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Journal of Chinese Integrative Medicine ›› 2012, Vol. 10 ›› Issue (2): 200-209.doi: 10.3736/jcim20120211

• Original Experimental Research • Previous Articles     Next Articles

Preventive effects of Citrus reticulata essential oil on bleomycin-induced pulmonary fibrosis in rats and the mechanism

Zhou Xian-mei1(),Zhao Yang1,He Cui-cui2,Li Jian-xin2   

  1. 1. Department of Respiratory Diseases, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing 210029, Jiangsu Province, China
    2. State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, Jiangsu Province, China
  • Received:2011-08-29 Accepted:2011-10-08 Online:2012-02-20 Published:2018-10-09

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OBJECTIVE: To investigate the effects of essential oil of Citrus reticulata (EOCR) on proliferation of human embryonic lung fibroblasts (HELFs), and to explore its protective effects on bleomycin (BLM)-induced lung fibrosis in rats.
METHODS: Routinely cultured HELFs during the logarithmic phase of growth were divided into control and treated groups, and applied for evaluation of inhibitory activity using methylthiazol tetrazolium (MTT) assay. A rat model of BLM-induced pulmonary fibrosis was used for the evaluation of antifibrotic effect of EOCR. Forty-two Sprague-Dawley rats were randomly divided into normal group, model group, prednisone group and different doses of EOCR groups. BLM was intratracheally instilled into all the rats except those in the normal group, and EOCR was orally given to BLM-treated rats at doses of 25, 50, 100 and 200 mg/kg once per day for four weeks. The rats in the normal group were intratracheally administered the same volume of saline. On the 28th day, rats were sacrificed under anesthesia, and the serum and lung tissues were collected. Superoxide dismutase (SOD) activities and malondialdehyde (MDA) contents in serum and lung tissues were analyzed with corresponding kits; type Ⅰ collagen (Col Ⅰ) content in lung tissues was evaluated with enzyme-linked immunosorbent assay; pulmonary fibrosis was assessed by lung histology; protein and mRNA expressions of connective tissue growth factor (CTGF) in lung tissues were measured with immunohistochemical and in situ hybridization semiquantitative image analyses, respectively.
RESULTS: The EOCR at different concentrations displayed inhibitory activity on proliferation of HELFs. In in vivo experiment, the weight gain of the rats in groups treated with EOCR at doses of 50, 100 and 200 mg/kg per day was significantly higher than those in the model group at the 7th, 14th, 21st and 28th day (P<0.05 or P<0.01). The scores of alveolitis and pulmonary fibrosis in the groups treated with EOCR at doses of 100 and 200 mg/kg per day were significantly lower than those in the model group (P<0.01); the SOD levels in serum and pulmonary tissues of the EOCR (50, 100 and 200 mg/kg) groups were markedly increased compared with the model group (P<0.01 ), while the MDA levels in both serum and pulmonary tissues were markedly reduced (P<0.05); the Col Ⅰ level in pulmonary tissues of the EOCR (100 and 200 mg/kg per day) groups were markedly lower than that of the model group (P<0.01); the protein and mRNA expressions of CTGF in the groups treated with EOCR at doses of 100 and 200 mg/kg per day were down-regulated compared with the model group (P<0.01).
CONCLUSION: The results indicate that EOCR has preventive effects on BLM-induced pulmonary fibrosis in rats. The mechanism may be via adjusting the unbalance of oxidation and antioxidation, down-regulating CTGF protein and mRNA expressions, and reducing collagen deposition and fibrosis.

Key words: Pericarpium citri reticulatae, essential oil, human embryonic lung fibroblasts, pulmonary fibrosis, bleomycin, rats

"

Group n Inhibition rate
Control 5 0.00±21.92
EOCR 300 μg/mL 5 46.64±1.12*
EOCR 150 μg/mL 5 49.74±5.75*
EOCR 15 μg/mL 5 54.52±0.45*
EOCR 5 μg/mL 5 44.19±2.71*
EOCR 0.5 μg/mL 5 41.86±6.71*

"

Group n Body weight gain
7th day 14th day 21st day 28th day
Normal 6 35.8±8.7 56.7±15.1 88.3±20.4 117.5±18.1
Model 6 –14.2±12.9△ 8.3±18.4△△ 24.2±27.1△△ 47.5±29.1△△
Prednisone 5 mg/kg 6 –24.2±10.7 –2.5±21.7 18.3±10.8 37.5±17.0
EOCR 25 mg/kg 6 –9.2±26.0 –7.5±28.9 23.3±38.5 54.2±24.8
EOCR 50 mg/kg 6 25.0±13.5▲▲ 31.7±22.1 60.0±25.2▲ 90.8±21.3▲
EOCR 100 mg/kg 6 23.3±18.4▲▲ 37.5±25.7▲ 74.2±24.0▲▲ 105.0±20.0▲
EOCR 200 mg/kg 6 22.5±28.8▲▲ 32.5±20.4 81.7±18.9▲▲ 105.0±20.5▲

Figure 1

Histological analysis of lung tissues observed under a light microscope (hematoxylin and eosin staining, ×100) A: Normal group; B: Model group; C: Prednisone group; D, E, F and G: Groups of rats treated with essential oil of Citrus reticulata at doses of 25, 50, 100 and 200 mg/kg per day, respectively."

"

Group n Alveolitis Fibrosis
– + ++ +++ – + ++ +++
Normal 6 4 2 0 0 4 1 1 0
Model 6 0 1 2 3△△ 0 0 1 5△△
Prednisone 5 mg/kg 6 1 3 1 1 0 2 3 1▲
EOCR 25 mg/kg 6 0 1 4 1 0 1 3 2
EOCR 50 mg/kg 6 1 2 2 1 0 1 4 1▲▲
EOCR 100 mg/kg 6 2 3 1 0▲ 1 2 3 0▲▲
EOCR 200 mg/kg 6 2 3 1 0▲ 1 3 2 0▲▲

"

Group n Superoxide dismutase
Serum (U/mL) Lung tissues (U/mg protein)
Normal 6 166.03±13.61 131.81±5.82
Model 6 137.09±13.16△ 93.32±6.78△
Prednisone 5 mg/kg 6 151.23±4.52▲ 107.79±16.34▲▲
EOCR 25 mg/kg 6 139.08±13.96 90.50±6.68
EOCR 50 mg/kg 6 155.59±7.11▲▲ 116.33±11.64▲▲
EOCR 100 mg/kg 6 159.48±9.01▲▲ 131.48±3.72▲▲
EOCR 200 mg/kg 6 157.40±9.06▲▲ 128.48±4.83▲▲

"

Group n Malondialdehyde
Serum (nmol/mL) Lung tissues (nmol/mg protein)
Normal 6 1.00±0.41 9.92±2.77
Model 6 2.75±0.80△ 24.35±9.10△
Prednisone 5 mg/kg 6 1.83±0.73▲ 16.51±3.58▲
EOCR 25 mg/kg 6 2.22±0.97 19.56±7.09
EOCR 50 mg/kg 6 1.63±0.75▲▲ 17.00±3.58▲
EOCR 100 mg/kg 6 1.14±0.56▲▲ 12.65±2.53▲▲
EOCR 200 mg/kg 6 1.17±0.46▲▲ 10.87±5.37▲▲

"

Group n Type Ⅰ collagen content (μg/g wet lung)
Normal 6 57.53±7.23
Model 6 83.05±9.84△△
Prednisone 5 mg/kg 6 71.41±12.73▲▲
EOCR 25 mg/kg 6 79.27±13.71
EOCR 50 mg/kg 6 74.22±4.65
EOCR 100 mg/kg 6 66.03±9.33▲
EOCR 200 mg/kg 6 65.64±6.62▲

Figure 2

Expression of connective tissue grouth factor protein in lung tissues observed under a light microscope (Immunohistological method, ×400) A: Normal group; B: Model group; C: Prednisone group; D, E, F and G: Groups of rats treated with essential oil of Citrus reticulata at doses of 25, 50, 100 and 200 mg/kg per day, respectively."

Figure 3

Expression of connective tissue grouth factor mRNA in lung tissues observed under a light microscope (In situ hybridization, ×400) A: Normal group; B: Model group; C: Prednisone group; D, E, F and G: Groups of rats treated with essential oil of Citrus reticulata at doses of 25, 50, 100 and 200 mg/kg per day, respectively."

"

Group n CTGF
Protein integral mRNA integral
Normal 6 37.07±4.27 42.94±6.86
Model 6 52.92±8.01△△ 77.09±9.61△△
Prednisone 5 mg/kg 6 44.23±4.41▲ 54.07±6.28▲▲
EOCR 25 mg/kg 6 49.50±8.45 70.08±8.32
EOCR 50 mg/kg 6 47.44±3.14 68.70±11.27
EOCR 100 mg/kg 6 38.08±8.38▲▲ 49.27±9.10▲▲
EOCR 200 mg/kg 6 35.92±5.11▲▲ 46.64±9.97▲▲
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