Animals and chemicals
Male Sprague-Dawley rats aged 7 weeks were purchased from Youngbio (Seongnam, Korea) and maintained 2~4 per cage at 20~25ºC with a 12-h light-dark cycle. Rat chow and tab water were allowed ad libitum. All procedures were approved by the institutional animal care and research committee of Korea University (KUIACUC-2016-153). Nicotine hydrogen tartrate salt, type II collagen, complete and incomplete Freund’s adjuvant, polyethylene glycol and linalyl acetate were obtained from Sigma-Aldrich (St Louis, MO, USA). Lavender oil was purchased from Aromarant Co. Ltd. (Rottingen, Germany).
Gas chromatography/mass spectrometry profiling of Lavandula angustifolia Mill. oil.
Gas chromatography/mass spectroscopy (GC/MS) profiling of the non-polar constituents in Lavandula angustifolia Mill. oil (Aromarant Co. Ltd., batch no. 180622, Rottingen, Germany) was done using a GC/MS (8890-5977B GC-MS, Agilent, USA). The extract was dissolved and transferred to GC- vials and run on the GC/MS. The instrument was blank with Supleco methanol. The chromatographic conditions were as follows: the samples were separated on a HP-5MS capillary columng (30 m x 0.25 mm x 0.25 μm, Agilent J&W Scientific, USA), and the injection volume was 1μL with a spilt ratio 100:1. The inlet temperature was 230°C, highly pure helium gas was used as the carrier gas, and the flow rate was 1.2 mL/min. The initial temperature was 40°C, which was maintained for 10 min, and the temperature was increased to 300°C at the rate of 5°C/min. The final temperature was held for 10 min. The mass spectrum conditions were as follows: ion source temperature of 230°C, transfer line temperature of 250°C, full scanning mode, mass scanning range of m/z 40-500. Identification of the non-polar phytoconstituents was by comparison with those available in the National Institute of Standards and Technology (NIST) library coupled to the instrument.
Experimental protocol
Fifty-two rats were randomly allocated to seven groups: a normal control (NC) group, a collagen-induced arthritis (CIA) group, a CIA plus chronic nicotine-induced HTN (H-CIA) group, and H-CIA groups treated with 10 mg/kg lavender, 100 mg/kg lavender, 10 mg/kg linalyl acetate, and 100 mg/kg linalyl acetate. CIA and chronic nicotine-induced HTN were induced simultaneously (Fig. 2). HTN was induced by intraperitoneal injection of 0.8 mg/kg/day nicotine for 3 weeks, starting on the first experimental day, followed by a boost of 3 mg/kg nicotine on day 22 [22]. CIA was induced by injection of 2 mg/ml type II collagen obtained from chicken sternal cartilage and dissolved in 0.01 N glacial acetic acid emulsified in an equal volume of complete Freund’s adjuvant, and a second injection of type II collagen emulsified in incomplete Freund’s adjuvant on day 19 [23].
Lavender and linalyl acetate were dissolved in polyethylene glycol and injected intraperitoneally twice per week during the entire follow up period. All groups of rats were followed-up for 35 days. Hind paw thickness, body weight, and BP were measured on days 0, 8, 15, 22, 29, and 35. At the end of the experimental protocol, blood was obtained from the inferior vena cava and the serum separated by centrifugation at 3,000 g for 20 min. The thoracic aorta and both gastrocnemius muscles were carefully removed from each rat for further evaluation.
Hind paw thickness and BP measurements
Joint edema was assessed as the average thickness on both sides of hind paw that measured using calipers (Mitutoyo, Japan), as previously described [24]. BP was measured using a volume-pressure recording tail cuff method (CODA-6 System, Kent Scientific, Torrington, CT). Rats were placed in a holding device maintained at 38°C on a heating plate for 10 minutes. BP was measured 15 times, and the average value were used.
Measurements of aorta endothelium thickness
Extracted aortae were fixed in 4% paraformaldehyde and sectioned into 4 um-thick slices. The slices were stained with hematoxylin and eosin (H&E) and imaged at 400x magnification with a Nikon DS-Ri2 fluorescence microscope (Nikon, Japan). Images were captured with a digital camera attached to the microscope, and endothelium thickness per 50 µm was determined by NIS Elements imaging software (Nikon, Japan), using the formula: total vessel wall diameter - (outer vessel wall diameter + middle vessel wall diameter). Arterial endothelium thickness was calculated as the average of three measurements along the wall of each artery.
Measurements of serum interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) concentrations
Concentrations of serum IL-6 and IGF-1 were measured using rat IL-6 (Komabiotech, Korea) and IGF-1 (Abcam, UK) Enzyme-Linked Immunosorbent Assay (ELISA) kits, respectively. Results were obtained by measuring absorbance at 405 nm using a microplate ELISA reader (BMG Labtech, Germany), according to the manufacturer's instructions.
Histopathological analysis of muscle tissue
The inflammatory status of gastrocnemius muscle tissue was assessed histopathologically. Muscle tissues fixed in 4% paraformaldehyde and embedded to paraffin blocks were cut into 4-μm–thick sections, which were mounted onto slides. The slides were stained with H&E and imaged at 400× magnification using a Nikon DS-Ri2 fluorescence microscope (Nikon, Japan). The threshold of each of the three images was measured the background, connective tissue, and total tissue by Image J software (National Institutes of Health, USA). Percentage of fibrosis in the tissue was calculated as follows: background area was subtracted from connective tissue and total tissue, then followed the formula: percentage of fibrosis = connective tissue/ connective tissue + total tissue x100 [25].
Measurement of mitochondrial membrane potential
Muscle tissue samples were homogenized and lysed for 30 min on ice with lysis buffer and centrifuged for 15 min at 13,000 g. Mitochondrial membrane potential was determined using a fluorescent JC1- Mitochondrial Membrane Potential Assay Kit (Abcam, UK). The fluorescence intensities of the aggregate and monomer forms were measured with a fluorescence microplate reader (PerkinElmer, Korea) operated at excitation/emission wavelengths of 535 and 485 nm, respectively. The results are presented as the ratio of aggregate/monomer fluorescence intensity.
Statistics
All data are presented as means ± SEM, with percentages reported relative to the H-CIA group. Multiple groups were compared by one-way ANOVA followed by Tukey's honestly significant difference (HSD) post hoc tests. All statistical analyses were performed using SPSS version 24.0 software (SPSS, Chicago, IL), with p < 0.05 considered statistically significant.