Female BALB/C mice (age, 4–6 weeks; weight, 15±1 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and bred and housed at the animal center at China Medical University. Mice were housed at 21°C, 55% humidity, under a 12-h light/dark cycle with free access to food and water. Mice were randomly divided into the following seven groups (n=10/group): Control group, MPP model group, Baicalin group, azithromycin group, miR-221 mimic group, miR-221 negative control group and Baicalin + miR-221 mimic group. Mice were housed and maintained in cages containing five mice/cage. To induce MP infection, each mouse was anesthetized by intraperitoneal injection of 40 mg/kg pentobarbital (Nembutal; Sumitomo Dainippon Pharma Co., Ltd.) (35). Once anesthetized, the mice in the control group were treated with saline. The mice in all other groups were intranasally inoculated with 50 µl MP solution containing 1×1010 CCU/l. This procedure was performed once a day for 3 consecutive days to prepare an MPP model (36). After the last MP inoculation, mice in the treatment groups were intragastrically injected with 80 mg/kg Baicalin or 22.5 mg/kg azithromycin. Mice in the MPP model group and the control group were intraperitoneally injected with the same amount of saline. Mice in the miR-221 mimic group, miR-221 negative control group and Baicalin + miR-221 mimic group received 100 µl miR-221 mimic lentivirus or miR-221 negative control lentivirus at concentration of 1×108 TU/ml by intranasal infusion for 7 consecutive days (37). The miR-221 mimic and negative control sequences are shown in Table I. Lentiviruses containing miR-221-mimics and miR-221-negative control were constructed and synthesized by Shanghai GenePharma Co., Ltd.. Blood samples were collected by cardiac puncture into ETDA-containing plasma separation tubes (BD Biosciences) after anesthesia with 40 mg/kg sodium pentobarbital. Subsequently, mice were euthanized with 120 mg/kg sodium pentobarbital (intraperitoneal) and necropsied immediately. The bronchoalveolar lavage fluid (BALF) and lung tissues of mice were collected and stored at −80°C for further experiments. The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (IACUC no. CMU2018309; Shenyang, China).

Sequences of miR-221 mimics and negative control.

miR-221, microRNA-221.

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