Inhibitory Effects of GGX on Lung Injury of Chronic Obstructive Lung Disease (COPD) Mice Model

Article information

J Korean Med. 2021;42(3):56-71

Publication date (electronic) : 2021 September 01

doi : https://doi.org/10.13048/jkm.21025

Tae Hyeon Kim ¹^,^†

, Won Kyung Yang ¹^,²^,^†

, Su Won Lee ¹

, Seung Hyung Kim ²

, Yee Ran Lyu ¹

, Yang Chun Park ¹^,²^,

¹Division of Respiratory Medicine, Dep. of Internal Medicine, College of Korean Medicine, Daejeon University, Daejeon, Korea

²Institute of Traditional Medicine and Bioscience, Daejeon University, Daejeon, Korea

Correspondence to: Yang-Chun Park, Korean Internal Medicine, Daejeon Korean Medicine Hospital of Daejeon University 75, Daedeok-daero 176-beongil, Seo-gu, Daejeon, Republic of Korea 35235, Tel: +82-42-470-9126 (clinic), 229-6763 (office, Munchang Campus), Fax: +82-42-470-9486, E-mail: omdpyc@dju.kr

^†

These authors contributed equally to this work

Received 2021 May 3; Revised 2021 June 7; Accepted 2021 July 28.

Abstract

Objectives

This study is aimed to evaluate the protective effects of GGX on lung injury of Chronic Obstructive Lung Disease (COPD) mice model.

Materials and Methods

C57BL/6 mice were challenged with lipopolysaccharide (LPS) and cigarette smoke extract (CSE) and then treated with vehicle only (Control group), dexamethasone 3 mg/kg (Dexa group), gam-gil-tang 200 mg/kg (GGT group), GGX 100, 200, and 400 mg/kg (GGX group). After sacrifice, its bronchoalveolar lavage fluid (BALF) or lung tissue was analyzed with cytospin, Enzyme-Linked Immunosorbent Assay (ELISA), real-time polymerase chain reaction (PCR) and hematoxylin & eosin (H&E), and Masson’s trichrome staining.

Results

In the COPD model, GGX significantly inhibited the increase of neutrophils, TNF-α, IL-17A, CXCL-1, MIP2 in BALF and TNF-α, IL-1β, IL-10 mRNA expression in lung tissue. It also decreased the severity of histological lung injury.

Conclusion

This study suggests the usability of GGX for COPD patients by controlling lung tissue injury.

Keywords: Chronic Obstructive Lung Disease; GGX; Herbal drug; Korean medicine

Fig. 1

Total particulate matter of cigarette smoke solution.

TPM: total particulate matter, WFHA: Weight of filter holder after smoke, WFHB: Weight of filter holder before smoke, N: Cigarette number of each trap.

Fig. 2

Experimental plan of repeated CSE+LPS exposure. CSE+LPS: Intranasal instillation of LPS 100 μg/μl and cigarette smoke extract 1 mg/ml.

Fig. 3

Effect of GGX on cytospin image (A) and neutrophils count (B) in BALF of COPD mice.

Mice were challenged by aspiration of LPS+CSE (Control), and then treated with Dexa (dexamethasone 3 mg/kg), GGT (200 mg /kg) and GGX (100, 200, 400 mg /kg) for 21 days (n=8). All values are presented as mean±SE. †: Significant difference with the non-treated group (††† p<0.001), *: Significant difference with the Control (*** p<0.001).

Fig. 4

Effect of GGX on TNF-α production in BALF of COPD mice.

Mice were challenged by an aspiration of LPS+CSE (Control), and treated with Dexa (dexamethasone 3 mg /kg), GGT (200 mg /kg) and GGX (100, 200, 400 mg /kg) for 21 days (n=8). Level of TNF-α was determined with ELISA. All values are presented as mean±SE. †: Significant difference with the non-treated group (††† p<0.001), *: Significant difference with the Control (** p<0.01, *** p<0.001).

Fig. 5

Effect of GGX on IL-17A production of BALF in COPD mice.

Fig. 6

Effect of GGX on MIP2 production in BALF of COPD mice.

Fig. 7

Effect of GGX on CXCL-1 production in BALF of COPD mice.

Fig. 8

Effect of GGX on TNF-α mRNA expression in lung tissue of COPD mice.

Mice were challenged by an aspiration of LPS+CSE (Control), and treated with Dexa (dexamethasone 3 mg /kg), GGT (200 mg /kg) and GGX (100, 200, 400 mg /kg) for 21 days (n=4). Level of TNF-α was determined with real time PCR. All values are presented as mean±SE. †: Significant difference with the non-treated group († p<0.05), *: Significant difference with the Control (* p<0.05).

Fig. 9

Effect of GGX on IL-1β mRNA expression in lung tissue of COPD mice.

Mice were challenged by an aspiration of LPS+CSE (Control), and treated with Dexa (dexamethasone 3 mg /kg), GGT (200 mg /kg) and GGX (100, 200, 400 mg /kg) for 21 days (n=4). Level of IL-1β was determined with real time PCR. All values are presented as mean±SE. †: Significant difference with the non-treated group (†† p<0.01), *: Significant difference with the Control (** p<0.01).

Fig. 10

Effect of GGX on IL-6 mRNA expression in lung tissue of COPD mice.

Fig. 10

Effect of GGX on IL-10 mRNA expression in lung tissue of COPD mice.

Mice were challenged by an aspiration of LPS+CSE (Control), and treated with Dexa (dexamethasone 3 mg /kg), GGT (200 mg /kg) and GGX (100, 200, 400 mg /kg) for 21 days (n=4). Level of IL-10 was determined with real time PCR. All values are presented as mean±SE. †: Significant difference with the non-treated group († p<0.05), *: Significant difference with the Control (* p<0.05, ** p<0.01).

Fig. 12

Effect of GGX on histopathological changes and histology scores in the lung of COPD mice.

Mice were challenged by an aspiration of LPS+CSE (Control), and treated with Dexa (dexamethasone 3 mg /kg), GGT (200 mg /kg) and GGX (100, 200, 400 mg /kg) for 21 days (n=4). (A) Representative sections from each treatment group are shown (Light microscope at 100×magnification). (B) Quantitative analysis of the degree of lung tissue damage in the sections. All values are presented as mean±SE. †: Significant difference with the non-treated group (††† p<0.001), *: Significant difference with the Control (** p<0.01, *** p<0.001).

Table 1

The Composition of GGX

Table 2

Oligonucleotide Sequence Used for Mouse Real-time PCR

References

1. Park YB, Rhee CK, Yoon HK, Oh YM, Lim SY, Lee JH, et al. 2018;COPD clinical practice guideline of the Korean Academy of Tuberculosis and Respiratory Disease: a summary. Tuberc Respir Dis (Seoul) 81:261–73.

2. Barnes PJ, Shapiro SD, Pauwels RA. 2003;Chronic obstructive pulmonary disease: molecular and cellular mechanisms. Eur Respir J 22(4):672–88.

3. Kim IA, Park YB, Yoo KH. 2004;Pharmacotherapy for chronic obstructive pulmonary disease. J Korean Med Assoc Sep. 61(9):545–551.

4. Anthonisen NR, Connett JE, Kiley JP, Altose MD, Bailey WC, Buist AS, et al. 1994;Effects of smoking intervention and the use of an inhaled anticholinergic bronchodilator on the rate of decline of FEV1: the Lung Health Study. JAMA 272:1497–505.

5. Singh S, Amin AV, Loke YK. 2009;Long-term use of inhaled corticosteroids and the risk of pneumonia in chronic obstructive pulmonary disease: a meta-analysis. Arch Intern Med 169:219–29.

6. Barnes PJ. 2013;New anti-inflammatory targets for chronic obstructive pulmonary disease. Nat Rev Drug Discov 12(7):543–59.

7. Lee ES, Han JM, Kim MH, Namgung U, Yeo Y, Park YC. 2013;Effects of inhalable microparticles of Socheongryong-tang on chronic obstructive pulmonary disease in a mouse model. J Korean Med 34(3):54–68.

8. Lee JG, Yang SY, Kim MH, Namgung U, Park YC. 2011;Protective effects of Socheongryong-tang on elastase-induced lung injury. J Korean Oriental Med 32(4):83–99.

9. Kim Y, Yang SY, Kim MH, Namgung U, Park YC. 2011;Effects of Saengmaekcheongpye-eum on LPS-induced COPD model. Korean J Oriental Int Med 2011;32(2):217–31.

10. Kim HW, Yang SY, Kim MH, Namgung U, Park YC. 2011;Protective effects of Maekmundong-tang on elastase-induced lung injury. J Korean Oriental Med 32(2):63–78.

11. Lee H, Kim Y, Kim HJ, Park S, Jang YP, Jung S, et al. 2012;Herbal Formula, PM014, Attenuates Lung Inflammation in a Murine Model of Chronic Obstructive Pulmonary Disease. Evid Based Complement Alternat Med 2012;:769830.

12. Han JM, Yang WK, Kim SH, Park YC. 2015;Effects of Sagan-tang and individual herbs on COPD mice model. J Korean Med Soc Herb Formula Study 23(2):171–87.

13. Park JJ, Yang WK, Lyu YR, Kim SH, Park YC. 2019;Inhibitory effects of SGX01 on lung injury of COPD mice model. Korean J Int Korean Med 40(4):567–81.

14. Yang WK, Lyu YR, Kim SH, Park YC. 2018;Effects of GHX02 on Chronic Obstructive Pulmonary Disease Mouse Model. J Korean Med 39(4):126–35.

15. Kim SH, Hong JH, Yang WK, Geum , et al. 2020;Herbal combinational medication of Glycyrrhiza glabra, Agastache rugosa containing Glycyrrhizic acid, Tilianin inhibits Neutrophilic lung inflammation by affecting CXCL2, Interleukin-17/STAT3 signal pathways in a murine model of COPD. Nutrients 12(4):926.

16. Yang L, Li J, Li Y, Tian Y, Li S, Jiang , et al. 2015;Identification of metabolites and metabolic pathways related to treatment with Bufei Yishen formula in a rat COPD model using HPLC Q-TOF/MS. Evidence-Based Complementary and Alternative Medicine, 2015

17. Hwang DY. 1986. Bang-yak-hap-pyeon Seoul: Namsandang. p. 240.

18. Lyu YR. 2020. Inhibitory effects of GGX in a particulate matter-induced lung injury mouse model. doctoral dissertation Daejeon university;

19. Hong HW. 2011. The Effects of Kamgiltang on Passive Smoking in Rats. doctoral dissertation Dong-eui University.

20. Mizutani N, Fuchikami J, Takahashi M, Nabe T, Yoshino S, Kohno S. 2009;Pulmonary emphysema induced by cigarette smoke solution and lipopolysaccharide in guinea pigs. Biol Pharm Bull 32(9):1559–64.

21. Lopez AD, Shibuya K, Rao C, Mathers CD, Hansell AL, Held LS, et al. 2006;Chronic obstructive pulmonary disease: current burden and future projections. Eur Rspir J 27(2):397–412.

22. Mathers CD, Loncar D. 2006;Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med 3(11):e442.

23. An TJ, Yoon HK. 2018;Prevalence and socioeconomic burden of chronic obstructive pulmonary disease. J Korean Med Assoc 61(9):533–8.

24. GBD 2015 Chronic Respiratory Disease Collaborators. 2017;Global, regional, and national deaths, prevalence, disability adjusted life years, and years lived with disability for chronic obstructive pulmonary disease and asthma, 1990–2015 a systematic analysis for the Global Burden of Disease Study 2015. Lancet Respir Med 5(9):691–706.

25. Jung YM, Lee H. 2011;Chronic obstructive pulmonary disease in Korea: Prevalence, risk factors, and quality of life. J Korean Acad Nurs 41(2):149–56.

26. Ministry of Gender Equality & Family (MOGEF). 2014. 2014 Comprehensive survey on the contact with the harmful environment for youth Available at: URL: http://www.mogef.go.kr . Accessed April 1.

27. Yoon J, Seo H, Oh IH, Yoon SJ. 2016;The Non-Communicable Disease Burden in Korea: Findings from the 2012 Korean Burden of Disease Study. J Korean Med Sci Nov. 31(Suppl 2):S158–S167.

28. Park SK. 2002;Chronic obstructive pulmonary disease - definition, severity, risk factors, etiology, pathology, diagnosis. J Korean Med 63(2):389–99.

29. Lurwidya F, Damayanti T, Yunus F. 2016;The Role of Innate and Adaptive Immune Cells in the Immunopathogenesis of Chronic Obstructive Pulmonary Disease. Tuberc Respir Dis(Seoul) 79(1):5–13.

30. Lee S, et al. 2007;Antielastin autoimmunity in tobacco smokinginduced emphysema. Nature Med 13:567–569.

31. Yoo CG. 2009;Pathogenesis and pathophysiology of COPD. Korean J Med 77(4):383–400.

32. Barnes PJ. 2004;Macrophages as orchestrators of COPD. COPD 1:59–70.

33. Majo J, Ghezzo H, Cosio MG. 2001;Lymphocyte population and apoptosis in the lungs of smokers and their relation to emphysema. Eur Respir J 17:946–53.

34. Barnes PJ. 2008;The cytokine network in asthma and chronic obstructive pulmonary disease. J Clin Invest 118(11):3546–56.

35. Rovina N, Koutsoukou A, Koulouris NG. 2013;Inflammation and immune response in COPD: where do we stand? Mediators Inflammation 2013;:413735.

36. Herbology Editorial Committee of Korean Medicine schools. 1991. Boncho-hak Seoul: Younglimsa. p. 124–5. p. 136–7. p. 214–5. p. 448–9. p. 534–5. p. 580–1. p. 588–9.

37. Park YC, Jin M, Kim SH, Kim MH, et al. 2014;Effects of inhalable microparticle of flower of Lonicera japonica in a mouse model of COPD. Journal of Ethnopharmacology 151(1):123–130.

38. Di Stefano A, Capelli A, Lusuardi M, Balbo P, Vecchio C, Maestrelli P, et al. 1998;Severity of airflow limitation is associated with severity of airway inflammation in smokers. Am J Respir Crit Care Med 158(4):1277–85.

39. Aaron SD, Angel JB, Lunau M, Wright K, Fex C, Le Saux N, et al. 2001;Granulocyte inflammatory markers and airway infection during acute exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 163(2):349–55.

40. Deveci Y, Deveci F, Ilhan N, Karaca I, Turgut T, Muz MH. 2010;Serum ghrelin, IL-6 and TNF-α levels in patients with chronic obstructive pulmonary disease. Tuberk Toraks 58(2):162–72.

41. Traves SL, Donnelly LE. 2008;Th17 cells in airway diseases. Curr Mol Med 8(5):416–26.

42. Levänen B, Glader P, Dahlén B, Billing B, Qvarfordt I, Palmberg L, et al. 2016;Impact of tobacco smoking on cytokine signaling via interleukin-17A in the peripheral airways. Int J Chron Obstruct Pulmon Dis 11:2109–16.

43. Lukacs NW, Hogaboam CM, Kunkel SL. 2005;Chemokines and their receptors in chronic pulmonary disease. Curr Drug Targets Inflamm Allergy 4(3):313–7.

44. Traves SL, Culpitt SV, Russell RE, Barnes PJ, Donnelly LE. 2002;Increased levels of the chemokines GROα and MCP-1 in sputum samples from patients with COPD. Thorax 57(7):590–5.

45. Culpitt SV, Rogers DF, Shah P, De Matos C, Russell RE, Donnelly LE, et al. 2003;Impaired inhibition by dexamethasone of cytokine release by alveolar macrophages from patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 167(1):24–31.

Article information Continued

Fig. 1

Total particulate matter of cigarette smoke solution.

TPM: total particulate matter, WFHA: Weight of filter holder after smoke, WFHB: Weight of filter holder before smoke, N: Cigarette number of each trap.

Fig. 2

Experimental plan of repeated CSE+LPS exposure. CSE+LPS: Intranasal instillation of LPS 100 μg/μl and cigarette smoke extract 1 mg/ml.

Fig. 3

Effect of GGX on cytospin image (A) and neutrophils count (B) in BALF of COPD mice.

Fig. 4

Effect of GGX on TNF-α production in BALF of COPD mice.

Fig. 5

Effect of GGX on IL-17A production of BALF in COPD mice.

Fig. 6

Effect of GGX on MIP2 production in BALF of COPD mice.

Fig. 7

Effect of GGX on CXCL-1 production in BALF of COPD mice.

Fig. 8

Effect of GGX on TNF-α mRNA expression in lung tissue of COPD mice.

Fig. 9

Effect of GGX on IL-1β mRNA expression in lung tissue of COPD mice.

Mice were challenged by an aspiration of LPS+CSE (Control), and treated with Dexa (dexamethasone 3 mg /kg), GGT (200 mg /kg) and GGX (100, 200, 400 mg /kg) for 21 days (n=4). Level of IL-1β was determined with real time PCR. All values are presented as mean±SE. †: Significant difference with the non-treated group (†† p<0.01), *: Significant difference with the Control (** p<0.01).

Fig. 10

Effect of GGX on IL-6 mRNA expression in lung tissue of COPD mice.

Fig. 10

Effect of GGX on IL-10 mRNA expression in lung tissue of COPD mice.

Mice were challenged by an aspiration of LPS+CSE (Control), and treated with Dexa (dexamethasone 3 mg /kg), GGT (200 mg /kg) and GGX (100, 200, 400 mg /kg) for 21 days (n=4). Level of IL-10 was determined with real time PCR. All values are presented as mean±SE. †: Significant difference with the non-treated group († p<0.05), *: Significant difference with the Control (* p<0.05, ** p<0.01).

Fig. 12

Effect of GGX on histopathological changes and histology scores in the lung of COPD mice.

Table 1

The Composition of GGX

Herb	Pharmacognostic name	Amount (g)
Gilgyeong	Platycodi Radix	14.00
Gamcho	Glycyrrhizae Radix	6.00
Geumeunhwa	Lonicerae Flos	14.00
Sangbaekpi	Mori Radicis Cortex	6.00
Total amount		40.00

Table 2

Oligonucleotide Sequence Used for Mouse Real-time PCR

Gene	Primer	Sequence
TNF-α	FAM	5′-CACGTCGTAGCAAACCACCAAGTGGA-3′
TNF-α	Forward	5′-CACCTTCTTTTCCTTCATCTT-3′
IL-1β	Reverse	5′-GTCGTTGCTTGTCTCTCCTTGTA-3′
IL-1β	Forward	5′-TACCCCCAGGAGAAGATTCC-3′
IL-6	Reverse	5′-TTTTCTGCCAGTGCC TCTTT-3′
IL-6	Forward	5′-GATGCCTTCAGCAGAGTGAAGA-3′
IL-10	Reverse	5′-CATGGCTTTGTAGATGCCTTTC-3′
G3PDH	VIC	5′-TGCATCCTGCACCACCAACTGCTTAG-3′