Home | Current Issue | Past Issues | Search | CollectionsRSS | PDA Services | FAQ | SHCIM Online | Chinese Updated Wednesday, April 23, 2014
 How to Show Chinese in English Operating System
Original Experimental Research
Journal of Chinese Integrative Medicine: Volume 10   February, 2012   Number 2

DOI: 10.3736/jcim20120211
Preventive effects of Citrus reticulata essential oil on bleomycin-induced pulmonary fibrosis in rats and the mechanism
1. Xian-mei Zhou (Department of Respiratory Diseases, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing 210029, Jiangsu Province, China E-mail: zhouxianmeijs@yahoo.com.cn)
2. Yang Zhao (Department of Respiratory Diseases, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing 210029, Jiangsu Province, China )
3. Cui-cui He (State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, Jiangsu Province, China )
4. Jian-xin Li (State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, Jiangsu Province, China )
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.
JCIM
Open Access
THIS ARTICLE
-  Abstract
-  Full text
-  Download PDF file
-  Send to a friend
-  Related articles in JCIM
-  Cited in JCIM
-  Reader's comments
-  Send a comment

Received August 29, 2011; accepted October 8, 2011; published online February 15, 2012.
Full-text LinkOut at PubMed. Journal title in PubMed: Zhong Xi Yi Jie He Xue Bao.

Correspondence: Xian-mei Zhou, MD, Professor; Tel: 025-86617141; E-mail: zhouxianmeijs@yahoo.com.cn

 

Jump to Section
-  Top
-  Article and Author Info
-  Introduction
-  Materials and methods
-  Results
-  Discussion
-  Acknowledgements
-  Competing interests
-  References
  

     Idiopathic pulmonary fibrosis (IPF) is characterized by progressive alveolar inflammation, fibroblast proliferation and collagen deposition. IPF is the most common interstitial pneumonia of unknown etiology and one of the most aggressive interstitial lung diseases[1,2]. Although many studies suggest that some pro-inflammatory mediators, growth factors and infiltrating cells may be involved in IPF development, the understanding of causes and biology of IPF remains relatively poor[3-5]. The common treatment uses corticosteroid in combination with immunosuppressant, anti-inflammatory, antifibrosis, antioxidant and anticoagulative drugs, but the evidence for the treatment is not persuasive[6]. Despite a number of advances in basic and clinical research, currently, IPF is still a progressive, fatal disease and no effective medical therapies are available to slow the decline of pulmonary functions or reduce the mortality[7]. Thus, there is an urgent need to identify or develop novel and effective therapeutic agents for IPF. In recent years, with progressive studies on interstitial lung diseases, the research concentrated on agents sourced from medicinal plants for IPF has become a focus of attention[8,9].
     Within a research project aimed at the discovery of active agents from traditional Chinese medicines (TCMs) against IPF, the authors have reported a Chinese herbal formula, named Huqiyin, possessing a protective effect on the bleomycin (BLM)-induced pulmonary fibrosis in rats[10]. Research on the ingredients of Huqiyin clarified that peel of Citrus reticulata Blanco (Rutaceae; common name, citrus) showed the most potent antifibrotic activity, which was consistent with the clinical use in TCM, as an important herbal drug for the treatment of lung-related diseases. Further effort revealed that the ethanol extract of the peel of C. reticulata significantly inhibited the proliferation of human embryonic lung fibroblasts (HELFs) in vitro and suppressed pulmonary fibrosis in rats[11]. The main chemical constituents in the citrus peel are flavonoids and essential oil[12,13], the former of which has been proved to be inactive on HELFs[11]; however, the function of the latter is still unclear. As a continuing research of the project, to clarify the active components responsible for the antifibrotic activity, we focused our studies on the essential oil. In the literature, we found that essential oil extracted from C. reticulata possesses a variety of bioactivities, such as relaxing isolated tracheal and intestinal smooth muscle, releasing acetylcholine-induced tracheal and intestinal smooth muscle spasm, protecting drug-induced asthma in guinea pig and playing anti-allergic effects by inhibiting the release of allergy mediators[14].
     In the present study, the essential oil of C. reticulata (EOCR) was tested for its anti-proliferation activity using HELF culture system. The in vivo effects of EOCR on BLM-induced pulmonary fibrosis in rats were also investigated.

 
  


1  Materials and methods
1.1  Experimental drugs 
The peel of C. reticulata Blanco (Rutaceae) was obtained from the Department of Medicinal Materials, Jiangsu Province Hospital of Traditional Chinese Medicine (No. TCM081219). Voucher specimens have been deposited in the hospital; BLM A5 hydrochloride (8 mg per bottle) was purchased from Tianjin Taihe Pharmaceutical Co., Ltd. (Tianjin, China, No. 080502); prednisone (5 mg/tablet) was purchased from Zhejiang Xianju Pharmaceutical Co., Ltd. (Hangzhou, China, No. 0807136).
1.2  Experimental regents and cell lines  Superoxide dismutase (SOD) and malondialdehyde (MDA) test kits and rat type Ⅰ collagen (Col Ⅰ) enzyme-linked immunosorbent assay (ELISA) kit were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China); rat connective tissue growth factor (CTGF) in situ hybridization kit was obtained from Wuhan Boster Biological Technology Ltd. (Wuhan, China); rat CTGF immunohistochemical detection kit was got from Fuzhou Maixin Technology Ltd. (Fuzhou, China). Dulbecco’s modified Eagle’s medium (DEME) was purchased from HyClone (USA). Fetal bovine serum (FBS) was got from Hangzhou Ever Green Organism Engineering Materials Co., Ltd. (Hangzhou, China). Human embryonic lung fibroblasts (HELFs) were obtained from Nanjing Keygen Biotech Co., Ltd. (Nanjing, China).
1.3  Experimental animals  Special pathogen-free mature male Sprague-Dawley rats weighing from 180 to 200 g (about 8 weeks) were procured from Laboratory Animal Center, Nanjing University of Traditional Chinese Medicine (scxk(jing)2006—0008). The study was approved by the Jiangsu Animal Care and Use Committee and followed the national and institutional rules considering animal experiments. Rats were housed in a climate-controlled room under a 12 h light/dark photoperiod. All the rats had free access to food and water. To adapt to the new environment, the rats were held for 1 week before the experiments.
1.4  Preparation of C. reticulatg essential oil  The essential oil was extracted by hydrodistillation method in accordance with the description of the Chinese Pharmacopoeia (2005)[15]. The dried and cut peel of C. reticulate (7.0 kg) was hydrodistilled for 4 h with 4 L/kg distillated water in an all-glass Clevenger apparatus (until no more essential oil was recovered). The essential oil was collected, extracted with ether and refrigerated prior to analysis. The yield of the oil was 0.84% (volume/weight) based on dry plant weight.
1.5  Cell culture and cell viability assay  HELFs were grown in DEME containing 10% FBS and 1% penicillin-streptomycin. The logarithmically growing cells were detached using 0.25% trypsin-phosphate-buffered saline (PBS) and centrifuged, then the cell concentration was adjusted to 6×104 cells/mL using culture medium. Solutions of the essential oil (100 μL) at the desired concentrations in combination with the HELFs (100 μL) were added to the 96-well culture plates and incubated for 24 h. After that, methylthiazol tetrazolium (MTT) method was applied for the cell viability analysis. All cultures were kept in an incubator under the moist condition of 5% CO2 in air at 37 ℃.
     The essential oil was dissolved in dimethyl sulfoxide (DMSO) followed by dilution with culture medium to desired concentrations, and the final concentration of DMSO was 0.1%. DMSO at 0.1% was added into control group and showed no effects on the cells.
1.6  BLM-induced pulmonary fibrosis and treatment  The animal model was prepared following our reported protocol[11]. Briefly, rats were anesthetized by intraperitoneal injection of pentobarbital followed by intratracheal instillation of 5 mg/kg BLM in 2.0 mL/kg PBS, and normal animals received an intratracheal injection of an equal volume of the sterilized saline instead of BLM.
     Forty-two rats were randomly divided into seven groups as follows: normal group, saline plus the 5% tween-80-water (w/w) dissolvent; model group, BLM plus the dissolvent; prednisone group, BLM plus prednisone at a dose of 5 mg/kg per day; essential oil groups, BLM plus essential oil at doses of 25 (EOCR-25 group), 50 (EOCR-50 group), 100 (EOCR-100 group) and 200 mg/kg (EOCR-200 group) per day, respectively. Prednisone and EOCR were dissolved in the 5% tween-80-water (w/w) dissolvent. The day of intratracheal injection of BLM or saline was designated as the 0th day. The normal and model groups were orally given the dissolvent and other groups were orally administered prednisone or EOCR at the designed doses respectively. The drugs were given once a day starting from the first day of the intratracheal injection for 4 weeks.
1.7  Sample collections  The rats in each group were sacrificed and dissected on the 28th day post-BLM treatment under pentobarbital anesthesia (from 40 to 50 mg/kg). The rat blood was collected and centrifuged at 1 500 r/min for 20 min (reactive centrifugal force: 5 000×g; radius of centrifugalization: 16 cm). The serum was kept at -20 ℃ until SOD and MDA levels were analyzed. The right lung was fixed in 10% neutral buffered formalin (pH 7.4) to assess the histopathologic changes via light microscope examination and the protein and mRNA expressions of CTGF. The left lung tissues were homogenized for 5 min in saline using a Teflon homogenizer, and the homogenates were centrifuged at 4 000 r/min for 15 min. All processes were carried out at 4 ℃. The supernatant was used for SOD, MDA and Col Ⅰ level measurements.
1.8  Analytical methods  The fixed right lung was dehydrated and embedded with paraffin. And then 3 to 4 μm-thick sections were cut and stained with hematoxylin and eosin (HE). For evaluating the degrees of alveolitis and fibrosis, a semi-quantitative grading system was applied[16]. The scores of alveolitis and fibrosis in lung specimens were graded from negative (-) to positive (+ to +++) and correspondingly numbered as 0 to 3. All of the score evaluations were conducted by three blinded pathologists.
     Total SOD activity was determined according to the hydroxylamine method[17] following the SOD test kit instruction. Absorbance was measured at 550 nm and the total SOD activity was expressed as units per milliliter (U/mL) in serum and units per milligram of protein (U/mg protein) in lung tissues.
     MDA content was assayed following the MDA test kit instruction, which was based on the thiobarbituric acid method[18]. Absorbance was determined at 532 nm and the level was defined as nanomoles per milliliter (nmol/mL) in serum and nanomoles per milligram of protein (nmol/mg protein) in lung tissues.
      Analysis of Col Ⅰ level in lung tissue was conducted with a double-antibody sandwich ELISA kit following the kit instruction and the content was determined as micrograms per gram (μg/g), wet weight of lung.
     Analyses of the protein and mRNA expressions of CTGF in lung tissues were conducted by immunohistochemical and in situ hybridization methods with commercial available kits, respectively. The experimental protocols followed the kit instructions.
1.9  Statistical analysis  Data are presented as mean±standard deviation (SD). Statistical analysis was done by one-way analysis of variance followed by appropriate post hoc tests including multiple comparison tests (LSD-t test). Alveolitis and fibrosis scores of lung tissues were evaluated by using the Mann-Whitney U-test. All analyses of data were performed using SPSS 11.5 software package and the probability values (P) of 0.05 or less were considered to be statistically significant.

Jump to Section
-  Top
-  Article and Author Info
-  Introduction
-  Materials and methods
-  Results
-  Discussion
-  Acknowledgements
-  Competing interests
-  References
  


2  Results
2.1  Effects of EOCR on the proliferation of HELFs 
The effect of EOCR on the proliferation of HELFs was assessed. As shown in Table 1, the EOCR displayed an inhibitory activity at each concentration. However, the EOCR did not show a dose-dependent effect, while the 15 μg/mL exhibited the most potent inhibitory activity.

 

Table 1  Effects of EOCR on the proliferation of HELFs

(x±s, %)

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*

*P<0.05, vs control group. EOCR: essential oil of Citrus reticulata; HELFs: human embryonic lung fibroblasts.

2.2  Effects of EOCR on rat body weight gain  The difference in body weight between days 0 and 28 was determined as the body weight gain. As shown in Table 2, the BLM-caused lung injury resulted in a marked decrease of rat body weight (model group) compared with normal rats during the experimental period (P<0.01). By the same data as we previously reported[11], treatment with prednisone, a clinical available IPF therapy drug, did not give any benefit on the weight loss caused by BLM. The rats treated by EOCR with dose of 25 mg/kg per day displayed no effect. However, the treatments with doses of 50, 100 and 200 mg/kg per day showed a significant body weight gain at the first week as compared to BLM-treated rats, and this weight increasing effect was exhibited even at the third and forth weeks (P<0.01 or P<0.05).

Table 2  Effects of EOCR on rat body weight gain

(x±s, g)
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

P<0.05, △△P<0.01, vs normal group; P<0.05, ▲▲P<0.01, vs model group. EOCR: essential oil of Citrus reticulata.


2.3  Histopathological changes in the lung tissues  To observe the histopathological changes, the lung tissues after 4-week treatment were stained with HE methods. In the normal group, there were no signs of septal oedema and inflammation, while a few light blue collagen deposition was observed in the alveolar septa in Masson stained section. In the BLM-treated group, the alveolar septa were markedly thickened, accompanied by infiltration of inflammatory cells and fibrotic changes. Furthermore, alveolar spaces became smaller due to the accumulation of collagen and fibroblast proliferation, and remarkable destruction of alveolar structure appeared. In the prednisone-treated group, alveolar walls were slightly thickened, and only some infiltration of inflammatory cells and fibrotic changes displayed. Although only some amelioration on histopathological changes were observed in the EOCR-25 and EOCR-50 groups, EOCR treatments at doses of 100 and 200 mg/kg per day significantly reduced the infiltration of inflammatory cells and collagen deposition, and markedly meliorated the thickened alveolar septa compared with the model group (Figure 1).

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.


     In the semi-quantitative assessment of lung sections, the grades of fibrosis of the seven groups were summarized in Table 3. The data demonstrated that the BLM-treated rats showed a great increase in scores of both alveolitis and fibrosis compared with the normal rats (P<0.01). As expected, prednisone treatment significantly reduced the scores of both alveolitis and fibrosis. In comparison with the BLM group, the alveolitis and fibrosis scores of the EOCR-100 and EOCR-200 groups indicated that these exacerbations were significantly reduced (P<0.01 or P<0.05).

Table 3  Effects of EOCR on the grades of lung alveolitis and fibrosis induced by bleomycin in rats
Group n Alveolitis Fibrosis
+ ++ +++ + ++ +++

Normal

6

4

2

0

4

1

1

Model

6

0

1

2

3△△

0

0

1

5△△

Prednisone 5 mg/kg

6

1

3

1

0

2

3

1 

EOCR 25 mg/kg

6

0

1

4

0

1

3

EOCR 50 mg/kg

6

1

2

2

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▲▲

△△P<0.01, vs normal group; P<0.05, ▲▲P<0.01, vs model group. EOCR: essential oil of Citrus reticulata. The results were analyzed by Kruskal-Wallis H test and Wilcoxon Rank sum test.


2.4  Effects of EOCR on SOD levels in serum and lung tissues  As shown in Table 4, treatment of rats with BLM caused a significant reduction of the SOD levels both in serum and in lung tissues (P<0.01). Prednisone-treated rats showed a significant increase of SOD levels. Although EOCR administration at dose of 25 mg/kg per day did not statistically increased the SOD level, treatments with EOCR at doses of 50, 100 and 200 mg/kg per day significantly elevated the SOD activity at the end of the 4th week compared with the BLM-treated group (P<0.01 or P<0.05).

 

Table 4  Effects of EOCR on superoxide dismutase activities in serum and lung tissues

(x±s)
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▲▲

P<0.05, vs normal group; P<0.05, ▲▲P<0.01, vs model group. EOCR: essential oil of Citrus reticulata.

2.5  Effects of EOCR on MDA contents in serum and lung tissues  As shown in Table 5, BLM treatment produced significant increases of the MDA levels both in serum and lung tissues (P<0.01). Prednisone caused a significant attenuation of the BLM-induced increase of MDA levels in serum and lung tissues. Similar to prednisone treatment, oral administration with EOCR markedly reduced the MDA level except for the dose of 25 mg/kg per day, in which no statistically significant change was observed.

 

Table 5  Effects of EOCR on malondialdehyde contents in serum and lung tissues

(x±s)
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▲▲

P<0.05, vs normal group; P<0.05, ▲▲P<0.01, vs model group. EOCR: essential oil of Citrus reticulata.


2.6  Effects of EOCR on Col Ⅰ content in lung tissues  As shown in Table 6, treatment of rats with BLM (the model group) caused a significant elevation of the Col Ⅰ level compared with the normal rats (P<0.01). The prednisone-treated rats displayed a decrease of Col Ⅰ level as expected. The rats treated with EOCR at doses of 100 and 200 mg/kg per day showed a notable reduction in the Col Ⅰ level (P<0.01 or P<0.05). The other two doses (25 and 50 mg/kg per day) lowered the Col Ⅰ content, however, no statistical difference was observed compared with the untreated group.


Table 6  Effects of EOCR on type Ⅰ collagen content in lung tissues

(x±s)

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

△△P<0.01, vs normal group; P<0.05, ▲▲P<0.01, vs model group. EOCR: essential oil of Citrus reticulata.

2.7  Effects of EOCR on the protein and mRNA expressions of CTGF  BLM-instillation resulted in significant up-regulations of the protein and mRNA expressions of CTGF compared with the dissolvent (P<0.01). Compared with the model group, treatment with prednisone markedly down-regulated the protein and mRNA levels of CTGF (P<0.01 or P<0.05). Oral administration of EOCR at doses of 100 and 200 mg/kg per day greatly reduced the BLM-induced over expressions of the protein and mRNA of CTGF in lung tissues (P<0.01) (Figures 2 and 3, Table 7).

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.

 

Table 7  Effects of EOCR on protein and mRNA expressions of CTGF in lung tissues

(x±s)
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▲▲

Jump to Section
-  Top
-  Article and Author Info
-  Introduction
-  Materials and methods
-  Results
-  Discussion
-  Acknowledgements
-  Competing interests
-  References
  

△△P<0.01, vs normal group; P<0.05, ▲▲P<0.01, vs model group. EOCR: essential oil of Citrus reticulata; CTGF: connective tissue growth factor.

3  Discussion
    
IPF is characterized by increases of fibroblastic proliferation and extracellular matrix remodeling, resulting in a loss of lung function and, eventually, respiratory failure[2]. Fibrosis is a repeatedly injured and reparative process characterized by the formation of excessive fibrous tissue. The accumulation of fibroblasts to the wound site is one of the key events in both normal repair and the development of fibrosis in lungs. In this context, an agent which inhibits the fibroblast differentiation or function might yield great potential therapeutic benefits for IPF[19]. In the present study, EOCR significantly inhibited HELF proliferation. The EOCR at concentration of 15 μg/mL showed the most potent inhibitory activity. This result suggested that the inhibitory activity of EOCR was not due to cytotoxicity.
     BLM is an anticancer agent prescribed for various cancers, including that of the lungs. However, this drug has a dose-dependent pulmonary toxicity, including lung fibrosis, which limits its clinical use[20]. The toxic effect of BLM has been utilized advantageously in a number of experimental approaches to induce pulmonary fibrosis in animal models. In BLM-treated animals, histological hallmarks, such as mural incorporation of collagen and obliteration of the alveolar space could be presented, which are similar to those of IPF patients[21,22]. In the current study, as expected, BLM treatment caused rat body weight loss and significant histopathological changes, such as thickened alveolar walls, smaller alveolar spaces, infiltration of inflammatory cells and collagen deposition. Prednisone did not benefit the body weight loss of rats, but improved the histopathological deterioration of lung tissues, which was in agreement with our previous results[11], and might be due to its side effects, such as inhibition of protein synthesis, reduction of immunological function. However, EOCR treatment not only suppressed the body weight loss, but also significantly improved scores of alveolitis and fibrosis of lung tissues.
     The oxidant/antioxidant balance in the lung is thought to be a key step in the development of pulmonary fibrosis. The SOD is a family of enzymes that play a pivotal role in protecting tissues from being damaged by oxidant stress[23]. Oxidant-induced lipid peroxidation causes a loss of membrane stability and integrity leading to increased transepithelial permeability. The lipid peroxidation could be monitored by measuring the MDA level which results from free radical damage to membrane components of the cells[24]. Thus, besides the histopathological examination, BLM-induced lung injury can be shown indirectly by measurements of antioxidants such as SOD and the indices of lipid peroxidation such as MDA. Data from the present study revealed that BLM significantly decreased SOD production and increased MDA levels in both serum and lung tissues, clearly suggesting that BLM exposure caused the marked decreases in antioxidant enzyme activity and an increase of lipid peroxidation. EOCR administration remarkably attenuated those changes, indicating that the effects of EOCR on lung fibrosis were associated with free radical scavenging and antioxidant activity. Because oxidant-induced damage gives rise to high level of inflammation, above results suggested that EOCR could also exhibit an anti-inflammatory activity indirectly, which should relate to antipulmonary fibrosis effect of EOCR.
     Collagen is the major extracellular matrix component of the lungs and its deposition directly reflects the grade of lung fibrosis. Fibroblasts persist at the sites of fibrosis in IPF, continuously adding to an environment of aberrant excessive collagen deposition. Col Ⅰ constitutes greater than 65% of the total lung collagen in normal human lungs[25], thus, a Col Ⅰ analysis was conducted. The data revealed that BLM treatment significantly promoted Col Ⅰ generation, and EOCR at doses of 100 and 200 mg/kg per day greatly reduced the collagen accumulation. The above results demonstrated that suppression of Col Ⅰ deposition should be one of the key paths that EOCR exerted its preventive effect on pulmonary fibrosis.
     As a downstream mediator of transforming growth factor-β1 (TGF-β1), CTGF, a prototypic member of the CCN (CTGF, cystein rich protein and nephroblastoma overexpressed gene) protein family playing a crucial role in TGF-β-induced connective tissue cell proliferation and extracellular matrix deposition, is capable of conferring susceptibility to BLM-induced fibrosis[26,27]. In the present study, the protein and mRNA expressions of CTGF in lung tissues were significantly up-regulated by BLM-treatment. While, oral administration of EOCR at doses of 100 and 200 mg/kg per day greatly reduced the BLM-induced overexpressions of CTGF protein and mRNA. The results suggested that down-regulation of CTGF is one of the important approaches that EOCR implemented its antipulmonary fibrosis activity.
     As a conclusion, the findings of the present study suggest that the essential oil extracted from the peel of C. reticulata may be a highly promising agent in preventing pulmonary fibrosis induced by BLM. The effects may exert via scavenging the reactive oxygen species produced from inflammatory cells and lung cells, reducing collagen deposition and down-regulating the overexpressions of CTGF protein and mRNA.

Jump to Section
-  Top
-  Article and Author Info
-  Introduction
-  Materials and methods
-  Results
-  Discussion
-  Acknowledgements
-  Competing interests
-  References
  


4  Acknowledgements
    
The authors would like to thank Mr. Ren-sheng Lai, a pathologist of Jiangsu Province Hospital of Traditional Chinese Medicine for his excellent support on the histopathological analyses of lung tissues of rats.

Jump to Section
-  Top
-  Article and Author Info
-  Introduction
-  Materials and methods
-  Results
-  Discussion
-  Acknowledgements
-  Competing interests
-  References
  


5  Competing interests
    
There are no competing interests within this research. The authors have no conflicts of interest to declare in regard to this study.

Jump to Section
-  Top
-  Article and Author Info
-  Introduction
-  Materials and methods
-  Results
-  Discussion
-  Acknowledgements
-  Competing interests
-  References
  
References
1. Han MK, Swigris J, Liu L, Bartholmai B, Murray S, Giardino N, Thompson B, Frederick M, Li D, Schwarz M, Limper A, Flaherty K, Martinez FJ. Gender influences Health-Related Quality of Life in IPF[J]. Respir Med, 2010, 104(5) : 724-730.
    
2. Agarwal R, Jindal SK. Acute exacerbation of idiopathic pulmonary fibrosis: a systematic review[J]. Eur J Intern Med, 2008, 19(4) : 227-235.
    
3. Bringardner BD, Baran CP, Eubank TD, Marsh CB. The role of inflammation in the pathogenesis of idiopathic pulmonary fibrosis[J]. Antioxid Redox Signal, 2008, 10(2) : 287-301.
    
4. Harari S, Caminati A. IPF: new insight on pathogenesis and treatment[J]. Allergy, 2010, 65(5) : 537-553.
    
5. Parra ER, Kairalla RA, Ribeiro de Carvalho CR, Eher E, Capelozzi VL. Inflammatory cell phenotyping of the pulmonary interstitium in idiopathic interstitial pneumonia[J]. Respiration, 2007, 74(2) : 159-169.
    
6. Dempsey DJ. Clinical review: Idiopathic pulmonary fibrosis — past, present and future[J]. Respir Med, 2006, 100(11) : 1871-1885.
    
7. Gomer RH, Lupher ML Jr. Investigational approaches to therapies for idiopathic pulmonary fibrosis[J]. Expert Opin Investig Drugs, 2010, 19(6) : 737-745.
    
8. Yang J, Cui Y, Kolb M. How useful is traditional herbal medicine for pulmonary fibrosis?[J]. Respirology, 2009, 14(8) : 1082-1091.
    
9. Jiang HD, Guan HS. MS80, a novel sulfated oligosaccharide, inhibits pulmonary fibrosis by targeting TGF-beta1 both in vitro and in vivo[J]. Acta Pharmacol Sin, 2009, 30(7) : 973-979.
    
10. Zhou XM, Zhang GC, Li JX, Hou J. Inhibitory effects of citrus extracts on the experimental pulmonary fibrosis[J]. J Ethnopharmacol, 2007, 111(2) : 255-264.
    
11. Zhou XM, Huang MM, He CC, Li JX. Inhibitory effects of citrus extracts on the experimental pulmonary fibrosis[J]. J Ethnopharmacol, 2009, 126(1) : 143-148.
    
12. Peterson JJ, Dwyer JT, Beecher GR, Bhagwat SA, Gebhardt SE, Haytowitz DB, Holden JM. Flavanones in oranges, tangerines (mandarins), tangors, and tangelos: a compilation and review of the data from the analytical literature[J]. J Food Compost Anal, 2006, 19(Suppl) : S66-S73.
  [ScienceDirect]  
13. Yu L, Li X, Liu S, Xu G, Liang Y. Comparative analysis of volatile constituents in Citrus reticulata Blanco using GC-MS and alternative moving window factor analysis[J]. J Sep Sci, 2009, 32(20) : 3457-3465.
    
14. Xu P. Comparison of pharmacological effects in water extract and essential oil of Citrus reticulata[J]. Jiangxi Zhong Yi Xue Yuan Xue Bao, 1998, 10(4) : 172-173. Chinese.
  
15. China Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China (part one). 2005 ed[M]. Beijing: Chemical Industry Press, 2005. 53. Chinese.
16. Szapiel SV, Elson NA, Fulmer JD, Hunninghake GW, Crystal RG. Bleomycin-induced interstitial pulmonary disease in the nude, athymic mouse[J]. Am Rev Respir Dis, 1979, 120(4) : 893-839.
  
17. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction[J]. Anal Biochem, 1979, 95(2) : 351-358.
    
18. Oyanagui Y. Reevaluation of assay methods and establishment of kit for superoxide dismutase activity[J]. Anal Biochem, 1984, 142(2) : 290-296.
    
19. Kohyama T, Yamauchi Y, Takizawa H, Itakura S, Kamitani S, Kato J, Nagase T. Clarithromycin inhibits fibroblast migration[J]. Respir Med, 2008, 102(12) : 1769-1776.
    
20. Chua F, Gauldie J, Laurent GJ. Pulmonary fibrosis: searching for model answers[J]. Am J Respir Cell Mol Biol, 2005, 33(1) : 9-13.
    
21. Moeller A, Ask K, Warburton D, Gauldie J, Kolb M. The bleomycin animal model: a useful tool to investigate treatment options for idiopathic pulmonary fibrosis?[J]. Int J Biochem Cell Biol, 2008, 40(3) : 362-382.
    
22. Gharaee-Kermani M, Ullenbruch M, Phan SH. Animal models of pulmonary fibrosis[J]. Methods Mol Med, 2005, 117: 251-259.
  
23. Gao F, Kinnula VL, Myllrniemi M, Oury TD. Extracellular superoxide dismutase in pulmonary fibrosis[J]. Antioxid Redox Signal, 2008, 10(2) : 343-354.
    
24. Boyaci H, Maral H, Turan G, Bayiit , Dilliolugil M, Yildiz F, Tugay M, Pala A, Erin C. Effects of erdosteine on bleomycin-induced lung fibrosis in rats[J]. Mol Cell Biochem, 2006, 281(1-2) : 129-137.
    
25. Cairns JA, Walls AF. Mast cell tryptase stimulates the synthesis of type Ⅰ collagen in human lung fibroblasts[J]. J Clin Invest, 1997, 99(6) : 1313-1321.
    
26. Kothapalli D, Frazier KS, Welply A, Segarini PR, Grotendorst GR. Transforming growth factor beta induces anchorage-independent growth of NRK fibroblasts via a connective tissue growth factor-dependent signaling pathway[J]. Cell Growth Differ, 1997, 8(1) : 61-68.
  
27. Scotton CJ, Chambers RC. Molecular targets in pulmonary fibrosis: the myofibroblast in focus[J]. Chest, 2007, 132(4) : 1311-1321.
    
Jump to Section
-  Top
-  Article and Author Info
-  Introduction
-  Materials and methods
-  Results
-  Discussion
-  Acknowledgements
-  Competing interests
-  References
  
JCIM
Open Access
THIS ARTICLE
-  Abstract
-  Full text
-  Download PDF file
-  Send to a friend
-  Related articles in JCIM
-  Cited in JCIM
-  Reader's comments
-  Send a comment
    
Reader's comments
1. Latest Pre Paid Mastercard Uk Auctions PrePaid Mastercard black bailey button uggs http://www.ask10.co.uk/black-bailey-button-ugg-boots-sale-p-1.html (2013-9-1 0:00:00)
2. are generally trapped simply by frantic graduated pupils and also busy school fellow workers, just about all besides the requirements of their pupils. More, they are often in charge of the treating one or more North Face Denali Fleece Jackets for Women MONTAGUE BLUE http://www.ogrizkov.net/north-face-denali-fleece-jackets-for-women-montague-blue-t-87.html (2013-10-29 0:00:00)
3. In ell, make up one's mind the features you would like to comprehend in your website. The next step is to judge a website layout and planning how to sort out cheap 200 dirt bike brake light switch http://cheap200dirtbikebrakelightswitch.atvpartsbest.com (2014-3-3 0:00:00)
4. In ell, take the features you would like to embody in your website. The next agreement with is to judge a website layout and planning how to arrange cheap 200 dirt bike brake calipers http://cheap200dirtbikebrakecalipers.atvpartsbest.com (2014-3-8 0:00:00)

Send a comment

 Home | Current Issue | Past Issues | Search | CollectionsRSS | PDA Services | FAQ | SHCIM Online | Chinese
Copyright © 2003-2012 by JCIM Press. All rights reserved. ISSN 1672-1977