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2TMR Integrative Medicine  2019, 3: e19002.       DOI: 10.12032/TMRIM201903002
    
Effect of the Tiaobufeishen decoction on Caveolin-1-p38 MAPK signaling pathway and mechanism of improving the tracheobronchomalacia in chronic obstructive pulmonary disease
Peng-Cheng Zhou1,*(), Wei Yu2, Ke-Ling Chen1, Wen-Jun Tang1, Wei Xiao1, Qian-Ming Xia1, Jun-Mei Ma1, Yan Dong1
11Department of Respiratory Medicine, Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, China
2Clinical Medical School, Chengdu University of Traditional Chinese Medicine, Chengdu, China.
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Highlights:

Tiaobufeishen decoction could decrease the expression of the caveolin-1, p-p38, MMP3, inhibit the activation of the caveolin-1-p38MAPK signaling pathway.

Abstract

Objective: Reliving the relationship of the Caveolin-1-p38 MAPK signaling pathway and COPD tracheobronchomalacia, and research the mechanism of Tiaobufeishen decoction improve the regression of the weasand cartilage cells.Methods Flow cytometry was used to analyze the apoptosis rate to determine the optimal concentration of Tiaobufeishen decoction and CSE, CCK8 assay was used to determine the optimal concentration of P38-MAPK specific inhibitor. The COPD cell model was created by tracheal chondrocyte which dispose by optimal concentration CSE, then add the IL-1β set up the chondrocyte degeneration model, use the method of toluidine blue staining and immunohistochemical authenticate degeneration of cartilage. This research included control group, model group, model-Tiaobufeishen group, model-blocker group. When the model was set up succeed, add the Tiaobufeishen decoction and P38-MAPK blocker in the model-Tiaobufeishen and model-blocker groups, respectively. Western Blot was used to detect the expression of caveolin-1 and p-p38 in the chondrocyte. RT-PCR was used to detect the expression of MMP3 and caveolin-1 in the matrix.Results The cell activity was not influence by the concentration of Tiaobufeishen decoction and blocker, the concentration of the CSE model was moderation. Compared with control group, the level of caveolin-1, p38MAPK, MMP3 in the model group was significant increase, moreover, the result of toluidine blue staining and immunohistochemical methods show that the chondrocyte has obvious regression. The expression of caveolin-1, p38MAPK, and MMP3 have significant decrease than the control group, and the reduction of chondrocyte degeneration.Conclusion The caveolin-1-p38MAPK signaling pathway play an important role in the morbidity of the tracheobronchomalacia. Tiaobufeishen decoction could decrease the expression of the caveolin-1, p-p38, MMP3, inhibit the activation of the caveolin-1-p38MAPK signaling pathway, therefore, it can improve the tracheobronchomalacia.



Key wordsCOPD      Tracheobronchomalacia      Caveolin-1-p38 MAPK signaling pathway     
Published: 21 January 2019
Fund:  Funding: This research have got support from the technology innovation fund of Hospital of Chengdu University of Traditional Chinese Medicine (NO. 2017-QYY-38), and don’t received any specific grant from commercial, or not-for-profit sectors.
Corresponding Authors: Zhou Peng-Cheng   
E-mail: zhoupengcheng@cdutcm.edu.cn
Cite this article:

Peng-Cheng Zhou, Wei Yu, Ke-Ling Chen, Wen-Jun Tang, Wei Xiao, Qian-Ming Xia, Jun-Mei Ma, Yan Dong. Effect of the Tiaobufeishen decoction on Caveolin-1-p38 MAPK signaling pathway and mechanism of improving the tracheobronchomalacia in chronic obstructive pulmonary disease. 2TMR Integrative Medicine, 2019, 3: e19002.  

URL:

https://www.tmrjournals.com/im/EN/10.12032/TMRIM201903002     OR     https://www.tmrjournals.com/im/EN/Y2019/V3/IVolume 3/88888

Figure 1 Tracheobronchomalacia
Note: Fig. 1A: Tracheal cartilage ring clear, smooth mucosa, unobstructed lumen。Fig. 1B: Tracheal cartilage annulus disappears, mucous membrane is swollen, lumen is narrow。Fig. 1C: Bilateral main bronchial lumens are unobstructed (inspiratory phase) at the red arrow。Fig. 1D: Bilateral main bronchial cavities collapse (end-expiratory) at the red arrow.
Figure 2 Rate of chondrocyte apoptosis for Tiaobufeishen decoction
Figure 3 Rate of chondrocytes at different concentrations of CSE
Groups Cell viability
Control group 100.0000 ± 1.74815
5umol/L 92.9754 ± 1.45461*
10umol/L 97.3914 ± 2.28608a
20umol/L 96.2526 ± 1.81477*
30umol/L 93.1276 ± 1.83695*
50umol/L 94.9153 ± 1.81232*
Table 1 Comparison of cell viability in different concentrations of P38-MAPK inhibitor (ヌ± s)
Figure 4 Toluidine blue staining
Note: In the normal control group, the cytoplasmic cyanosis of chondrocytes was observed, and the nuclei were dark blue. There were a few pale blue cyanosis around the cells, and 1-2 nucleoli were clearly visible. The chondrocytes in the model group showed obvious pale staining, long and narrow cells, mostly monolayer growth, more vacuolar formation, nucleolar deformation and irregular cell morphology. Chondrocytes in the model group + p38-mapk blocker group were stained deeply, some nuclei were thick, and a few vacuoles were observed in the cells, with uneven cell morphology and size. The cytoplasm of chondrocytes in the model group and the traditional Chinese medicine group was light blue with clear nucleoli and close to normal cell morphology.
Figure 5 Immumohistochemical staining
Note: Immunohistochemical staining showed collagen type Ⅱ antibody staining is stronger in the blank group, all of the cytoplasm are visible in the expression of collagen type Ⅱ. Model group Ⅱ type collagen expression decreased significantly, the basic negative. Model + p38 lightning - MAPK blockers group can see Ⅱ expression of collagen type is abate, only a few cells expressed. Model + Chinese traditional medicine group Ⅱ expression of collagen type is abate, more cartilage cytoplasm is visible.
Figure 6 The expressions of MMP3 and Caveolin-1 mRNA were detected by RT-PCR
Note: *Comparison between two groups, P < 0.05(Figure 6B, P = 0.0188); **Comparison between two groups, P < 0.01 (Figure 6A, P = 0.0014, P = 0.0019; Figure 6B, P = 0.0081, P=0.0063); ***Comparison between two groups, P < 0.001 (Figure 6A, P = 0.0006). a, b, c and d were compared with the blank control group, respectively, P < 0.01 (aP = 0.0081; bP = 0.0063; cP = 0.0019, dP = 0.0006, Unpaired t test)
Figure 7 Western blot revealed caveolin-1 and p-p38 expression in chondrocytes of each group
Note: *Comparison between two groups, P < 0.05 (Figure 7A, P = 0.0346, P = 0.0120, Figure 7B, P = 0.0297); ** Comparison between two groups, P < 0.01 (Figure 7A, P = 0.0076; Figure 7B, P = 0.0074, P = 0.0057); a, b were compared with the blank control group, respectively, P < 0.01 (aP = 0.0074; bP = 0.0057).
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