Grade 5

BALB/c mice (n=6–8 per group) were immunized on days 0 and 7 by intraperitoneal (i.p.) injection of OVA/alum (10 μg OVA emulsified in 1 mg aluminium hydroxide; grade V; Sigma-Aldrich, St Louis, MO, USA) and were exposed to OVA aerosol challenges (grade III) on days 17–19; aerosol challenges were generated from a jet nebulizer delivering 1% OVA in phosphate-buffered saline (PBS) for 30 min. To examine the effect of MSC transplantation, 24 h prior to the first OVA aerosol challenge, a number of the mice received an intravenous injection of 10⁶ MSCs. The positive control mice received PBS instead of MSCs. The negative control mice were neither sensitized with OVA nor given MSC treatment prior to OVA aerosol challenges. Twenty-four hours after the last OVA exposure, bronchoalveolar lavage fluid (BALF) was collected with 1-ml washes of Ca²⁺- and Mg²⁺-free Hank’s balanced salt solution (HBSS; Invitrogen Life Technologies) supplemented with 0.1 mM EDTA, and slides were prepared with Cytospin III (Shandon Scientific, Pittsburgh, PA, USA) and stained with May-Giemsa (Sigma-Aldrich) for determination of cell counts. Immediately after the collection of BALF, lungs were resected and stored in optimum cutting temperature freezing medium. In certain experiments, MLNs and lungs were digested with collage-nase/DNAse (Sigma-Aldrich) as previously described (25 (link)).

The protocol of experimental model establishing was adopted as previously described [12 (link)]. Mice were sensitized IP with 20 μg chicken OVA (Grade V, Sigma Aldrich Co., St. Louis, MO) emulsified in 2.0 mg of alum (Shanghai No.4 Reagent & H.V. Chemical Industries Ltd., Shanghai, China) in a total volume of 100 μl of 0.9% sterile saline on days 1 and 14. Non-sensitized mice just received 2.0 mg of alum in 0.9% saline. On day 24, 25, and 26, mice received three aerosol challenges with 5% OVA (wt/vol) (Grade II, Sigma Aldrich Co., St. Louis, MO) in 0.9% endotoxin-free saline (non-sensitized mice received saline only) for 30 min daily. Using a plastic box (50 × 30 × 40 cm³) as exposure chamber, where the air was kept at 20 – 22°C and relative humidity 50 – 60%, the aerosol was generated by a sidestream jet nebulizer (PARI BOY, Starnberg, Germany) into the chamber and mice were put into stainless cages inside the chamber with irradiated food and acidified water provided ad libitum. One hour before each challenge, mice received dexamethasone (2 mg/kg) IP in 0.1 ml endotoxin-free PBS or PBS alone. The dosage of dexamethasone has been adopted by multiple publications [16 (link)].

The model of choice for allergic pulmonary inflammation induction was previously reported [21 (link), 22 (link)] and is detailed in Figure 1. Animals were immunized intraperitonially on days 0 and 14 using 50 μg of OVA (Grade IV, Sigma Aldrich, St. Louis, MO) with 6 mg of Al (OH)3 adjuvant (Pepsamar, Sandei-Synthelabo SA, Rio de Janeiro, Brazil) diluted in 0.2 mL saline. Eight days after the second immunization, the mice were placed in a Plexiglas box (30 x 15 x 20 cm) coupled to an ultrasonic nebulizer (US-1000, ICEL, São Paulo, Brazil), and an aerosol of OVA solution (1%) (Grade V, Sigma Chemical Co., St. Louis, MO) diluted in 0.9% sterile NaCl solution (saline) was generated for 30 minutes 4 times every other day. Control mice received the adjuvant intraperitoneally and were exposed to nebulized aerosol comprised of saline (0.9% NaCl).
Treated mice received intraperitoneal injections containing 2 mg/kg of CrataBL administered in 0.2 mL for each dose over seven consecutive days, two hours after inhalations exposures (days 22 to 28) (Figure 1). The administered dose was the same as that used in an experimental model of thrombosis [23 (link)], similar to the elastase inhibitor BbCI (Bauhinia bauhinioides Cruzipain Inhibitor) which is also a proteinase inhibitor used in asthma model [24 (link), 25 ].

A rat model of allergic asthma was used as previously described [32 (link)]. Briefly, male Brown Norway rats were sensitised on day 0, 14 and 21 with chicken ovalbumin (OVA) (100 μg/rat, i.p., Grade V, Sigma, UK.) administered with Alum (20 mg/rat aluminium hydroxide and 20 mg/rat magnesium hydroxide, i.p., Alum™ Thermo Scientific, UK). Rats were exposed to room air or CS for 1 h, twice a day (4 h apart) on day 21, 22, 23, 24, 25, 26, and 27. On day 28 the rats were exposed to air/smoke in the morning and in the afternoon the rats were challenged with vehicle (saline, aerosolised for 30 min) or OVA (1% w/v, aerosolised for 30 min). The LAR was monitored in conscious BN rats for 1 to 6 h after challenge as previously described [31 , 35 ]. The following day the animals were euthanised with pentobarbitone (200 mg/kg, i.p., Centaur Services, UK). Bronchoalveolar lavage (BAL) was carried out by injecting 3 ml of RPMI culture medium (Invitrogen, UK) via a cannula inserted into the trachea, waiting 30 s and then removing it. This was repeated and the collected BAL fluid (BALF) pooled. Total and type of white cells in the BALF were determined as previously described [32 (link), 35 ].

Forty mice were randomly divided into the control group (NC), the control + acupoint catgut-embedding group (NA), the asthma group (AST), the asthma + acupoint catgut-embedding group (ASA), and the asthma + sham-acupoint catgut-embedding group (ASS), n = 8 in each group. To establish a model of allergic asthma, mice were sensitized and challenged with ovalbumin (OVA) by intraperitoneal injection and atomization inhalation as mentioned previously [20 (link)]. In brief, on day 0 and day 7 of model building (sensitization stage), mice in groups AST, ASS, and ASA were given a mixture of OVA (20 μg, Grade V; Sigma-Aldrich) and aluminum hydroxide (2 mg, Thermo Scientific) by intraperitoneally injection. From day 14 to 16 (challenge stage), mice were given an inhalation of 3% OVA solution (w/v) for 30 min through an ultrasonic nebulizer (402AI, Yuyue Medical Equipment Co., Ltd., Jiangsu, China). Then, from day 21 to 48, mice were challenged daily. Additionally, mice in the NC and NA groups were sensitized and challenged with saline.

WT BALB/c mice or CD1d^-/- mice were intraperitoneally sensitized on day 0 and 14 with 20 μg of chicken ovalbumin (OVA; grade V, Sigma, St Louis, MO, USA) emulsified in 2 mg of aluminum hydroxide (Thermo Scientific Pierce, Rockford, Rockford, IL, USA) in 200 μL of phosphate-buffered saline (PBS). Intranasal OVA challenges (100 μg/50 μL in PBS) were administered on days 25, 26, and 27. Age- and sex-matched control mice were similarly treated with PBS alone. The mice were sacrificed 24 h after the final challenge and their sera, BALF, lungs, and spleen were harvested and analyzed.