Evaluation of Two herbal formulation for cigarette smoke extract induced pulmonary inflammation in C57BL/6 mice.

 

STUDY TITLE

Evaluation of Two herbal formulation for cigarette smoke extract induced pulmonary inflammation in C57BL/6 mice.

 

Experimental animals

RAT

Species	:	Mus musculus
Strain	:	C57BL/6
Source	:	Hylasco Pvt Ltd, Hyderabad, India
Sex	:	male
Age	:	6-8 weeks
Body weight range	:	20-25  g
No. of animals/group	:	08
Randomization	:	Animals will be randomized based on body weight
Acclimatization	:	Animals will be acclimatized for one week
Identification of animals	:	By body marking and cage labelling

 Allocation of Animals

Groups	Treatment	Dose & Regimen	No. of Animals
G1	Normal Control	0.5% MC; p.o., b.i.d	8
G2	Disease Control	0.5% MC; p.o., b.i.d.	8
G3	Reference Control-Roflumilast	10 mg/kg; p.o, q.d.	8
G4	Test Formulation-1	60 mg/kg; p.o., b.i.d.#	8
G5	Test Formulation-1	200 mg/kg; p.o., b.i.d.#	8
G6	Test Formulation-1	600 mg/kg; p.o., b.i.d,#	8
G7	Test Formulation-1	1200 mg/kg; p.o., b.i.d,#	8
G8	Test Formulation-2	60 mg/kg; p.o., b.i.d.*	8
G9	Test Formulation-2	200 mg/kg; p.o., b.i.d.*	8
G10	Test Formulation-2	600 mg/kg; p.o., b.i.d,*	8
G11	Test Formulation-2	1200 mg/kg; p.o., b.i.d,*	8

 

#, * Animal doses have been calculated based on human therapeutic dose; TF-1, TF-2, 82 g/person per day

Experimental Procedure(S)

Anti-inflammatory activity of two herbal formulation using cigarette smoke extract (CSE) induced pulmonary inflammation will be carried out using 88 female C57BL/6 mice. Their weights will be recorded and randomly divided into a set of 11 groups. Animal doses of two herbal formulation will be calculated based on human dose i.e. 2g/day. Animals will receive vehicle/test substance orally two-week prior to CSE administration. Pulmonary inflammation will be induced by cigarette smoke extract in the disease & treatment group for 4 weeks (6 days/week). For CSE induction, mice will be anaesthetized by exposure to isoflurane, held in vertical position and CSE will be placed on edge of nostril and inhaled by mouse. Along with the CSE, animals will be treated with test drug daily and continued till the end of the experiment. Mice will be daily monitored for clinical signs. Body weight will be measured weekly twice. At the end of CSE induction and treatment, on termination day, mice will be anaesthetized, In-vivo imaging & AHR will be performed and Bronchoalveolar lavage (BAL) and blood will be collected for determination of total BAL cells, % macrophages, Total macrophages, PMN, total PMNs, BAL neutrophils, BAL cytokines (IL-6, IL-8, IL-10, IL-1β, IL-17, TNF-α, TGF-β1), serum cotinine level and Serum mucin TNF-α. Animals will be sacrificed; lung will be excised and weighed, portions of the left lung will be used for histology & fixed in 10% neutral buffer formalin and the right will be frozen in liquid nitrogen and stored at -80°C, until use. Antioxidant parameters such as SOD, catalase, GSH, GPx, MDA and hydroxyproline will be measured in lung tissue. Quantification of BACH-1, MCP-1, TNF-α, IFN-γ, IL-6, MMP-9 & MMP-12, TIMP-1, Caspase-3, β-actin, FAS, Collagen-α, NRF-2, VGEF, HO-1, MUC5AC, MUC5B & TGF-β1 will be performed in right lung tissue using RT-PCR.

      Animals procurement & randomization
88 male C57BL/6 mice, aged 6-8 weeks & weighing between 20-25g will be procured from CCSEA approved vendor in India and quarantined for 1 weak. After the quarantine of animals, animals will be transferred to the experiment room and housed under the standard housing conditions. Animals will be randomly divided into 11 different groups and acclimatized for one week. The animal will receive water and standard rodent chow ad libitum for the entire course of the study
Cigarette smoke extract (CSE) preparation and administration
Cigarette smoke extract (CSE) will be prepared by bubbling the cigarette smoke from 10 University of Kentucky 1R6F reference cigarette through 10 mL of sterile, pyrogen-free PBS (pH = 7.4). Smoke will be drawn through the medium using a standard vacuum pump. One cigarette will be burned for 6 minutes. CSE will be made fresh before each treatment and administered within 30 minutes. Mice will be anaesthetized by exposure to isoflurane in a jar until effect. Mice will be then held in a vertical position and 50 uL of CSE will be placed on the edge of the nostril and inhaled by the mouse. Mice will be treated for 6 days/ week for 4 weeks.(Elliott et al., 2006), (J. Lamb et al., 2012).
 Vehicle, Test and Reference Substance Administration

Reference drug Roflumilast will be procured from the medical shop and test drugs two herbal formulation and doses will be calculated from their human dose i.e. 2g/day. Animals will receive vehicle/test substance orally two-week prior to CSE administration. CSE instillation will be started after two week of test drug administration and continued till the end of the experiment along with vehicle/test & reference drug. Animals will receive vehicle/test substance/reference substance orally in a dose volume of 5 ml/kg. The dosing details of individual groups are: -

·       Group G1 will serve as normal control and will be administered 0.5% MC, b.i.d.

·       Group G2 will serve as disease control and will be treated with 0.5% MC, b.i.d.

       Animals of group G3 will be treated with reference substance- Roflumilast at the dose of 10 mg/kg, q.d. (J. Lamb et al., 2012).

·       Animals of groups G4, G5, G6 and G7 will be treated with TF-1 at the dose levels of 10, 30, 100 and 300 mg/kg, b.i.d. respectively.     

·       Animals of groups G8, G9, G10 and G11 will be treated with TF-2 at the dose levels of 10, 30, 100 and 300 mg/kg, b.i.d. respectively.

Blood collection and serum separation

At the end of the CSE induction & treatment period and on termination day, mice will be anaesthetized with 50mg/kg thiopentone sodium. Blood will be collected and incubated for 1 h at 37°C, and serum will be obtained by centrifugation at 3000 rpm, 4℃ for 10 min and stored at -80°C till further analysis. Serum cotinine level and mucin TNF-α will be estimated.
LUNG FUNCTION MEASUREMENT
Lung function will be assessed with a ventilator for small animals (flexivent™; SCIREQ). After 04 weeks of exposure to CSE, mice will be anesthetized with thiopentone sodium (50 mg/kg, ip) and tracheotomised. Then, a 18G catheter will be inserted into the trachea and a catheter will be used to connected to flexiVent instrument equipped with a small animal ventilator (Emka-Scireq) for the measurement of in vivo respiratory mechanics and airway hyperresponsiveness. Succinylcholine (7.5 mg/kg) will be administered to animals, intraperitoneally prior to pulmonary function testing to prevent respiratory drive artifacts. Baseline measurements of respiratory mechanics will be assessed prior to challenge with increasing concentration of aerosolized methacholine. The tidal volume of the ventilator will be 10 ml/kg, and respiratory rate will be 120 breaths/min. Lung function will be expressed as Newtonian resistance (Rn), respiratory system resistance (Rrs), respiratory system compliance (Crs), and respiratory system elasticity (Ers).(Li et al., 2017).
 
Preparation OF BRONCHOALVEOLAR LAVAGE FLUID (BALF) FLUID AND CELL COUNTS
After 04 weeks of CSE exposure & treatment, mice will be anaesthetized by using 50mg/kg thiopentone sodium and Bronchoalveolar lavage fluid (BALF) will be obtained by cannulating the trachea and lavaging it five times with 01 mL of Hank's Balanced Salt Solution (HBSS). BALF cells will be centrifuged with HBSS at 3500 RPM for 10 min at 4 °C. Supernatants will be collected and stored at -80°C to measure different cytokines & inflammatory factors. Pelleted BALF cells will be resuspended in 1 ml HBSS, and erythrocytes, neutrophils, and monocytes will be quantified by using a Hematology analyser. Cell smear will be prepared by centrifuging the 50µL of resuspended cells in a cyto-centrifuge and differential cell count will be performed using an inverted microscope. (Li et al., 2017). Total BAL cells, % macrophages, Total macrophages, PMN, total PMNs, BAL neutrophils, BAL cytokines (IL-6, IL-8, IL-10, IL-1β, IL-17, TNF-α, TGF-β1) will be determined. 

Histopathological analysis  

The left lung tissues will be cut into 3 mm-thick slices and immersed in a 10% neutral buffer formalin solution to allow complete fixation. Thereafter, the slices will be embedded in paraffin, and 4-μm thick sections will be cut and stained with haematoxylin and eosin (H&E) for inflammation observation and Masson trichome (MT) staining for evaluation of collagen deposition. Then light microscopy will be used to observe the morphological changes. Paraffin-embedded lung sections will be deparanized, washed with PBS and antigen retrieval will be performed by autoclave (120°C for 20 min) in 10 mM citric acid buffer (pH 9.0). Then immunofluorescence staining will be performed. Immunofluorescence images will be taken using an inverted microscope. For the quantitation of positive cells, multiple fields containing more than a hundred cells will be analysed in each sample. The right lung portion will be stored at -80°C for the further analysis. (Wang et al., 2019), (Mikawa et al., 2020).

Antioxidant parameters and gene expression analysis

Antioxidant parameters such as SOD, catalase, GSH, GPx, MDA, hydroxyproline, HDAC will be measured in frozen lung tissue. Quantification of BACH-1, MCP-1, TNF-α, IFN-γ, IL-6, MMP-9 & MMP-12, TIMP-1, Caspase-3, β-actin, FAS, Collagen-α, NRF-2, VGEF, HO-1, MUC5AC, MUC5B, TGF-β1, Smad2 and Smad3 will be performed in frozen right lung tissue using RT-PCR.

PARAMETERS TO BE EVALUATED

·       Clinical signs

·       Bodyweight (weekly twice)

·       Airway hyperresponsiveness (AHR)

·       Serum parameters – Serum cotinine level and mucin TNF-α.

·       BAL Contents: Total BAL cells, % macrophages, Total macrophages, PMN, total PMNs, BAL neutrophils, BAL cytokines (IL-6, IL-8, IL-10, IL-1β, IL-17, TNF-α, TGF-β1).

·       Lung contents- SOD, catalase, GSH, GPx, MDA, hydroxyproline, HDAC.

·       Gene expression - BACH-1, MCP-1, TNF-α, IFN-γ, IL-6, MMP-9 & MMP-12, TIMP-1, Caspase-3, β-actin, FAS, Collagen-α, NRF-2, VGEF, HO-1, MUC5AC, MUC5B, TGF-β1, Smad2 and Smad3.

Histopathological staining- H&E staining, Masson trichome (MT) staining & Immunofluorescence staining.

 

REFERENCES

a.     Elliott, M., Sisson, J., West, W., & Wyatt, T. (2006). Differential in vivo effects of whole cigarette smoke exposure versus cigarette smoke extract on mouse ciliated tracheal epithelium. Experimental Lung Research, 32(3–4), 99–118. https://doi.org/10.1080/01902140600710546

b.     J. Lamb, D., Parker, N., Ulrich, K., Walsh, R., & Yeadon, M. (2012). Characterisation of a Mouse Model of Cigarette Smoke Extract-Induced Lung Inflammation. Journal of Pulmonary & Respiratory Medicine, 02(03). https://doi.org/10.4172/2161-105x.1000125

c.      Li, Y., Yu, G., Yuan, S., Tan, C., Lian, P., Fu, L., Hou, Q., Xu, B., & Wang, H. (2017). Cigarette Smoke-Induced Pulmonary Inflammation and Autophagy Are Attenuated in Ephx2-Deficient Mice. Inflammation, 40(2), 497–510. https://doi.org/10.1007/s10753-016-0495-z

d.     Mikawa, R., Sato, T., Suzuki, Y., Baskoro, H., Kawaguchi, K., & Sugimoto, M. (2020). P19arf exacerbates cigarette smoke‐induced pulmonary dysfunction. Biomolecules, 10(3). https://doi.org/10.3390/biom10030462

e.      Wang, W., Zha, G., Zou, J. jing, Wang, X., Li, C. nian, & Wu, X. jun. (2019). Berberine Attenuates Cigarette Smoke Extract-induced Airway Inflammation in Mice: Involvement of TGF-β1/Smads Signaling Pathway. Current Medical Science, 39(5), 748–753. https://doi.org/10.1007/s11596-019-2101-8

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