After inhalation, the area under the curve (AUC) of baicalin and oroxyloside in tissues  was 7.9–115.3 and 9.5–16.0 times that observed after intravenous administration, respectively. Baicalin and oroxyloside maintained high concentrations 4 h after inhalation, but only 1 h after intravenous injection. The mean lung-to-plasma concentration ratios of baicalin and oroxyloside were 287.6 and 49.9 times higher than with intravenous administration. Inhaled TRQ achieved the same effect against lipopolysaccharide-induced lung inflammation in mice at doses of only 1/16–1/8 of those administered intravenously. The results indicate that TRQ inhalation is a promising alternative to intravenous injections for the treatment of respiratory infection.To avoid adverse drug reactions associated with injection, off-label nebulization of Tanreqing (TRQ) injection is often used in China to treat respiratory diseases. However, the aerodynamic properties and lung availability of TRQ aerosols remain largely uninvestigated. This study aimed to investigate the size distribution of TRQ aerosols and to compare the pharmacokinetics and tissue distribution of two compounds from TRQ  after transnasal aerosol inhalation and intravenous administration. Furthermore, this study aimed to evaluate the efficacy of TRQ against lipopolysaccharide-induced lung inflammation. The Dv(50) and transmission of TRQ aerosols were 2.512 μm and 74.867%, respectively. The C Scutellaria baicalensis Georgi , Selenaretos thibetanus Cuvier , Naemorhedus goral Hardwicke , Lonicera japonica Thunb. , and Forsythia suspensa (Thunb.) Vahl  (Tanreqing (TRQ) injection is a Chinese medicinal preparation derived from five traditional Chinese medicines, namely, ; 47.1%] . The bot; 47.1%] . The che; 47.1%] . TRQ pos; 47.1%] . TRQ was; 47.1%] . However; 47.1%] .Compared to intravenous (IV) administration, inhalation does not cause pain and can increase patients’ comfort and compliance, improving the effectiveness of treatment of respiratory diseases. Aerosol inhalation in traditional Chinese medicine has been widely used to clinically treat respiratory diseases, especially the medicine with heat-clearing and detoxifying effects. Lung and respiratory diseases, such as pneumonia, respiratory infections, tracheitis, and bronchitis, can be treated by single or combined aerosolization , demonstThe clinical aerosolization application of TRQ is beyond that indicated on the package insert. Nonetheless, the particle size of the TRQ nebulized aerosols has not been systematically studied, and the absorption and metabolism of multiple components in the lungs after aerosolization are not clear. Based on the chemical composition of TRQ, seven analytes were selected to conduct a preliminary study on pharmacokinetics (PK) after transnasal aerosol inhalation (TI). These analytes include baicalin (BAI) and oroxyloside , chlorogenic acid (from JYH), forsythoside D and forsythoside E (both from LQ), and ursodeoxycholic acid and chenodeoxycholic acid (both from XD). The preliminary study showed that BAI, ORO, ursodeoxycholic acid, and chenodeoxycholic acid could be detected in rat plasma after TI of TRQ. Ursodeoxycholic acid and chenodeoxycholic acid are endogenous components and have high blood concentrations. The concentration changes of the two compounds after TI administration were difficult to distinguish from the blank. Therefore, BAI and ORO from HQ were selected as the target compounds in the current study. This study evaluated the particle size distribution of TRQ aerosols generated by a nebulizer. The pharmacokinetic characteristics of BAI and ORO were compared in plasma and tissues, including the lung and trachea after TI and IV administration, and the anti-inflammatory effects were investigated in mice lungs.BAI  was purchased from the National Institutes for Food and Drug Control . ORO  was purchased from Chengdu SinoStandards BioTech Co., Ltd. . Paeoniflorin  was obtained from Shanghai Macklin Biochemical Co. Ltd. . The chemical structures of the two analytes and IS are shown in The animal procurement and experiments were approved by the Animal Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences . Specific pathogen-free (SPF) Sprague–Dawley (SD) rats (200–240 g) and SPF ICR mice (20–24 g) were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. . All animals were maintained on a 12/12 h light/dark cycle in an environmentally controlled SPF room . The animals were acclimated to the laboratory for at least 5 days and were unfed 12 h before the experiment, but with free access to water.A high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) system  equipped with an ExionLC HPLC tandem with a Qtrap 5500 mass spectrometer was used for analysis and determination. The Shimadzu Shim-pack Velox SP-C18 LC column  was used as the chromatographic column at a temperature of 40°C. The autosampler temperature was 6°C. After 5 μl injection, mobile phases A (water) and B (methanol: acetonitrile = 1:1) containing 0.05% formic acid were used for elution at a flow rate of 0.3 ml/min according to the following procedure: 0–0.5 min, 95% A; 0.5–0.6 min, 95%–60% A; 0.6–2.0 min, 60% A; 2.0–2.5 min, 60%–5% A; 2.5–3.0 min, 5% A; 3.0–3.1 min, 5%–95% A; 3.1–6.0 min, 95% A.m/z 447.3→271.0, m/z 461.2→284.9, and m/z 498.2→179.2, respectively. The declustering potential (DP) of BAI, ORO, and IS was 53.1, 91.9, and 147.0 V, respectively. The collision energy (CE) of BAI, ORO, and IS was 26.9, 26.1, and 23.0 V, respectively. The dwell time and collision exit potential (CXP) for all compounds were 100 ms and 15 V, respectively.In the positive ion mode of the electrospray ionization (ESI) source, multiple reaction monitoring scans were used to detect the analytes and IS. The ion spray voltage and curtain gas pressure were maintained at 5500 V and 35 psi, respectively. The collision gas was set at 6 psi, the turbo spray temperature was maintained at 500°C, and the nebulizer (gas 1) and heater gases (gas 2) were set at 45 psi using nitrogen. The precursor/product pairs of BAI, ORO, and IS were After anesthesia, blood samples of six rats were collected from the abdominal aorta into heparinized tubes. Plasma was harvested by centrifuging blood samples at 10,000 rpm for 2 min at 4°C. The lung, trachea, and brain were dissected rapidly and harvested. The tissues were rinsed thoroughly, weighed, and processed by homogenization with cold saline in a 1:4, 1:9, and 1:4 (w/v) radio for the lung, trachea, and brain, respectively. Blank plasma and tissue homogenates were stored at −80°C until use.Biological samples were pre-treated via protein precipitation as follows: 50 μl biological samples were vortex-mixed with 400 μl IS working solution (the plasma samples needed to add 2 μl formic acid first) for 1 min, ultrasound for 2 min, and centrifuged at 14,000 rpm for 5 min at 4°C. The supernatant (400 μl) was centrifuged and concentrated at 1,700 rpm at 47°C until it becomes dry. The residue was redissolved in 100 μl of 70% methanol (0.05% formic acid) followed by a 1 min vortex and a 2 min ultrasound. The supernatant was injected into the HPLC-MS/MS system for analysis after centrifuging at 14,000 rpm for 5 min at 4°C.For the preparation of working and sample solution, and method validation of HPLC-MS/MS, refer to TRQ was nebulized using a TurboBoy nebulizer with a red nozzle from PARI Pharma GmbH . A Spraytec laser diffractometer  was used to evaluate the size distribution of TRQ aerosols generated by the nebulizer at a flow rate of 15 L/min. The experiments were carried out by placing 2 ml of the TRQ solution into the nebulizer cup. The nebulizer cup was then connected to the laser diffractometer and measured. The aerosol transmission volume and particle size distribution were recorded in real-time. The experiment was repeated three times.The actual aerosol inhalation dose was calculated using l volume . The expFor the pharmacokinetic experiment, SD rats were randomly divided into four groups (0.03 ml/kg), a low dose group of TI (0.03 ml/kg), a medium dose group of TI (0.06 ml/kg), and a high dose group of TI (0.12 ml/kg), each group consisted of six rats. The animals in each group underwent a jugular vein catheterization the day before the experiment. They were unfed overnight but had free access to water. For the IV group, rats were intravenously injected with TRQ through the tail vein. For the three TI groups, rats were placed in the FinePointe oral and nasal exposure system. A TurboBoy N nebulizer with a red nozzle was used to nebulize TRQ for 5, 10, and 20 min. A photometer was used to record aerosol concentrations at dynamic equilibrium. Before administration and 0.083, 0.167, 0.333, 0.5, 0.75, 1, 2, 4, 6, 8, 10, and 24 h after administration, approximately 0.15 ml of blood was collected from the jugular vein into a 1.5 ml centrifuge tube with heparin anticoagulation followed by centrifuging at 10,000 rpm for 2 min at 4°C. After blood collection at each treatment time, an equal volume of 0.9% saline was added through the jugular vein catheterization to the rat. The 2 μl of formic acid was added to a 50 μl plasma sample and stored at −80°C until analysis. All rats were euthanized with carbon dioxide after sample collection.For the tissue distribution experiment, 60 SD rats were randomly divided into the IV group  and the TI group . The inhalation system was the same as that used in the pharmacokinetic experiments. Six rats for each treatment time were anesthetized at 0.083, 0.333, 1, 4, and 8 h after administration, respectively. Blood samples were collected from the abdominal aorta into heparinized tubes. The right lung was clamped, and the left lung was lavaged with 1.5 ml cold saline twice to collect bronchoalveolar lavage fluid (BALF). Subsequently, the right lung, trachea, and brain were dissected rapidly and harvested, and homogenized according to “2.4.”Seventy male ICR mice were randomized and divided into the normal, model, positive, IV, and low-, medium-, and high-dose groups of TI. There were 10 mice in each group. Except for the normal group, mice in the other six groups were placed in an inhalogic NIES inhalation tower . A 4 mg/ml LPS solution was nebulized for 30 min using a TurboBoy N nebulizer with a red nozzle for modeling. The first intervention was immediately initiated in each group after modeling, and the second intervention was initiated 15 h thereafter. The normal and model groups were nebulized with saline for 20 min. The positive and IV groups were injected with 5.2 mg/kg dexamethasone sodium phosphate and 2.6 ml/kg TRQ  through the tail vein, respectively. The TI groups were nebulized with TRQ for 5, 10, and 20 min through the inhalation tower. One hour after the last administration, mice in each group were sacrificed. The lungs were dissected rapidly and harvested, and rinsed thoroughly with cold saline. Subsequently, 100 mg of lung tissue was weighed and homogenized in an ice bath after adding 20× (by weight) RIPA lysis buffer. The homogenate was centrifuged at 12,000 rpm for 15 min at 4°C. The level of IL-6 in the lung homogenate was determined by using a mouse IL-6 ELISA kit with a Spectramax i3x reader  at 450 nm.max and Tmax were obtained directly from the experimental process.Data acquisition and analysis for the concentration of BAI and ORO were performed using Analyst 1.7 software . Plasma and tissue homogenate concentrations of BAI and ORO were determined using the calibration curve of each analysis batch. Pharmacokinetic parameters were calculated using a noncompartment model with the MaS Studio 1.5.2.14 stable software . Ct-tests. p < 0.05 was considered to be statistically significant.Data were expressed as mean ± standard deviation (SD), with relative standard deviation (RSD). Multiple data sets were subjected to independent 2 weighting was 2–200 ng/ml and 1–100 ng/ml, respectively. The lower limit of quantification (LLOQ) of BAI and ORO in the two matrices was 2 ng/ml and 1 ng/ml, respectively. The intra-day and inter-day precision (RSD) and accuracy (RE) in plasma and lung homogenate samples of BAI ranged from 0.47% to 6.25% and 92.23%–114.4%, respectively. The intra-day and inter-day precision and accuracy in plasma and lung homogenate samples of ORO ranged from 0.34% to 7.63% and 90.41%–107.50%, respectively. The dilution procedure showed excellent precision (RSD < 3.74%) and accuracy (RE: 95.80–103.85%). The RSD of relative matrix effects for all analytes ranged from 1.38% to 13.07%, and the average extraction recovery was 37.81%–48.14%. The RSD values ranged from 0.79% to 10.42%, and the deviation values ranged from –13.53% to 6.78%, indicating that BAI and ORO were stable in the autosampler at 6°C for 24 h, at room temperature (25°C) for 1 h, after three freeze/thaw cycles from –80°C to room temperature, and in long-term storage at −80°C for 30 days in plasma and lung samples from rats. The detailed results of the method validation are shown in Mass spectrometry parameters of BAI, ORO, and PAE (IS) were optimized by using 100 ng/ml methanol solution of the three compounds. In the positive ion mode, the two analytes and IS were more sensitive, which made them suitable for detection in the ESI (+) mode. In ESI (+) mode, DP, CE, CXP, gas 1, gas 2, spray voltage, and curtain gas were optimized to determine the precursor ions and product ions of the three compounds. The optimized chromatographic column was a Shimadzu Shim-pack Velox SP-C18 LC column. The chromatographic separation was performed using water-methanol-acetonitrile containing 0.05% formic acid as the mobile phase system. Representative chromatograms of BAI, ORO, and IS in plasma and lung homogenates are shown in The Spraytec laser diffraction system  was used to study changes in particle size and percentage transmission of TRQ aerosols nebulized by the PARI TurboBoy N nebulizer with a red nozzle over time, where Dv(10) and Dv(50) remained stable for 3 min, as shown in Validated analytical methods were used to determine the rat plasma concentration of BAI and ORO after IV (0.03 ml/kg) and TI  of TRQ. The plasma concentrations of BAI and ORO at different times after administration are shown in max at 5, 10, and 30 min and were slowly eliminated  was 4.51 h and 3.03 h, respectively. The T1/2 of BAI (5.17–7.98 h) and ORO (4.89–9.16 h), and the MRT0-t of BAI (6.20–8.12 h) and ORO (5.69–6.73 h) after TI administration were longer than after IV administration. The Cmax of BAI , and Cmax of ORO  in plasma after low, medium, and high doses TI of TRQ increased with increasing dose (0-∞) of BAI (226.565 h·ng/ml) after medium dose TI of TRQ was slightly lower than that after low dose TI (257.171 h·ng/ml), but the AUC0-∞ showed an increasing trend with the increase of TI doses in the range of 0.03-0.12 ml/kg  in plasma after TI did not increase with increasing dose  were studied. Concentrations of BAI and ORO for biological samples at different sampling times are shown in max of 5 min (As shown in of 5 min . Subsequof 5 min . It was of 5 min . This stof 5 min . The lunp < 0.001). After administration of dexamethasone, the content of IL-6 in mouse lung homogenate decreased significantly (p < 0.001). These results indicated that LPS-induced mice models experienced acute lung injury. Compared to the model group, the content of IL-6 in the IV group (2.6 ml/kg), and in the medium and high dose groups of TI  decreased significantly (p < 0.01), as shown in A mouse pneumonia model was established by aerosol inhalation of LPS at 4 mg/ml. The level of IL-6 in lung homogenates was used as an index to evaluate the model and the anti-inflammatory effect of TRQ . CompareThe TRQ injection extracted and processed from five botanical drugs has been reported to achieve curative effects in the clinical treatment of acute bronchitis, acute pneumonia, chronic obstructive pulmonary disease, and other respiratory infections. In recent years, numerous respiratory diseases have been treated by aerosol inhalation. Although aerosol inhalation administration of TRQ can reduce adverse reactions caused by IV administration, it is still an off-label drug use. For more efficient and safer clinical use, it is necessary to systematically study its inhalable properties, including component analysis, preparation process, pharmacological effects, safety evaluation, pharmacokinetics, and tissue distribution. This study focused on the characteristics of TRQ aerosols, the pharmacokinetics and tissue distribution, and anti-inflammatory effects after TI administration of TRQ.Based on our preliminary experimental results, the representative flavonoids BAI and ORO in TRQ were selected as the target compounds. BAI, ORO, and IS standard solutions were used to optimize the respective precursor ions and product ions. The analytical conditions of HPLC-MS/MS for BAI and ORO were optimized. This established method has high specificity and sensitivity and can be used for the quantitative analysis of BAI and ORO in biological samples from rats.Compared to oral administration, TI can avoid the liver first-pass effect and increase drug concentration in the bronchi and alveoli. After delivery to lung tissue, the target organ of respiratory diseases, drug ingredients are absorbed into the blood through the air–blood barrier in the lung alveoli. Subsequently, the active ingredients are distributed to various organs and tissues through blood circulation. The concentration of ingredients entering the systemic circulation after TI is markedly reduced, thus avoiding adverse reactions caused by IV administration.The size and distribution of aerosol particles are key parameters that influence the nebulization process and the therapeutic effect. Due to the particularity of the respiratory system, aerosol particles of different sizes are deposited at different positions. Moreover, only aerosol particles with a size of 1–5 μm can enter and deposit in the bronchi and alveoli . In addimax  of BAI and ORO in rat lung, BALF, and trachea (max and AUC after TI (in vivo after absorption. The high distribution of drug ingredients in the lungs indicated that TI administration has better lung availability than IV administration. Compounds with small molecules can be rapidly absorbed into the lung and their absorption rate depends on the lipophilicity of the drug. The Tmax of hydrophilic compounds is within 30 min (The local curative effect on respiratory diseases is directly related to the concentration of drugs in the respiratory tissues. Aerosol inhalation can target drug delivery to the lungs. Therefore, administration via inhalation has unique advantages over other routes, such as oral and IV administration, as it avoids the liver first-pass effect and allows the drug to accumulate in the lung or trachea. It compensates for the low concentration of drug components in lung tissue after oral and intravenous administration . After T trachea  was highafter TI  were lown 30 min . The pern 30 min . As showThis study showed that TRQ aerosols produced by the jet nebulizer have good aerodynamic characteristics, which is beneficial for lung deposition. Compared to IV administration, drug exposure in the rat lungs, BALF, and trachea was increased significantly and the local anti-inflammatory efficacy was better after TI administration. In conclusion, according to the analysis of pharmacokinetics, tissue distribution, and anti-inflammatory effects in animals, transnasal aerosol inhalation administration of TRQ has advantages over intravenous administration in the treatment of respiratory diseases. Further clinical studies are needed to confirm the prospects of TRQ inhalation administration.


Dolichos trilobus Linn (Leguminosae) is often used in Yi ethnic medicine to treat pain, fracture, and rheumatism.D. trilobus and its disodium salt (DBDS) and the underlying mechanism in pain.To explore the therapeutic potential of doliroside B (DB) from In the writhing test, Kunming mice were orally treated with DB and DBDS at doses of 0.31, 0.62, 1.25, 2.5, and 5 mg/kg. Vehicle, morphine, indomethacin, and acetylsalicylic acid were used as negative and positive control on the nociception-induced models, respectively. In the hot plate test, mice were orally treated with DB and DBDS at doses of 2.5, 5, 10, and 20 mg/kg. In the formalin test, mice were orally treated with DB and DBDS at doses of 2.5, 5, 10, and 20 mg/kg. In the meanwhile, lipopolysaccharide-induced inflammatory model in RAW264.7 macrophages was adopted to study the mechanism of pain alleviation for DBDS.A1 receptor.DBDS (5 mg/kg) inhibited the writhing number by 80.2%, which exhibited the highest antinociceptive activity in pain models. DBDS could selectively inhibite the activity of COX-1. Meanwhile, it also reduced the production of NO, iNOS, and IL-6 by 55.8%, 69.0%, and 49.9% inhibition, respectively. It was found that DBDS also positively modulated the function of GABADBDS displayed antinociceptive activity by acting on both the peripheral and central nervous systems, which may act on multitargets. Further work is warranted for developing DBDS into a potential drug for the treatment of pain. Pain causes a negative consequence on health status and influences the quality of life (Williams and Craig Dolichos trilobus Linn (Leguminosae) have been widely used to treat pain, fracture, and rheumatism for many years  in the present research. Meanwhile, the probable antinociceptive mechanisms involved in DBDS activity were also explored.Traditional Yi medicine, as an ancient and still vital holistic system, has made great contributions to the health of the Yi people distributed mainly over Yunnan, Sichuan and Guizhou provinces, China, receiving much wider recognition for its own unique theories of human health and therapeutics  were purchased from Beyotime Biotechnology Co., Ltd.Acetic acid was purchased from Guangdong Guanghua Sci-Tech Co., Ltd. Acetylsalicylic acid (ASA), indomethacin (IND), and morphine (MOP) were purchased from National Institutes for Food and Drug. Naloxone hydrochloride (NAL) and formalin were purchased from J&K Scientific Ltd. Materials for cell culture were purchased from Yunnan Chien Technology Co., Ltd. ELISA Kit were purchased from Bio-Techne China Co., Ltd. Griess reagent and NG-Monomethyl-D. trilobus were collected at Chuxiong City, Yunnan Province, China. It was authenticated by Prof. Qing-Song Yang at the Yunnan Minzu University. The voucher specimen (No. 20190928) was deposited at the Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, School of Ethnic Medicine, Yunnan Minzu University.During September 2019, the roots of D. trilobus were macerated in 95% ethanol (3 × 60 L) for 48 h at room temperature. After filtering and evaporating the extract to remove ethanol, the remaining dark brown residue was retained (1.15 kg). DB  was isolated from the D. trilobus extract by recrystallization (methanol). DB (20 g) was dissolved in methanol at room temperature followed by addition of 2 equiv. of NaOH for the production of DBDS. After NaOH was dissolved completely, the solution was concentrated under reduced pressure to provide the desired product DBDS . The 1H and 13C NMR spectra of DB and DBDS were shown in Supplementary material.According to previously reported procedures by our group  and noise (≤ 60 db), and maintained under a 12 h light-dark cycle. After the experiment is completed, mice were euthanized humanely. All the animal experiments were approved by the Institutional Animal Care and Use Committee of Yunnan Minzu University (permit no. 20200608).Female Kunming mice (20 ± 2 g) from Chengdu Dossy Experiment Animal Co., Ltd.  were used for antinociceptive experiments. These animals were kept under a constant temperature (23 ± 1 °C), humidity (40–70%), airflow speed , each group was pretreated with vehicle (distilled water), MOP (1 mg/kg), IND (10 mg/kg), ASA (100 mg/kg), and D. trilobus extract  by oral or intraperitoneal administration, respectively. After intragastric (i.g.) or intraperitoneal administration of 30 min, mice were intraperitoneally (i.p.) injected with 0.6% acetic acid (0.2 mL). Following acetic acid injection, the number of writhing induced by acetic acid were counted cumulatively for 15 min. Employing the above mentioned procedures, mice were randomly divided into six groups (n = 10), we evaluated the antinociceptive effect of DB, DBDS, IND, ASA, and MOP by treating mice with vehicle and different dose of test compounds, respectively. Among the tests in the absence and presence of NAL (5 mg/kg), i.p. injection of NAL was performed 40 min before acetic acid injection. The inhibition was calculated using the formula: inhibition (%) = (average of control group – average of test group)/average of control group × 100%.Acetic acid-induced writhing test Koster  was empln = 10), each group was pretreated with vehicle (distilled water), MOP , IND , ASA , DB , and DBDS  by oral or intraperitoneal administration, respectively. After administration of the test compounds, the pain threshold was recorded at 0, 30, 60, 90, and 120 min. Among the tests in the absence and presence of NAL (5 mg/kg), i.p. injection of NAL was performed 10 min before administration of testing compounds. After administration of the test compounds, the pain threshold was recorded at 0, 30, 60, 90, and 120 min.This was performed according to a previously reported procedure (Eddy and Leimbach n = 6), each group was pretreated with vehicle (distilled water), MOP , IND , ASA , DB , and DBDS  by oral or intraperitoneal administration, respectively, 1 h before the formalin injection. 2.5% Formalin (20 μL) was injected in the plantar surface of the right hind paw. The indicator of pain was evaluated as the time spent licking paw. It was recorded from 0 to 5 min (early phase) and from 15 to 30 min (late phase) after the injection of formalin. Among the tests in the absence and presence of NAL, i.p. injection of NAL (5 mg/kg) was performed 10 min before administration of testing compounds.The test was performed as described by Ayumi et al. . Female 2, 37 °C).The RAW264.7 cell line (purchased from Kunming Institute of Zoology. Chinese Academy of Sciences) was cultured in DMEM (Dulbecco’s modified Eagle medium) supplemented with 10% FCS  under a humidified atmosphere -HEK293 cells. All the ion channel-expressed cell lines were made in Haibo Yu’s lab. Among the parental cells, T-REx™-CHO, CHO-FlpIn, and HEK-FlpIn were obtained from Thermo Scientific; CHO-K1 and HEK293 cells were obtained from ATCC. CHO-derived cells were cultured in DMEM and nutrient F-12 mixture, and HEK293-derived cells were cultured in DMEM supplemented with 10% FCS. For GABA (γ-aminobutyric acid) cells, the following antibiotics were added, blasticidin (10 μg/mL), hygromycin (300 μg/mL), zeocin (100 μg/mL), and puromycin (1 μg/mL). For Nav1.2-CHO-FlpIn and Nav1.7-HEK-FlpIn, hygromycin (300 μg/mL) was added. For Nav1.4-CHO-K1, Nav1.5-HEK293, and TRPV1-HEK293 cells, geneticin (500 μg/mL) was added. Stable cell lines with highly-expressed channels or receptors are cultured in the designated medium, and cultured in a humidified incubator . Cells were passaged every 3–4 days.To study the antinociceptive mechanism of the test compound, the following cells were used, including GABA50  values were determined from dose-response curves for 8 distinct concentrations in quadruplicates. The concentration-response curves were fitted by Hill equation, according to the standard procedure.On the day before screening, the cells were seeded in 384-well plates  and cultured overnight. The assay protocol was adapted from the manufacturer’s recommended protocol. Briefly, the medium was removed; cells were loaded with the designated dye, 20 μL/well, and incubated for 30 min [FMP  dye] and 60 min  at room temperature in the dark. After dye loading, the test compounds were added into the cell plate and incubated for 20 min. Cell plates were loaded to FLIPR machine . After establishing fluorescence baseline by 1 Hz scanning for 10 s, the channels were activated by the addition of agonist or test compounds , and fluorescence measurement was continued at 1 Hz for another 120 s. EC5 cells/well (volume of the well is 100 μL). Under a humidified atmosphere , the cells were cultured for 24 h following treatment with vehicle and DBDS . After 24 h, 20 μL of MTT solution was added to each well and kept under the same environment for 4 h again. Subsequently, 150 μL of DMSO was added to each well after removing the culture supernatant, vibrated and kept under a darkroom at room temperature for 15 min. The optical density (OD) of the solution was measured with a microplate reader at 490 nm. The survival rate was calculated using the formula, survival rate (%) = OD of the sample-treated group/OD of the control group × 100%.The MTT -2,5-diphenyltetrazolium bromide) colorimetric method was used to determine cytotoxicity of DBDS. The logarithmic phase cells were seeded in 96-well plates with 1 × 105 cells/well (volume of the well is 100 μL). Under a humidified atmosphere , the cells were cultured for 24 h. Subsequently, 50 μL of lipopolysaccharide  was added to each well and continued to culture these cells under the same environment for an additional 1 h. Next, 50 μL of different concentrations of DBDS  and L-NMMA  were added to each well and cells were continued to be cultured under the same environment for an additional 24 h, and three repeated wells were set at each concentration. NO production was determined by measuring the accumulation of nitrite in the supernatant of culture medium using the Griess reagent. Inhibition (%) was calculated using the following equation:A modified method, according to previous report by Banskota et al. , was empA: NO concentration of LPS-treated group; B: NO concentration of sample-treated group; C: NO concentration of control group.5 cells/well (volume of the well is 400 μL). Under a humidified atmosphere , the cells were cultured for 24 h. Subsequently, 50 μL of LPS (1 μg/L) was added to each well and cells were continued to be cultured under the same environment for an additional 1 h. Next, 50 μL of different concentrations of DBDS  were added to each well and incubated with cells for an additional 24 h, and three repeated wells were set at each concentration. Then, the supernatant of culture medium was collected and used for measuring iNOS (inducible NO synthase), interleukin6 (IL-6), tumor necrosis factor-α (TNF-α), COX (cyclooxygenases)-1, and COX-2 by ELISA kits. Absorbance was measured in an ELISA reader (Thermo Multiskan GO) at 450 nm. Measurements performed in triplicate. The inhibition on iNOS, IL-6, TNF-α and COX releases was calculated.The logarithmic phase cells were seeded in 24-well plates with 1 × 10t-test. For GABA-induced dose-response curves, statistical differences were determined using Student’s two tailed t-test  < 0.05 indicated a statistically significant difference between two groups.All the data are expressed as mean ± s.e.m. All statistical analyses were carried out using the Origin 6.0 software and the GraphPad Prism 6.0 software . The data obtained were evaluated by One-way ANOVA and unpaired Student’s 6, 63 = 86.12, p < 0.0001). The extract of D. trilobus exhibited a significant antinociceptive activity at the dose of 200, 400, and 800 mg/kg. The inhibition of 22.2, 43.7, and 57.4% were obtained, respectively (p < 0.0001). For the compounds DB and DBDS, when their doses were increased from 0.31 to 5 mg/kg, the number of writhing was progressively decreased  and 80.2% , respectively. DB exhibited an ED50 (median effective dose) value of 4.68 mg/kg, which is nearly 4-fold weaker than that of DBDS (ED50 = 1.1 mg/kg). As a comparison, IND and ASA were tested in the same model as DBDS with an ED50 value of 5.91 and 70.09 mg/kg, which are approximately 5 and 64-fold weaker than that of DBDS, respectively. IND at the dose of 20 mg/kg inhibited the writhing number by 76.1% . ASA at the dose of 200 mg/kg inhibited the writhing number by 73.3% . Meanwhile, as positive control, MOP was also tested in the same model with an ED50 value of 0.22 mg/kg, which was stronger than that of DBDS. MOP at the dose of 1 mg/kg inhibited the writhing number by 80.6% .Initially, we performed the acetic acid-induced writhing test. As shown in ecreased . DB and 11, 108 = 0.47, p > 0.05), 30 min , 60 min , 90 min , and 120 min . MOP (1 mg/kg) exhibited outstanding antinociceptive effect, and ASA (100 mg/kg) exhibited particular antinociceptive effect. However, DB and DBDS could only increase withdrawal threshold at the dose of 20 mg/kg (see p < 0.01).Next, we carried out the Eddy’s hot plate test to further evaluate the antinociceptive effect of DB and DBDS. Compared to control group, IND (10 mg/kg) did not exhibited antinociceptive effect with increased paw withdrawal threshold at 0 min . However, DB exhibited poor antinociceptive effect. In the late phase, DB and DBDS showed a significant inhibition at the doses ranging from 2.5 to 20 mg/kg . As expected, IND and ASA mainly acted at the peripheral nervous system (PNS), they could not distinctly shorten the licking time of mice in the first phase, whereas it exhibited remarkable antinociceptive activity in the late phase with 57.3% (p < 0.0001) and 64.8% (p < 0.0001) inhibition, respectively. As an effective analgesic, MOP showed high antinociceptive activity in both phases with 55.3% (p < 0.0001) and 75.9% (p < 0.0001) inhibition, respectively.As shown in 5, 12 = 0.01, p > 0.05). Due to the fact that DBDS did not induce significant cytotoxicity in RAW264.7 macrophages, test of DBDS on inflammatory mediators and COX production in LPS-stimulated RAW264.7 macrophages were further performed. As shown in 5, 12 = 18.69, p < 0.001). However, it could be effectively reduced by the treatment of DBDS at higher concentrations  with 67, 111.9, and 107% inhibition (p < 0.01), respectively. After the treatment of DBDS , no distinct effects were observed on COX-2 production in LPS-stimulated RAW264.7 macrophages . These results showed that DBDS had effective anti-COX-1 activity with high selectivity.The cytotoxicity of DBDS on RAW264.7 macrophages was firstly tested. The RAW264.7 macrophages were treated with different dose of DBDS. DBDS did not exhibit toxic effects on RAW264.7 macrophages at the concentrations ranging from 12.5 to 200 μmol/L  and 55.8% (p < 0.0001) inhibition, respectively. Similarly, DBDS could effectively inhibit the production of iNOS at higher concentrations (10 and 40 μmol/L) with 32.2%  and 69% (p < 0.0001) inhibition, respectively , 25.5 (p < 0.01), and 49.9% (p < 0.0001) inhibition, respectively. However, the treatment of DBDS  could not show significant inhibitory effects on TNF-α production in LPS-stimulated RAW264.7 macrophages .We also investigated the effects of DBDS on NO, iNOS, IL-6 and TNF-α production in LPS-stimulated RAW264.7 macrophages with an ELISA assay. As shown in ectively . As showt = 11.3, p < 0.0001). Only a minor difference was observed when IND (t = 0.28), ASA (t = 0.05), DB (t = 0.8), and DBDS (t = 0.35) were tested in the absence and presence of NAL (p > 0.05). Similarly, the antinociceptive effect of MOP was removed by NAL in hot plat and formalin tests, and the antinociceptive effect of IND, ASA, DB, and DBDS was not effectively removed by NAL (see As shown in  NAL see .A1 channel at higher concentrations (10 µM and 30 µM). However, when an EC20 concentration of GABA (0.2 µM) was presented, it was found that DBDS dramatically increased GABA-induced activation of GABAA1 channel in a dose-dependent manner with EC50 value of 12.3 ± 3.0 µM . As shown in 50 = 0.28 ± 0.04 µM) with a similar level to GABA itself (EC50 = 0.29 ± 0.08 µM). When the concentration of DBDS was increased to 30 µM, the efficacy of GABA was increased to a higher level by around 150% (p < 0.01 vs. vehicle control), and the affinity of GABA-induced activation was significantly enhanced by 1.75-fold, with EC50 = 0.16 ± 0.05 µM (p < 0.01 vs. vehicle control), as shown in A1 receptors, diazepam was used as the allosteric control and could shift the EC50 value of GABA to 0.12 ± 0.03 µM , central nervous system (CNS) subtype Nav1.2 (IC50 = 140.67 µM), and stronger inhibition on skeletal muscle subtype Nav1.4 (IC50 = 3.29 ± 2.12 µM). Fortunately, DBDS has no effect on Nav1.5 subtype of cardiac sodium channel (50 = 7.3 ± 0.06 µM).Similar to GABA channel, FMP assay was used for the functional test of sodium channels. As shown in  channel , a potenD. trilobus, we prepared the ethanol extract and carried out the isolation of extract according to previously reported procedures by our group . As one of them, PGEIn the process of inflammatory pain, inflammatory mediators, such as NO and cytokines, play important roles between the disease process and pain. NO can directly damage the function of normal cells through binding with other superoxide radicals, which is an important inflammatory mediator during the inflammatory process , cyclooxygenases and their metabolites  is the most abundant isoform among GABA receptors. Therefore, the effect of DBDS on GABAA1 receptor was tested by using FMP-Blue-Dye method. For the FMP assay in the recombinant GABAA1 receptors expressed in T-REx™-CHO cells, as reported (Joesch et al. A receptor agonists also depolarized the membrane potential, in contrast to hyperpolarizing in mature cells. These might be caused by intracellular Cl− accumulation due to the absence of Na-K-2Cl cotransporter (Plotkin et al. A1 receptor.Ion channels also play an important role in the occurrence of pain, DBDS exhibited antinociceptive activity which was possibly related to ion channels, whereupon we decided to investigate its possible antinociceptive mechanism on several ion channels, such as GABASodium channels are important targets of pain. Genetic studies of patients and animals indicated that neuronal sodium channel Nav1.7 also plays key role in pain sensitization (Cox et al. A1 receptor. As a potential candidate drug for the treatment of pain, compound DBDS has displayed a great potential for pain alleviation, while the metabolism and toxicity of the compound need to be further explored.We demonstrated that compound DBDS exhibited potent antinociceptive activity acting on both the PNS and CNS. We found that DBDS had highly selected anti-COX-1 activity. Meanwhile, it plays an important role in relieving pain by reducing the production of NO, iNOS, and IL-6. Through the investigation of ion channels, DBDS could also positively modulate the function of GABAClick here for additional data file.


Bronchiectasis are abnormal permanently dilated bronchi which lead to chronic cough and other respiratory symptoms. Though tuberculosis (TB) is a common cause of bronchiectasis, data on this association are scarce. The objective of this study was to describe the profile of patients with post-TB bronchiectasis at a tertiary hospital in the southern region of Brazil. This was a retrospective study with data from patients in follow-up at our hospital from January 2005 to December 2020. We included patients 14 years of age or older who had bronchiectasis on chest computed tomography and a history of pulmonary TB. We excluded patients with bronchiectasis due to other causes or with confirmed non-tuberculous mycobacteria infection. We included 54 of the 204 non-cystic fibrosis bronchiectasis patients seen at our hospital during the study period. Most of the patients were female, older, and non-smokers. Less than a third had chronic bronchial infection by some agent. More than two thirds had some type of ventilatory defect, the most common being obstruction. More than half had upper-lobe impairment. Severity of the disease seemed to be equally distributed from mild to severe. Treatment was varied, including bronchodilators, inhaled corticosteroids, and azithromycin. We found that the profile of patients in our hospital is similar to that described in other studies, with slight differences in regard to microbiology and treatment. This form of bronchiectasis is poorly described in medical literature, and not much is known about how it relates to other forms of non-cystic fibrosis bronchiectasis (NCFB) In order to elucidate this gap in current knowledge about post-TB lung disease, we conducted an epidemiological study with patients in follow-up at the NCFB outpatient clinic of the Clinics Hospital of the Federal University of Paraná.2This was a retrospective study performed at the NCFB outpatient clinic of the Clinics Hospital of the Federal University of Paraná from January 2005 to December 2020, approved by the institution’s Committee for Ethics in Research on Human Beings under the opinion 28874720.0.0000.0096. We included patients 14 years of age or older who had bronchiectasis on chest computed tomography (CT) and a history of pulmonary TB. We excluded patients with bronchiectasis due to other causes (such as immunodeficiencies or autoimmune diseases) or with confirmed non-tuberculous mycobacteria infection. Bronchial infection was evaluated through periodic sputum samples, collected every 6 to 12 months. All chest CTs were analyzed by a single thoracic radiologist. Patients were classified as having mild, moderate or severe disease using the Bronchiectasis Severity Index (BSI), a score that combines clinical, radiological, and microbiological features. Treatment was categorized by medications in use at the last visit. Categorical variables were described as frequencies and percentages, and quantitative variables as means ± standard deviation or median (interquartile range [IQR]).3During the study period, 204 NCFB patients were seen in our clinic, 54 of which met inclusion criteria . Other cPseudomonas aeruginosa in 5 of them .Sputum samples were obtained from 41 patients (75.9 %). Chronic bronchial infection was found in only 8 patients (19.5 %), with identification of 1/FVC ratio of 59.7 ± 18.3, 75.0 %±23.3 % predicted, and a median FEV1 of 1.39 L (0.58–3.78 L), 57.2 % (26.8 %-135.4 %) predicted. Reduced FVC, either isolated or associated with obstruction, was found in 17 patients (37.8 %). In addition, 7 patients (17.1 % of those tested) had positive bronchodilator response. All patients had undergone chest CT, revealing a predominance of upper lobe impairment .Of the 45 patients who underwent spirometry, 34 (75.6 %) had impaired lung function. The most common defect was obstruction, found in 31 patients (68.9 %), with an FEVThe Bronchiectasis Severity Score was assessed in 41 patients, and ranged from 0 to 15 points, with a median of 6 points. Of these 41 patients, 14 (34.1 %) were considered as having mild disease (0–4 points), 13 (31.8 %) as moderate (5–8 points) and 14 (34.1 %) as severe (9 or more points).2- agonists and inhaled corticosteroids prescribed to 40 patients (74.1 %), followed by six using long-acting muscarinic antagonists (11.1 %) and two using isolated long-acting beta2-agonists (3.7 %). Azithromycin was prescribed to 18 patients (33.3 %), of which 2 (11.1 %) had chronic infection by P. aeruginosa. Four patients had indication of long-term supplemental oxygen therapy (7.4 %).Regarding treatment, the most common choice was the association of long-acting beta4The demographic profile in our study was compatible with that of other studies. A predominance of women was also observed in patients with post-TB bronchiectasis in China P. aeruginosa in those with positive cultures, in agreement with our findings. However, they found different microorganisms in the remaining patients, with non-tuberculous mycobacteria in 13.6 %, Enterobacteriaceae in 4.2 % and Haemophilus influenzae in 1.7 %. Fong et al. P. aeruginosa in 4.3 % and Klebsiella pneumoniae in 4.3 %.Our findings were slightly different from what is described in literature in regard to microbiology. Choi et al. 1 in our study (57.2 % predicted) was close to the ones found in studies from Saudi Arabia 1 of 62 % predicted), Korea 1 of 57.6 % predicted) and Singapore The functional and tomographic characteristics of our sample were comparable to those described by other authors. The median FEVWhen analyzing lung disease severity, we found similar number of patients with mild, moderate, and severe disease, unlike other studies, which had a predominance of severe cases. Wang et al. 2-agonists and inhaled corticosteroids. Though there is no indication to routinely use these medications in the treatment of these patients, Lee et al. P. aeruginosaP. aeruginosa infection. We did not find studies addressing the use of azithromycin in this specific group.Finally, we observed that most of the patients in our sample were using a combination of long acting betaIn conclusion, we found that the profile of patients in our hospital is similar to that described in other studies, with slight differences in regard to microbiology and treatment. Despite these similarities, there is a lack of consensus on the best treatment for these patients. Thus, more studies addressing this population are needed.Vitor Loureiro Dias: Conceptualization, Investigation, Data curation, Formal analysis, Visualization, Methodology, Supervision, Writing – original draft, Writing – review & editing. Mariane Gonçalves Martynychen Canan: Conceptualization, Investigation, Visualization, Supervision, Writing – review & editing. Cleverson Alex Leitão: Investigation, Data curation, Formal analysis, Visualization, Methodology. Elissa Ayumi Okuno: Investigation, Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Giulia Rafaela Dainez de Sant'Ana: Investigation, Data curation, Formal analysis, Visualization, Writing – original draft, Writing – review & editing. Juan Vitor Miranda: Investigation, Data curation, Visualization.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


P < .001; LDL-C 1.81 ± 0.73mmol/L vs 2.18 ± 1.49mmol/L, P < .001). Mean HOMA-β increased after management . After 1 year of management, patients in high school or above group achieved higher control rate of body mass index than those in middle school or below group . high density lipoprotein cholesterol control rate was higher in high income group , while LDL-C control rate was higher in low-income group . fasting plasma glucose control rate in new diagnosis group was higher than that of the middle and long course groups . The comprehensive control rate increased from 9.83% at baseline to 26.15% after 1 year MMC management. The metabolic outcomes and their control rate in T2DM patients were improved after 1 year MMC management. It indicated that patients may achieve more benefits with MMC management.The Chinese Medical Doctor Association has initiated metabolic management center (MMC) program for 6 years since 2016 nationwide. It is worth investigating the level of control metabolic outcomes in patients with type2 diabetes (T2DM) after MMC model in Yan’an, northwest China. Patients with T2DM was admitted to MMC in Yan’an University Affiliated Hospital from November 2018 to July 2021. They were asked to revisit hospital every 3 months. Blood glucose, blood pressure and blood lipids at baseline were compared to its counterparts after 1 year MMC management. Glycosylated hemoglobin and low density lipoprotein cholesterol (LDL-C) level in T2DM patients after 1 year management were lower than their baseline level (glycosylated hemoglobin 7.74 ± 1.94% vs 8.63 ± 2.26%,  Individually, patients with T2DM failed to adopt a healthy lifestyle and stick to regular treatment may account for it. In addition, the disparity in level of T2DM diagnosis and treatment in different areas of China attributed to it.With the rapid development of China’s economy and the westernization of people’s lifestyle, the prevalence of type 2 diabetes mellitus (T2DM) has risen to 11.2% in 2017, but the overall control rate of diabetes is still at a very low level. Studies have shown that MMC management could improve metabolic level for both outpatients and in-patients with (T2DM) in China’s developed areas, especially for blood glucose and glycosylated hemoglobin (HbA1c).–7In order to achieve the homogenization management of diabetes and raise the control rate of diabetes, Chinese Medical Doctor Association launched the Metabolic Management Center (MMC) program in 2016, which combined advanced diagnosis and treatment technology with big data sharing patients’ information online and offline among different hospitals. It is a new mode for T2DM management adapted to China’s grading treatment.Yan’an University Affiliated Hospital, located in Northwest China, is the largest medical service center in Northern Shaanxi, a less developed area. It joined MMC in November 2018 and was officially awarded the MMC sub-center in August 2019. The purpose of the study is to analyze the prognosis of T2DM patients after MMC management, and further to understand the current management status of T2DM patients in less developed areas of Northwest China. Accepted and adhered to MMC management for more than 1 year. Was able to download and use MMC app. Patients with the following conditions were excluded from the study: diabetic ketoacidosis, severe infection, acute cardiovascular and cerebrovascular diseases, acute pancreatitis, Type 1 diabetes mellitus, gestational diabetes mellitus. The study was approved by the Ethics Committee of Yan’an University Affiliated Hospital and was conducted in accordance with the principles of the Declaration of Helsinki.Patients with T2DM who were managed in sub-MMC center from November 2018 to July 2021 were recruited in the study. Patients were included as the following criteria: Patients aged 18 to 70 who were diagnosed with type 2 diabetes according to the World Health Organization criteria in 1999.Patients who meet the criteria above were guided to download and use the MMC app for appointments, follow-up and self-management. Their demographic information was collected and physical measurements recorded by MMC specified nurses. Their clinical examinations, diagnosis and treatment plan were completed by doctors. Patients went through a 60-minute diabetes education in MMC. Patients are requested to visit the hospital every 3 months for physical measurements, clinical examinations and diabetic education. After 1 year, all the patients returned to the hospital for health examination and data collection.Gender, date of birth, current address, education level, family income and other demographic information were collected when patients joined MMC. Physical measurements including height, weight, waist circumference, systolic blood pressure (SBP), diastolic blood pressure (DBP), and body mass index (BMI) were recorded when patients joined and revisited MMC.We categorized the patients based on their demographic information in this study education level was divided into 2 groups, junior middle school or less and senior high school or above. Family income levels were divided into low-income group , middle-income group  and high-income group . Duration of diabetes was divided into < 1 year, 1 to 5 years and > 5 years. In this study, duration of diabetes < 1 year was also defined as a new diagnosis, 1 to 5 years was defined as a medium course of T2DM, and > 5 years was defined as a long course of T2DM.Venous blood was collected after at least 8 hours’ fasting. Fasting blood glucose (FBG), 2-hour postprandial blood glucose (2hPG),HbA1c, total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) were tested in the central laboratory of the hospital. Plasma glucose level was determined by glucose oxidase method. Serum insulin level was examined by radioimmunoassay. Blood lipid profile was measured by standard enzymatic test.We conducted insulin release test among those whose blood glucose have been controlled. Blood glucose, insulin and C-peptide were examined for fasting level and postprandial level after breakfast. Oral glucose tolerance test was performed on those who need to make a definite diagnosis. The improved steady-state model  was adopted to calculate the insulin resistance index and the formula as following: HOMA-IR (CP) = 1.5 + fasting blood glucose × fasting C peptide/2800. The modified steady-state model was used to calculate islets β cell function index  and its formula HOMA-β (CP) = 0.27 × fasting C-peptide/ (fasting blood glucose – 3.5).2.According to the guideline for prevention and treatment of type 2 diabetes in China by Chinese Diabetes Society, patients with T2DM were considered to be controlled as the following: fasting plasma glucose (FPG) 4.4 to 7.0 mmol/L, non fasting plasma glucose < 10.0mmol/L, HbA1c < 7.0%; SBP < 130 mm Hg, DBP < 80 mm Hg; TC < 4.5 mmol/L, TG < 1.7mmol/L; HDL-C > 1.0mmol/L for male, 1.3 mmol/L for female; LDL-C < 2.6 mmol/L for patients without atherosclerotic cardiovascular disease and 1.8 mmol/L for patients with atherosclerotic cardiovascular disease; BMI < 24.0 kg/mt-test for 2 groups and analysis of variance for 3 or more groups. Continuous data distributed abnormally were described as median  and tested by Wilcoxon test for 2 groups and Kruskal–Wallis test for 3 groups. Categorical variables are presented as frequency and percentages and tested by χ2 test. P < .05 was considered statistically significant in the study.In the study, we compared physical measurement and laboratory tests among patients before and after MMC management for 1 year. Continuous data distributed normally were described as mean ± standard deviation, tested by A total of 363 patients with T2DM were included in the study, in which 276 (76.03%) were males and 87 (24.97%) were females. More than 80% of male patients had high school or above education level. The average level of HbA1c and HOMA-IR in patients with high school or above was lower than those in patients with middle school or less. More male patients were in high-income group (83.84%). The average level of HbA1c, HOMA-IR, HOMA-β in high-income group was lower than that in low-income group. The average SBP of patients with long course of T2DM was higher than that of moderate course and newly diagnosis group . FPG (10.21 ± 14.31mmo/L), HbA1c (9.51 ± 2.61%), TC (4.76 ± 1.06mmol/L) and LDL-C (2.82 ± 2.62mmol/L) in the newly diagnosed patients were higher than those in the other 2 groups. Table P < .001) in patients before and after MMC management for 1 year, LDL-C was 2.18 ± 1.49 mmol/L versus 1.81 ± 0.73 mmol/L (P < .001), Waist circumference was 90.28 ± 9.04cm versus 88.65 ± 9.37cm (P < .05), HOMA-β was 65.89 ± 90.81% versus 128.38 ± 293.93% (P < .05). However, body weight, BMI, FPG, 2hPG, DBP, TC, TG and HDL-C showed no significant changes before and after the management (P > .05) . The control rate of HDL-C in patients with high, medium and low family income were 42.86%, 34.97% and 16.28% , while the control rate of LDL-C were 78.57%, 84.51% and 97.67% , respectively. Compared with patients with medium or long course of T2DM, the control rate of FPG in patients with newly diagnosis was the highest . Table The control rate of BMI in patients with high school or above was higher than that in middle school or less group  before and after 1 year of management, the rate of blood pressure was 44.78% versus 56.32% , TG was 58.67% versus 69.23% , TC was 54.12% versus 69.78% , LDL-C was 73.08% versus 88.46% , BMI was 45.33% versus 57.14% . The overall control rate increased from 9.83% before management to 26.15% after management   model simulation. Our study added evidence that MMC program could improve the control rate of HbA1c and other metabolic outcomes in patients with T2DM. We proposed that clinical modes, especially for non-chronic disease, providing knowledge and skills with strict follow-ups for patients could enhance clinical outcomes. The modes may adopt information platform or new media.Control of blood glucose is critical for T2DM management. Results in the study showed the control rate improved overall after MMC management. HbA1c is one of the main metabolic indicators reflecting long-term glycemic control. The average HbA1c for majority of the patients with T2DM aimed to be less than 7%. However, compared with the middle and long course of duration groups, the FPG control rate of newly diagnosed T2DM patients was higher. The newly diagnosed patients might be younger, have better recovery of their islet β cell function after treatment. It was consistent with the results of MMC in Shanghai No.10 Hospital. Besides FPG, the study found that other indicators have no obvious differences among different demographics. On one hand, the management of MMC, including receiving the same diabetic education, standardization of diagnosis and treatment, and regular follow-up, might improve the overall conditions of the patients. On the other hand, it may also because the study ruled out patients with severe acute, chronic diabetic complications and patients with life expectancy < 5 years.There was no significant difference in HbA1c, waist circumference, BMI, blood pressure, TC, TG and other indexes of T2DM patients with different demographics after MMC management, indicating that demographic characteristics might have no significant impact on metabolic outcomes of the patients. The findings were accorded with the results from the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2010.This study has some limitations. The study only had patients who joined MMC program and compared their treatment effects before and after MMC. A control group of patients from conventional management mode should be involved in the future study. All the patients were those who visited the hospital for diabetic treatment. Sample size calculation omitted from the study. The patients in the study were often male and with high income, and this might have caused a bias in selection.The management of MMC emphasized providing knowledge and skills for patients with diabetes and has strict follow-up plans and procedures. Health education and close doctor-patient relationship greatly improved the compliance of patients with diabetes, which is conducive to delaying complications and reducing medical costs. Based on our study, we found MMC management benefited patients with T2DM in this area. It is necessary to continue to assess the treatment and control effect and expand this management model to other parts of China.Conceptualization: Xiaogang Bai, Jing Wang, Ting Bai, Yali Zhu, Sheli Li.Data curation: Yali Zhu.Formal analysis: Xia Li.Investigation: Jing Wang, Ting Bai, Sheli Li.Methodology: Xia Li, Ting Bai, Sheli Li, Xiaoling Niu.Project administration: Xiaogang Bai, Jing Wang, Yali Zhu.Resources: Wenjing Ren, Xiaoling Niu, Jiayin Zheng, Changqin Kou.Supervision: Sheli Li, Wenjing Ren, Xiaoling Niu, Jiayin Zheng.Software: Wenjing Ren.Validation: Wenjing Ren, Xiaoling Niu, Changqin Kou.Writing – original draft: Xia Li.Writing – review & editing: Xia Li, Xiaoling Niu, Jiayin Zheng, Changqin Kou.


For patients with ST-elevation myocardial infarction (STEMI), early reperfusion with primary percutaneous coronary intervention (PPCI) or thrombolytic treatment is essential to prevent major adverse cardiac events. The aim of the study is to compare the current status of managing STEMI patients at **** with European Society of Cardiology guidelines recommendations. Prospective cohort of all patients presenting with ST-elevation myocardial infarction (STEMI) between March 2020 and February 2021 in Alexandria University hospitals. Reporting patterns, causes of delay, and reperfusion status for all STEMI patients were noted. MACE:  was reported and compared among different management strategies.P < 0.001). Overall in-hospital mortality was 5.5%, and total MACE was 27.3%. A statistically significant difference was observed between the three management groups as regards MACE rate, being 15%, 28.1%, and 54.8% in PPCI, thrombolysis, and conservative groups, respectively.The study was conducted over one year on 436 patients, 280 (64.2%) of them underwent PPCI, 32 (7.3%) received thrombolysis, and 124 (28.5%) had a conservative strategy. Patients’ mean age was 55.2 years, 72.2% were smokers and 80.9% were men. Family history was positive in 14.2% of patients, 33.5% had diabetes, 7.3% had renal impairment, and 41.5% had hypertension. The median pre-hospital waiting time was 360 min; the mean pre-hospital waiting time was 629.0 ± 796.7 min. The median Emergency Room waiting time was 48.24 ± 89.30 min. The median time from CCU admission to wire crossing was 40.0 min with a mean value 53.86 ± 49.0 min. The mean ischemia duration was 408 min, while the total ischemic time was 372 min. All patients who presented within 12 h received reperfusion therapy either a PPCI or thrombolysis at a rate of 71.5%, with 35.0% of those patients achieving prompt reperfusion in accordance with ESC guidelines. The PPCI group mortality rate was 2.9%, in comparison to 12.9% in the conservative group, which was statistically significant (Despite financial and technical constraints, appropriate, timely reperfusion was near to achieving the ESC guidelines for the management of STEMI. The most common reperfusion strategy was PPCI, with an in-hospital death rate of less than 5% in the PPCI group. There was a concern about the increase in the total ischemia time due to some financial and technical constraints. An ST-elevation myocardial infarction (STEMI) is a major cause of morbidity and mortality worldwide . The acun = 124), by thrombolysis (n = 32), or by Primary Percutaneous Coronary Intervention (PPCI) (n = 288). Eight patients from the PPCI group were excluded due to the presence of pre-specified exclusion criteria. Admission within 12 h of the onset of chest discomfort qualified patients for inclusion, while those who were admitted later (with no ongoing pain) were excluded from the reperfusion strategy. Reporting patterns, causes of delay, and reperfusion status for all STEMI patients were noted. MACE events  were reported and compared among different management strategies.A total of 444 patients were admitted to Alexandria University Hospitals Cardiology department between March 2020 and February 2021 with STEMI. Patients were treated either conservatively  and Clopidogrel (loading dose of 600 mg) or Ticagrelor (loading dose of 180 mg) together with high-intensity statins and anticoagulation (intravenous unfractionated heparin 70–100 IU/kg) regularly. Radial or femoral access was used to perform PPCI. The culprit lesion was bridged with a guide wire, and the infarct-related artery was engaged with an adequately sized guiding catheter. Standard procedures were used to insert stents or dilate balloons. All patients received drug-eluting stents (DES). PPCI was restricted to Infarct Related Artery (IRA) in the event of multi-vessel illness. The TIMI flow grade was reported at baseline and following the procedure.Detailed history and physical examination were applied to all subjects, time variables and delays either pre-hospital, ER or CCU were documented. Intra-procedural and post-procedural complications were also reported. Follow up within hospital stay to detect any in-hospital events .With the aid of the IBM SPSS software package version 20.0, data were uploaded into the computer and evaluated. . Numbers and percentages were used to represent qualitative data. The normality of the distribution was examined using the Kolmogorov–Smirnov test. Interquartile range (IQR), mean, standard deviation, median, and range (minimum and maximum) were used to characterize quantitative data. At a 5% significance level, the obtained results were considered significant.Baseline characteristics were summarized in Table According to the management strategy, subjects were distributed into three groups; PPCI group, the thrombolytic group, and the conservative group. Table As mentioned in Table In contrast, the average ER delay time was 48.24 ± 89.30 min. With a mean value of 53.86 ± 49.0 min, the median time from CCU admission to wire crossing was 40.0 min. The mean ischemia duration was 408 min (6.8 h), while the overall ischemic time was 372 min (6.2 h). All STEMI patients who presented within 12 h received reperfusion therapy (PPCI or thrombolysis) at a rate of 71.5 percent, with 35.0% achieving prompt reperfusion in accordance with ESC guidelines for the management of STEMI (Tables P < 0.001), bleeding complications (P = 0.010), heart failure (P < 0.001) and in hospital Major adverse cardiac events (MACE) (P < 0.001).Figure Regression analysis for different parameters affecting in-hospital MACE is depicted below Table .Table 7UHealthcare system performance, as well as patient education and behavior, are the cornerstone in the management of STEMI and improving clinical outcomes. The emerging need to conduct this study is clear with the lack of a STEMI network in Alexandria. Reperfusion delays are the most easily audited index in STEMI management of quality care. A patient’s delay or a healthcare system’s delay is what delays the reperfusion strategy. Delay in the healthcare system is the period between FMC and reperfusion. A delay in the healthcare system can occur at many stages: Emergency Medical Services (EMS) delay, ER delay and CCU delay.As regarding baseline characteristics, Zeymer et al.  describeThe mean value of pre-hospital delay in our study was 629.0 ± 796.7 min (10.4 h) Table . The maivs. 292 min; P < 0.001) and a higher reperfusion rate . Choosing not to use the ER also decreased five-day mortality rates .ER delay was appointed as the time from ER admission to CCU admission. Upon analysis, the main factor of delay was transfer delay (61.5%) due to deficient numbers of transporting equipment and personnel. ER high volume admissions with the lack of ER beds and equipment also play an important role as it delays reaching to diagnosis. Steg et al.  studied As the primary PCI center, Door to balloon was calculated from the first medical contact in our emergency department through CCU to Cath lab. Mean door to crossing time was 92.86 ± 54.66 min, and the median time was 70.0 (60.0–110.0). ESC latest guidelines for the management of STEMI described Timely PPCI in PPCI capable hospital as less than 60 min from door to balloon, Zeymer et al. [n = 436) irrespective of the type of management, while in the PPCI group, 2.9% (n = 280), and 12.9% (n = 124) in the conservative management group, with a statistically significant difference (P < 0.001). Within one-month mortality rate was 3.4% irrespective of the type of management, with the highest in the conservative group, 8.2%, with a statistically significant difference from other groups (MCp = 0.001). MACE rate was 27.3% (n = 436) irrespective of the type of management, while the MACE rate was 15% in the PPCI group, 28.1% in the thrombolytic group, and 54.8 in the conservative group with a statistically significant difference (P < 0.001). One month follow-up MACE rate was 17.9% irrespective of the type of management, with the highest in the conservative group at 34.1%, with a statistically significant difference from other groups (P = 0.001)  irrespective of the type of management, while mortality occurred in 3.1% of the PPCI group (n = 8275), 4.4% (n = 2160) in thrombolytic group and 14.1% (n = 1027) conservative management group, which is consistent with our study.In-hospital mortality, in our study was 5.5% n = 43 irrespecIn-hospital mortality was observed by Shaheen et al.  to be 4.Despite financial and technical constraints, timely reperfusion was near to achieving the ESC guidelines for the management of STEMI. The most common reperfusion strategy was PPCI, with an overall in-hospital death rate of less than 5%. There was a concern about the increase in the total ischemia time due to some financial and technical constraints.This study emphasized the essential need for the expansion of public awareness and patient education and sufficient training of general practitioners and ER physicians to improve STEMI management. Design and application of the STEMI network while improving the EMS performance in **** are a must.


Diospyrus lotus L.) is an edible fruit from the Ebenaceae family, rich in nutrients, and having tremendous medicinal properties. This paper attempted to show the influence of different parameters of convective drying such as temperature  and air velocity  on the shrinkage and microstructure, rehydration properties, antioxidant activity, and phenolic compounds of date plum. The drying caused significant changes in the color, actual size, and distribution of the fruit cells of date plum. The total phenolic content (TPC), total flavonoid content (TFC), ferric reducing antioxidant power (FRAP), and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) of fresh date plum were 0.81 ± 0.00 mg GAE/g, 0.23 ± 0.10 mg ECE/g, 7.15 ± 1.09 mmol ISE/g, and 14.92 ± 0.88 mmol/TE, respectively. The drying at 70°C had the highest values of TPC, TFC, gallic acid, chlorogenic and syringic acids, catechin, quercetin‐3‐glucoside, resveratrol, and DPPH. The drying air velocities showed no significant effects on the antioxidant contents and the antioxidant activity. Of the models applied to the drying kinetics, the Midilli model was found as the best model to describe the drying kinetics of date plum. In addition, the Weibull model was found as the most successful among the models applied to the rehydration kinetics of date plum. According to the achieved findings, the convective drying temperature of 70°C is the optimum temperature to produce the dehydrated date plum.Date plum (D. lotus L. The study found the optimum drying conditions, and Midilli and Weibull models were the most fitted models to describe the drying and rehydration behaviors of D. lotus L. fruits, respectively. The drying provides a reasonable value of the possibility of continuous consumption of the fruits dried afforded on off‐seasons. The dried fruits are widely used for multipurpose and have been extensively used in food industries due to their rich nutraceutical and antioxidant compounds.This work has revealed the drying conditions responsible for preserving the phenolic compounds, total flavonoid content, and antioxidant features of  Date plum is cultivated in several countries for its edible fruits, owing higher nutritive and medicinal properties. The consumption of fruits is recommended for their beneficial effects on human health including sedative, antiseptic, antidiabetic, antitumor, laxative, antidiarrhea, dry cough reliever, and tension regulator  fruits were collected from Samsun (Turkey) in 2019 during the Winter harvest season. Fruits were sorted, filled in PTE bags (ca. 300 g), and kept in the refrigerator.Date plum , 2,4,6‐tris(2‐pyridyl)−1,3,5‐triazine (TPTZ), acetone, Trolox, sodium nitrite, hydrochloric acid, methanol, sodium hydroxide, Folin‐Ciocalteau reagent, (−)‐ epicatechin, hesperidin, catechin, fumaric acid, and quercetin were purchased from Sigma‐Aldrich. Gallic acid (GA) and sodium carbonate were purchased from Riedel‐de Haen. Sodium acetate and glacial acetic acid were bought from Carlo Erba. Aluminum (III) chloride and iron (III) chloride were acquired from Merck.2.3L*a*b* scale was measured. The total soluble solids were evaluated at 25°C using an Abbe refractometer , and pH was measured with a pH meter.The moisture content was measured in an oven at 70°C for 24 h according to the preliminary tests. The color was determined using a colorimeter  and the CIE 2.4The drying system was a convective regime cabinet dryer  described in our previous study  was determined using Equation :(1)MRR2), chi‐square (χ2) and root mean square error (RMSE) were generated by MATLAB software (R2016d) to validate the fitness of the models  and expressed as mg gallic acid equivalent per g (mg GAE/g).The TPC was determined by the Folin‐Ciocalteu method adopted from Zannou and Koca . Briefly2.82 and left to stand for 5 min, followed by the addition of 0.5 ml of 5% AlCl3. The mixture was kept for 6 min before adding 0.5 ml of 1 M NaOH. After 10 min, the absorbance was read at 510 nm. The TFC was estimated based on a calibration curve using epicatechin as standard. The results were given as mg epicatechin equivalents (ECE)/g dw.The TFC was determined using the protocol of Zannou and Koca . One mil2.9cA is the absorbance of the control and sA is the absorbance of extract.The DPPH radical scavenging was determined using the method described in Zannou and Koca . Briefly2.103:10 mM TPTZ . The assembly was shaken for about 5 min, and the absorbance was read at 593 nm against a blank. The FRAP values of the extracts were calculated from the calibration curve using FeSO4 as a standard. The results were given as mmol FeSO4 equivalents (mmol ISE/g dw).Ferric reducing antioxidant power (FRAP) assay was performed according to the procedure of Zannou and Koca . Briefly2.11cM) and rehydration ratio (rR) were calculated according to Pashazadeh, Zannou, Koca –(4).The rehydration was conducted at room temperature 25°C), and the rehydration features such as moisture content  as described in Zannou et al. . The liq2.13The fresh and dried samples’ microstructures were obtained using scanning electron microscope (SEM) (JEOL JSM‐7001F) , adjusted coefficient of determination (adj. R2), coefficient of variation (CV), and Fisher's test value . The regression coefficients were considered significant at p < 0.05. The optimum parameters were estimated considering the desirability function. The analyses were carried out in triplicate, and the one‐way ANOVA with post hoc Duncan's test was used . The significance of the results was given at p≤ 0.05.The MATLAB software (R2016d) was used for the modelling and the Design‐Expert software 9.0  to generate the optimization models and one‐factor graphics. The statistical significance of independent variables and the correlation between them was evaluated using ANOVA. The adequacy of the optimization model was determined based on the coefficient of determination  with a flesh/seed ratio of 2.81 and the number of grains is about 220 for each kilogram. As shown in Table In this study, the TPC, TFC, and antioxidant activities (FRAP and DPPH) were determined Table . Evidenc3.2MR, t) obtained for the different temperatures indicated that the Midilli model was the well fitted with the experimental data giving the highest coefficient of determination (R2), the lowest RMSE and lowest chi‐square (χ2)  Table . These r3.3R2 higher than 0.99, lower χ2 (0.0005), and lower RMSE (0.0243) . As can be seen in Table Thirteen phenolic compounds include gallic acid, catechin, chlorogenic, caffeic acid, vanillic acid, epicatechin, syringic, p‐coumaric acid, ferulic acid, sinapic acid, quercetin‐3‐glucoside, salicylic acid, and resveratrol were identified from date plum samples Table . Previou4The present study attempted to examine the characterization of dried date plum and the influence of the drying process on its total phenolic compound, rehydration, microstructure, antioxidant activity, and phenolic compounds. The results showed that the convective drying of the date plum increased the total phenolic content, flavonoid content, antioxidant activity, and individual phenolic compounds when increasing the temperature up to 70°C. Additionally, the drying process showed significant effects on microstructure, drying, and rehydration kinetics. The best mathematical models of drying and rehydration kinetics were found to be Midilli and Weibull models, respectively. These findings have significant implications for understanding how to assess the drying parameters of rehydration, antioxidant activity, and phenolic compounds of date plum. This study revealed that the date plum is rich in bioactive ingredients, and the dried product can be used for multipurpose in food, cosmetic, and drug industries. Further research should focus on other drying methods to produce interesting findings on the best drying conditions for date plums.Awadalgeed M. A. Hassan: conceptualization; data curation; methodology; visualization; writing – original draft. Oscar Zannou: conceptualization; data curation; methodology; visualization; writing – original draft; writing – review & editing. Hojjat Pashazadeh: Conceptualization; Data curation; Methodology; Visualization; Writing – original draft; Writing – review & editing. Ali Ali Redha: data curation; funding acquisition; validation; visualization; writing – original draft; writing – review & editing. Ilkay Koca: conceptualization; data curation; funding acquisition; methodology; project administration; supervision; validation; visualization; writing – original draft; writing – review & editing.None to declare.


Rana pirica. Our experimental approach revealed that R. pirica tadpoles exhibit cell death in their limbs that is likely to be induced by oxidative stress associated with their frequent air–breathing behavior.Amphibians shape their limbs by differential outgrowth of digits and interdigital regions. In contrast, amniotes employ cell death, an additional developmental system, to determine the final shape of limbs. Previous work has shown that high oxygen availability is correlated with the induction of cell death in developing limbs. Given the diversity of life histories of amphibians, it is conceivable that some amphibians are exposed to a high–oxygen environment during the tadpole phase and exhibit cell death in their limbs. Here, we examined whether air–breathing behavior underlies the cell death in limbs of aquatic tadpoles of the frog species The online version contains supplementary material available at 10.1186/s40851-022-00199-x. The shapes of the hands and feet are crucial for tetrapods to adapt to their habitats. In amphibians, the balance between the growth of the digits and interdigital regions determines whether the hands and feet are webbed , 2. HoweDespite the importance of cell death in the interdigital regions, the molecular mechanisms promoting it remain largely unclear. Bmp signaling is one of the critical pathways required for cell death to shape amniote limbs –13. ReceDesmongnathus aeneus) [Eleutherodactylus coqui) [Xenopus laevis, suggesting that the interdigital regions of amphibians have the molecular background to induce cell death in response to increased levels of environmental oxygen which is supplied via interdigital blood vessels [It has long been appreciated that cell death does not occur in the interdigital regions of amphibians , 2. Howe aeneus)  and the s coqui) . These a vessels .In amniote eggs, the highly vascularized extraembryonic membranes exchange oxygen with the air to provide an abundant oxygen supply to growing embryos. Thus, interdigital tissue of amniote embryos is supplied with sufficient levels of oxygen to produce ROS required for the induction of cell death . HoweverRana pirica and leads to the induction of cell death in developing limbs. Rana pirica is a typical frog species that develops through an aquatic tadpole stage into an adult, and its tadpoles seemed to rise to the water surface to breath relatively frequently  tadpoles were kept in 0.1% sea–salt solution at 16 °C and staged according to a developmental table [R. pirica and X. laevis were photographed with a single–lens reflex camera  .The frequency of air–breathing behavior of es Fig. S and 2 waX. laevis and coqui frogs [2, 3.3 mM MgCl2) for 2 hours, washed and photographed with na LSM780 confocal microscope (Zeiss). Some of the LysoTracker Red-stained tadpoles were then stained with 0.01% Nile blue in filtered water for 20 minutes, washed, and photographed. For TUNEL staining, hindlimb buds from stage 36 tadpoles were cryosectioned at 8–12 μm as described [Cell death was detected using LysoTracker, Nile Blue, or TUNEL, as previously described , 26, 27.ui frogs , 30. Briescribed , and staOxygen incubation of tadpoles were carried out as described . Brieflyhttps://fiji.sc). Images were converted to 8–bit images, adjusted for contrast, nonlinearly transformed with the gamma function, and binarized with the threshold. We considered the particle size from 5 to 8 to 200 pixels as cells, which were counted using the Analyze Particles function.We counted the number of the LysoTracker-positive cells as described , with moBlood vessels were visualized as previously described , 31. BriR. pirica tadpoles at early stage 36 were reared overnight in a 500 ml beaker without a plastic net (control), or with a plastic net to prevent the tadpoles from rising to the water surface  or in a 15 ml plastic tube covered with a plastic net for 3 days in a 1.5 L tank .Statistical analyses were performed with Prism 8.0.2 (GraphPad). The data are presented as the mean ± SEM. Significance was determined using two–tailed unpaired Images were captured using an LSM780 confocal microscope (Zeiss), stereomicroscope  with microscope digital camera , or single–lens reflex camera . Pseudocolor images were generated using Zen Black software (Zeiss). The following panels are maximum intensity projections of confocal image stacks: Figs. X. laevis and R. pirica tadpoles at stages 54–56 and 36–37, the stages in which all five digits of hindlimbs are formed, respectively  and are filled with air by stage 58 (when hindlimb morphogenesis is complete), the latter visualized by the presence of air bubbles [R. pirica tadpoles, 45% contained air bubbles. At stage 36, of the 12 lungs dissected from tadpoles, 75% contained air bubbles . These observations suggest that R. pirica tadpoles use their lungs for gas exchange at least by early stage 37.Next, we examined whether pulmonary respiration occurs during hindlimb development stages of  bubbles , 32, 33.X. laevis tadpoles, is correlated with high oxygen availability [R. pirica tadpoles by labelling them with LysoTracker . LysoTracker–positive cells were found preferentially in the interdigital regions, as well as in the limb margins . When we used the TUNEL assay, a few apoptotic cells were detected in the limb margin (n = 6/6) and the interdigital region (n = 4/6) at stages 35–37 , late stage 36 (n = 2), and early stage 37 (n = 2), high production of ROS was observed in LysoTracker–positive cells in both the limb margins and the interdigital regions  resulted in an approximately 1.7–fold increase in the amount of dissolved oxygen in the water . After 3 hours of incubation, the number of LysoTracker–positive cells was increased in the interdigital regions at late stage 36, but not at other stages under hyperoxia , consistent with the distribution of LysoTracker–positive cells ), the following stages were used: early stage 36 (L < 400 µm), mid stage 36 (400 µm < L < 500 µm), late stage 36 (500 µm < L < 700 µm), and early stage 37 (700 µm < L < 900 µm). Dashed lines indicate the outlines of hindlimbs. Anterior is to the left. Scale bars, 1 mm. b Schematic diagram of the R. pirica hindlimb with the length of the fourth digit (D4) defined as “L”. D1-5: digits 1-5; Dist.: distal; Post.: posterior.Fig. S2.X. laevis and R. pirica frogs and their hindlimbs. Interdigital webbings were observed in hindlimbs of both X. laevis and R. pirica post-metamorphic froglets. Fig. S3. Air breathing and lung blood vessels. a Frequency of air-breathing behavior of X. laevis and R. pirica tadpoles. Each symbol represents an individual specimen. Mean ± SEM. Two-tailed unpaired t-test. ∗p< 0.05. b A lung dissected from a stage 37 R. pirica tadpole. The presence of a large blood vessel (arrowheads) and thin capillary vessels (an arrow) were recognized. Scale bars, 200 µm. c Frontal section through the lungs of stage 37 R. pirica lungs. c’, c” Higher magnifications of the rectangles in (c). Blood vessels were defined in the lung wall (arrows). Scale bars, 500 µm in (c) and 100 µm in . Fig. S4. Cell death detection in R. pirica hindlimbs. a, a’ A hindlimb of stage 36 R. pirica tadpoles stained with LysoTracker Red and Nile Blue sulfate simultaneously. Note that both LysoTracker- and Nile Blue-signals were recognized in the same cells (arrowheads). b An optical  section of stage 36 R. pirica hindlimbs stained with LysoTracker. A few LysoTracker positive cells were detected in the interdigital region and the ectoderm (arrowheads). c, d TUNEL staining of stage 36 R. pirica hindlimbs. TUNEL positive cells were detected in the ectoderm (arrowheads in (c)) and the interdigital region (arrowheads in (d)). Scale bars, 100 µm. D4: digit 4; ID3-4: interdigital region 3-4. Fig. S5. Cell death, ROS staining and blood vessel pattern. a LysoTracker Green and CellROX staining of R. pirica hindlimbs at mid stage 36 and early stage 37. The arrowheads point to both LysoTracker- and CellROX-positive cells. The asterisks indicate pigments, not stained cells. b Vasculature (injected with fluorescent ink) of R. pirica hindlimbs stage 36. Vessels were observed in interdigital regions, limb margin edges (arrowheads), and surface ectoderm (arrows). c Vasculature (injected with fluorescent ink) of R. pirica hindlimbs (stage 37). Scale bars, 100 µm. D3: digit 3; ID3: interdigital region 3.thods of  with sliugulosus . BrieflyAdditional file 2: Movie 1. Air-breathing behavior of X. laevis tadpoles. Additional file 3: Movie 2. Air-breathing behavior of R. pirica tadpoles.