red regions) were counted. PBS: phosphate-buffered saline; LPS: lipopolysaccharidei; CuSO4: Copper sulfate; M: model (treated with LPS); Dex: dexamethasone. aP < 0.001 vs the control group; bP < 0.001, cP < 0.05, dP < 0.01 vs the CuSO4 group.
Figure 3 Inhibitory effects of 17 diterpenoid lactones on inflammation induced by tail transection in 3-dpf zebrafish larvae A: overview of the experimental timeline and location of tail transection; B: representative images of tail-cut zebrafish treated with different diterpenoid lactones; i: PBS group, ii: LPS group, iii: Dex group, iv-xx: AP-1-AP-17, respectively; C: neutrophils in the region of interest (red boxes) were counted. Ctl: control; M: model (treated with LPS); PBS: phosphate-buffered saline; LPS: lipopolysaccharidei; Dex: dexamethasone. aP < 0.001 vs the control group; bP < 0.001 vs the tail-cut group.
Figure 3 Inhibitory effects of 17 diterpenoid lactones on inflammation induced by tail transection in 3-dpf zebrafish larvae A: overview of the experimental timeline and location of tail transection; B: representative images of tail-cut zebrafish treated with different diterpenoid lactones; i: PBS group, ii: LPS group, iii: Dex group, iv-xx: AP-1-AP-17, respectively; C: neutrophils in the region of interest (red boxes) were counted. Ctl: control; M: model (treated with LPS); PBS: phosphate-buffered saline; LPS: lipopolysaccharidei; Dex: dexamethasone. aP < 0.001 vs the control group; bP < 0.001 vs the tail-cut group.
Figure 4 Protective effects of AP-5 on zebrafish larvae stimulated by LPS Zebrafish larvae were microinjected with 0.5 mg/mL LPS and followed by AP-5 or Dex treatment. A: the mortality was observed for 72 hpi; B: after 12 hpi, the zebrafish larvae were harvested and stained with hematoxylin and eosin. Inflammatory cells are indicated by black arrowheads. Expressions of IL-6 (C), TNF-α (D), NF-κB (E), IκBα (F), MyD88 (G) and STAT3 (H) at the mRNA level were analyzed by quantitative real-time PCR. i: PBS group, ii: LPS group, iii: Dex group, iv: AP-5 group. M: model (treated with LPS); PBS: phosphate-buffered saline; LPS: lipopolysaccharidei; Dex: dexamethasone; IL-6: interleukin-6; TNF-α: tumor necrosis factor-alpha, NF-κB: nuclear factor-kappa B; IκBα: inhibitor of kappa B alpha; MyD88: myeloid differentiation primary response 88; STAT3: signal transducer and activator of transcription 3; PCR: polymerase chain reaction. aP < 0.001, cP < 0.01 vs the PBS-treated group; bP < 0.001, dP < 0.05, eP < 0.01 vs the LPS-treated group.
Figure 4 Protective effects of AP-5 on zebrafish larvae stimulated by LPS Zebrafish larvae were microinjected with 0.5 mg/mL LPS and followed by AP-5 or Dex treatment. A: the mortality was observed for 72 hpi; B: after 12 hpi, the zebrafish larvae were harvested and stained with hematoxylin and eosin. Inflammatory cells are indicated by black arrowheads. Expressions of IL-6 (C), TNF-α (D), NF-κB (E), IκBα (F), MyD88 (G) and STAT3 (H) at the mRNA level were analyzed by quantitative real-time PCR. i: PBS group, ii: LPS group, iii: Dex group, iv: AP-5 group. M: model (treated with LPS); PBS: phosphate-buffered saline; LPS: lipopolysaccharidei; Dex: dexamethasone; IL-6: interleukin-6; TNF-α: tumor necrosis factor-alpha, NF-κB: nuclear factor-kappa B; IκBα: inhibitor of kappa B alpha; MyD88: myeloid differentiation primary response 88; STAT3: signal transducer and activator of transcription 3; PCR: polymerase chain reaction. aP < 0.001, cP < 0.01 vs the PBS-treated group; bP < 0.001, dP < 0.05, eP < 0.01 vs the LPS-treated group.
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