Animals and models
Thirty-five healthy male rats were used in this experiment. 6–8 weeks old SD (weight 200–300 g) rats were got from Hunan Slaughter Jingda Laboratory Animal Co., Ltd.). The experimental mice can get food and water normally, and can have 12 h of light with the stable environment temperature (25 ± 2 ° C) and 55 ± 10% relative humidity. Before the experiment, the rats were acclimatized for 7 days. Importantly, in this study, the animals were divided into control group, model group, Qilongtian low dose group, Qilongtian medium dose group, Qilongtian high dose group, Qilongtian high dose + OE-HMGB1 group, Qilongtian high dose + OE-HMGB1 + AMPKi group, with 5 animals in each group.
Model establishment: COPD rat model was established by passive smoking and intratracheal LPS injection. Using intraperitoneal ketamine (35 mg/kg) to anesthetize the rats, and LPS (0.2 ml, 1 mg/ml) was given twice on the 1st day and 14th day, and 200 µL saline was injected in the control group. From the 2nd to the 13th day and from the 15th to the 30th day, using a self-made plexiglass fumigation box to place the rats, and the rats were smoked cigarettes every day, burning 20 cigarettes each time, and removed after 30 minutes of fumigation, while the control group was not fumigated.
Administration
From the 8th day of modeling, using 0.27, 0.54, 1.08 G/kg Qilongtian doses to gavage the low, medium and high doses of groups every day, and the control group was not treated. The high dose Qilongtian + OE-HMGB1 group was additionally injected with OE-HMGB1 plasmid (120 nM/kg) into the trachea. High-dose Qilongtian + OE-HMGB1 + AMPKi group received 120 nM/kg OE-HMGB1 plasmid and AMPKi (AMPK inhibitor, 4 mg/mL, 50 µL) intratracheally for three weeks. The rats were sacrificed on the 29th day, and collecting the bronchoalveolar lavage fluid (BALF) and lung tissue for related detection.
Bronchoalveolar Lavage Fluid Extraction
Ligating the right main bronchus, and lavaging the left lung with 3 ml of sterile phosphate buffered saline (PBS) via tracheal intubation for three times, and BALF was collected. At 1200 rpm and 4°C centrifuge, BALF samples were treated immediately for 10 minutes, and collect the supernatant and stored at − 80 ° C for subsequent analysis.
Rett-giemsa Staining
A small amount of BALF was randomly placed in a hematocrit plate to make smears. Through the Wright-Giemsa staining kit, using formaldehyde to fix the specimens for 10 min. Then using distilled water to wash it, and then the Giemsa staining solution was dropped on the cells for 10–15 min. After washing with distilled water, cover glass was added while wet, and using glycerol to seale the slides and using a microscope to observe it.
He Staining
Using paraformaldehyde to fixe the lung tissues for 24 h, then the lung tissues were immersed in ethanol for dehydration, and then paraffin-embedded for sectioning. After dewaxing with xylene, soak the dewaxed slides in PBS for 5 min, drain the excess liquid and put it in hematoxylin and eosin solution for staining, and observe the staining under a microscope (Eclipse 80i, Nikon ) with an appropriate magnification.
Masson Staining
After conventional dewaxing in water, the slices were dyed with hematoxylin for 5 minutes and washed with tap water until the color of the slices turned purple. Subsequently, in order to remove cytoplasmic staining, we used 1% hydrochloric acid alcohol for color separation for 2 s, and washed with tap water until the color turned blue. In addition, the slices were stained with Masson ponceau acid magenta solution for 10 minutes. After soaking in 2% glacial acetic acid solution for a period of time, the slices are differentiated in 1% phosphomolybdic acid solution for 3–5 minutes. Cut aniline blue and dye for 5 minutes without washing, then soak in 0.2% glacial acetic acid solution for 1 minute. Finally, using absolute ethanol to dehydrate the slices, using xylene to transparent it, using neutral glue yo seale it, observed and photographed by optical microscope.
Immunohistochemistry
After dewaxing and hydrating, the slices were sealed and infiltrated with permeable liquid, and the antigen was repaired by microwave. Blocking some nonspecific binding sites with serum from the same source as the secondary antibody; adding the diluted primary antibody dropwise, overnight at 4 ℃; drop diluted secondary antibody Incubated at 37 ℃ for 2 H; DAB-H2O2 was used for color development, followed by staining with Mayer hematoxylin, washing with water, differentiation with hydrochloric acid and alcohol, dehydration, adding neutral gum to seal the slides, and taking photos for observation.
Western Blot
The proteins were extracted utilizing RIPA lysis buffer (Sangon Biotech, Shanghai) and a lysate containing Phenylmethanesulfonyl fluoride (PMSF) was added. Using BCA assay to determine the protein concent. 20% SDS-Page gel electrophoresis was for the target protein with the initial voltage of 90 v and the adjustment voltage of 120 v. Using skim milk (5%) to blocke the film for 3 h, when the transferred isolated proteins to PVDF membranes, and the PVDF membrane were incubated with the special antibodies: Parkin, Beclin1, P62, LC3, p-AMPK, AMPK, p-mTOR, p-ULK1, ATG5, PINK1, ULK1, GAPDH, all 1:1000), and incubating at 25℃ for 2 h. HRP-labeled secondary antibody (1:2000) was added and incubated at 25℃ for 1 h. ECL color development, Image J analysis of band gray values.
ELISA
In this study, mouse ELISA kit (ThermoFisher, USA) was used to detect the levels of TNF-α and IL-1β levels in BAL solution. Simply, in order to obtain bronchoalveolar lavage fluid (BAL), we inserted 23 G catheter into trachea. Then, we washed the lungs with 0.8mL PBS, and centrifuged the collected BAL solution for 5 minutes at 4℃ at 1500 rpm. Count the total cells with a blood cell counter. Cell needles on microscope slides were used for differential cell counting, and cells were stained by Diff-Quick staining (Sysmax, Japen). To identify different populations of inflammatory cells, we counted 400 cells in randomly selected sections. Finally, glutathione (GSH), peroxidase (SOD), and malondialdehyde (MDA) levels in tissue lysates were determined using a commercial ELISA kit (Abcam, USA).
Rt-qpcr
Using Total RNA kit to get the total RNA and treated with DNase I (Takara, Japan). Taking 1 µL RNA sample and conduct RNA integrity detection on 1% agarose gel electrophoresis, and taking 1 µl RNA sample after dilution to measure OD value, through the ratio of OD260/OD280 Identify total RNA purity, and the RNA sample was subsequently stored to -80°C. For miRNA quantification, the internal reference gene was U6, and the level was detected on an ABI7500 real-time PCR system. qRT-PCR conditions: 95 ℃ for 30 s, 95 ℃ for 3, followed by annealing at 60℃ for 30 s for 40 cycles. With the design of Premier 5.0, all primers used in this study were designed. The results were calculated by 2-ΔΔCt method after repeated over 3 times at least. Primers for RT-qPCR: HMGB1: F: 5 '-ATGGGCAAAGGAGATCCTAAAAAG-3 3', R: 5 '-TTATTCATCATCATCATCTTCTTCTTCAT-3 3'; GADPH: F: 5′- ATGCTGCCCTTACCCCGG-3′,
R 5′- TTACTCCTTGGAGGCCATGTAGG-3′;
Cell Culture, Transfection, And Cse Treatment
In this study, the Human bronchial epithelial cells Beas-2B were got from the American Type Culture Collection (ATCC, USA). RMPI-1640 medium (Gibco, USA) medium containing FBS (10%), penicillin (1%) and streptomycin (1%) was used to culture these cells at 37 ° C in 5% CO2 incubator. When the cell density has 80%, passage was performed. Subsequently when the cell density reached 2–3 passages, these experiments were performed. Using Lipofectamine 2000 reagent to transfecte OE-HMGB1 or non-targeted control (Sangon Biotech, China) into cells (Invitrogen, USA). Beas-2B cells were then exposed to CSE at a concentration of 2% for 24 h. Subsequently, cells were washed with serum-free RPMI and treated with Qilongtian at a concentration of 25 ng for 6 H at 37 ° C in 5% CO2 after CSE treatment. In addition, using H2O to wash the cultures twice to remove unabsorbed Qilongtian.
Mtr (Mitotracker Red) And Lc3 Co-localize Mitochondrial Autophagy
The culture plate was added the appropriate amount of culture medium to cover the coverslip for slide-climbing culture. MTR (MitoTracker red) staining solution preheated at 37℃ was incubated at 37℃ for 30 min. Using TBST to wash the cells, using paraformaldehyde (4%) to fixed it for 30 min, using Triton X-100 to permeabilize (0.2%) for 30 min, and using TBST to wash it at 25℃. Using bovine serum albumin (5%) to blocking the cells and using the rabbit polyclonal antibody LC3B to incubate 12 h at 4 ° C. Then, It was then using 2 µg/ml of goat anti-rabbit IgG to incubate it for 30 min at 37 ° C. Subsequently, using DAPIFine stain to stain the cells at 25 ° C with for 5 min and passFluorescence microscopy(Olympus, Japan) observed and photographed.
Mitosox Red Staining
In this study, using MitoSOX Red (5 µM) to incubate the cells at 37°C for 10 min to detect the level of mtROS in the cells.Fluorescence microscopy(Olympus, Japan) to detect the fluorescence of MitoSOX Red.
Jc-1staining
In this study, we measured mitochondrial membrane potential using JC-1 staining (Thermo Fisher Scientific, USA). Briefly, cells were incubated for 30 min in dark at 37°C using JC-1 solution. Subsequently, the supernatant was discarded and the cells were washed twice with PBS. Finally, it was imaged under a fluorescence microscope (Olympus, Japan).
Detection Of Mitochondrial Complex Enzyme Activity
Through UV spectrophotometry, we evaluated the activity of mitochondrial respiratory complex enzymes I, II, III, and IV by the mitochondrial complex I, II, III, and IV activity assay kit (Solarbio, China).
Atp Synthesis Assay
In the cells, we used ATP assay kit (Beyotime, China) to detect the ATP content. Briefly, using RIPA lysis buffer (Beyotime, China) to lyse the cells. And at 4°C, the cells were centrifuged at 13000 × G for 10 min, and the supernatant was collected. At 25°C, the supernatant was then transferred to the prepared wells and allowed to stand for 5 min. Then, using a microplate reader ro detect the luminescence of ATP, and on the basis of the standard curve, the ATP concentration was calculated.
Statistical Analysis
In this study, all experiments in this paper were repeated 3 times, and GraphPad Prism 8.0 was used for all statistical analyses. The statistical significance value was 0.05. The mean value and standard deviation (SD) was showed in this study. Expect that, using one-way analysis and Student's t test to analyze the data.