Empagliflozin improves renal ischemia-reperfusion injury by reducing inflammation and enhancing mitochondrial fusion through AMPK-OPA1 pathway promotion.

BACKGROUND: Renal ischemia-reperfusion injury (IRI) is one reason for renal transplantation failure. Recent studies have shown that mitochondrial dynamics is closely related to IRI, and that inhibition or reversal of mitochondrial division protects organs against IRI. Optic atrophy protein 1 (OPA1), an important factor in mitochondrial fusion, has been shown to be upregulated by sodium-glucose cotransporter 2 inhibitor (SGLT2i). Also, the antiinflammatory effects of SGLT2i have been demonstrated in renal cells. Thus, we hypothesized that empagliflozin could prevent IRI through inhibiting mitochondrial division and reducing inflammation. METHODS: Using hematoxylin-eosin staining, enzyme linked immunosorbent assay (ELISA), flow cytometry, immunofluorescent staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining, real-time PCR, RNA-sequencing, and western blot, we analyzed renal tubular tissue from in vivo and in vitro experiments. RESULTS: Through animal experiments and sequencing analysis, we first confirmed the protection against IRI and the regulation of mitochondrial dynamics-related factors and inflammatory factors by empagliflozin pretreatment. Then, through hypoxia/reoxygenation (H/R) cellular experiments, we confirmed that empagliflozin could inhibit mitochondrial shortening and division and upregulate OPA1 in human renal tubular epithelial cell line (HK-2) cells. Subsequently, we knocked down OPA1, and mitochondrial division and shortening were observed, which could be alleviated by empagliflozin treatment. Combined with the previous results, we concluded that OPA1 downregulation leads to mitochondrial division and shortening, and empagliflozin can alleviate the condition by upregulating OPA1. We further explored the pathway through which empagliflozin functions. Related studies have shown the activation of AMPK pathway by empagliflozin and the close correlation between the AMPK pathway and OPA1. In our study, we blocked the AMPK pathway, and OPA1 upregulation by empagliflozin was not observed, thus demonstrating the dependence of empagliflozin on the AMPK pathway. CONCLUSION: The results indicated that empagliflozin could prevent or alleviate renal IRI through antiinflammatory effects and the AMPK-OPA1 pathway. Ischemia-reperfusion injury is an inevitable challenge in organ transplantation. It is necessary to develop a new therapeutic strategy for IRI prevention in addition to refining the transplantation process. In this study, we confirmed the preventive and protective effects of empagliflozin in renal ischemia-reperfusion injury. Based on these findings, empagliflozin is promising to be a preventive agent for renal ischemia-reperfusion injury and can be applied for preemptive administration in kidney transplantation.

Serum periostin and TNF-α levels in patients with obstructive sleep apnea-hypopnea syndrome.

PURPOSE: Obstructive sleep apnea-hypopnea syndrome (OSAHS) may cause pulmonary diseases, and periostin plays an important role on the development of pulmonary diseases. In addition, periostin and pro-inflammatory cytokine TNF-α can regulate each other in vivo. This study aimed to observe the changes of serum periostin and TNF-α levels in patients with OSAHS compared with healthy volunteers and to investigate their correlation. METHODS: A convenience sample of 67 patients with OSAHS in our hospital from December 2018 to December 2019 was selected and categorized into mild, moderate, and severe groups according to apnea-hypopnea index by polysomnography. In addition, 21 healthy volunteers were selected as the control group. Serum levels of periostin and TNF-α were determined by enzyme-linked immunosorbent assay (ELISA). Results were analyzed using the SPSS software. RESULTS: Both serum periostin and TNF-α levels in all the three OSAHS groups were higher than those of the control group and increased with severity of OSAHS. The severe group had significantly higher serum periostin and TNF-α levels than the mild and moderate groups (p < 0.05). For patients with OSAHS, serum periostin and TNF-α levels positively correlated with the apnea-hypopnea index (AHI) (p < 0.01) and negatively correlated with the lowest saturation oxygen (LSaO2) and mean saturation oxygen (MSaO2) (both p < 0.01). In addition, there was a positive correlation between serum periostin and TNF-α levels in patients with OSAHS (p < 0.001). CONCLUSIONS: Serum periostin and TNF-α levels were significantly increased in patients with OSAHS and may serve as a potential biomarker for severity of OSAHS. These findings suggest that it may be fruitful to study the role of periostin and TNF-α in OSAHS-induced pulmonary diseases.

Elevated levels of interleukin-35 and interleukin-37 in adult patients with obstructive sleep apnea.

BACKGROUND: Systemic inflammation has a critical role in the pathogenesis of obstructive sleep apnea (OSA). Interleukin (IL)-35 and IL-37 have been identified as novel immune-modulating cytokines with anti-inflammatory activities in numerous types of inflammatory disease. The present study aimed to examine the serum levels of IL-35 and IL-37 in patients with OSA, and to investigate their associations with the severity of OSA. METHODS: A total of 97 patients, including 67 cases of OSA and 30 age- and gender-matched healthy control subjects, were enrolled in the present study. All subjects were evaluated by overnight polysomnography. Serum IL-35, IL-37, and pro-inflammatory cytokine IL-1ß levels were examined by ELISA. RESULTS: Compared with those in the control subjects, serum IL-35, IL-37, and IL-1ß levels were significantly elevated in patients with mild, moderate, or severe OSA. Furthermore, a severity-dependent increase in serum IL-35 and IL-37 levels was observed in patients with OSA. IL-35 and IL-37 levels were positively correlated with the apnea-hypopnea index (r = 0.742 and 0.578, respectively; both p < 0.001), while they were negatively correlated with the mean oxygen saturation (r = -0.461 and -0.339, respectively; both p < 0.001) and lowest oxyhaemoglobin saturation (r = -0.616 and -0.463, respectively; both p < 0.001) in patients with OSA. In addition, a positive correlation was observed between IL-35 or IL-37 and IL-1ß levels (all p < 0.001). CONCLUSION: The serum levels of IL-35 and IL-37 were significantly increased in patients with OSA and associated with the severity of OSA, implying that IL-35 and IL-37 may have a protective role in OSA by counteracting inflammatory responses.

[Association of obstructive sleep apnea hypopnea syndrome with carotid atherosclerosis and the efficacy of continuous positive airway pressure treatment].

OBJECTIVE: To evaluate the association of obstructive sleep apnea hypopnea syndrome (OSAHS) with carotid atherosclerosis and the efficacy of continuous positive airway pressure (CPAP) treatment. METHODS: A total of 93 OSAHS patients diagnosed by polysomnography (PSG) were selected from Sleep Disorders Center at Affiliated Hospital of Xuzhou Medical College between March 2013 and December 2014. Based on the results of apnea-hypopnea index (AHI), they were divided into mild (n=22), moderate (n=37), and severe OSAHS group (n=34). Meanwhile, 28 healthy adult individuals matched for age and body mass index (BMI) were enrolled as the control group. The carotid intima-mesa thickness (IMT) was measured by color Doppler uhrasonography, and plasma levels of tumor necrosis factor-α (TNF-α), endothelin-1 (ET-1) and nitric oxide (NO) were determined by Enzyme-Linked Immunosorbent Assay (ELISA). The correlations between carotid IMT and plasma levels of TNF-α, ET-1 and NO were analyzed. A total of 24 patients with moderate to severe OSAHS underwent CPAP treatment and the carotid IMT, plasma levels of TNF-α, ET-1 and NO were compared before and after CPAP treatment. RESULTS: OSAHS patients had significant increase of carotid IMT with the increasing disease severity, and the carotid IMT in mild, moderate and severe OSAHS groups were all significantly higher than that in the control group ((0.73 ± 0.31), (0.86 ± 0.07), (1.07 ± 0.14) vs (0.65 ± 0.10) mm, all P<0.05). The plasma levels of TNF-α and ET-1 in mild to severe OSAHS group were significantly higher than those in controls ((17.45 ± 3.02), (23.81 ± 2.91), (35.16 ± 3.43) vs (12.53 ± 3.48) ng/L and (0.81 ± 0.13), (1.06 ± 0.21), (1.66 ± 0.30) vs (0.64 ± 0.12) ng/L, all P<0.05 ), whereas plasma levels of NO in the three OSAHS groups were significantly decreased compared with the control group ((35.46 ± 10.12), (29.32 ± 9.47), (20.16 ± 7.41) vs (45.43 ± 7.92) µmol/L, all P<0.05). Furthermore, there were significant differences in plasma levels of TNF-α, ET-1 and NO among the three OSAHS groups (all P<0.05). Carotid IMT was positively correlated with plasma TNF-α and ET-1 (r=0.56 and 0.51) and negatively correlated with plasma NO (r=-0.46) (all P<0.05). After 3 months of CPAP treatment, plasma levels of TNF-α and ET-1 in OSAHS patients were significantly reduced ((19.64 ± 5.28), (0.94 ± 0.21) vs (28.72 ± 5.36), (1.36 ± 0.36) ng/L), and plasma NO was markedly increased ((33.57 ± 6.32) vs (24.34 ± 4.46) µmol/L, all P<0.05). However, CPAP treatment did not have a significant effect on carotid IMT ((0.91 ± 0.21) vs (0.96 ± 0.14) mm), P>0.05). CONCLUSIONS: Systemic inflammation and vascular endothelial dysfunction may play an important role in pathogenesis and development of carotid artery atherosclerosis in OSAHS. Short-term CPAP therapy alleviates systemic inflammation and improves endothelial function, but does not influence the increased carotid IMT in OSAHS patients.

Patient-reported outcomes for the phase 3 FURLONG study of furmonertinib versus gefitinib as first-line therapy for Chinese patients with locally advanced or metastatic EGFR mutation-positive non-small cell lung cancer.

Background: Furmonertinib showed superior efficacy compared with gefitinib as first-line therapy in patients with epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC) in the FURLONG study. Here we present prespecified secondary endpoints of patient-reported outcomes (PRO). Methods: In this multicentre, double-blind, double-dummy, randomised phase 3 study, patients were 1:1 randomly assigned to receive furmonertinib 80 mg once daily or gefitinib 250 mg once daily. PROs assessed by the European Organization for Research and Treatment of Cancer Quality-of-Life Questionnaire Core 30 and Quality-of-Life Questionnaire Lung Cancer 13 were analysed using a mixed model for repeated measures and time-to-event analyses. A difference in score of 10 points or more was deemed clinically relevant. Findings: Three hundred and fifty-seven patients (furmonertinib group, n = 178; gefitinib group, n = 179) received at least one dose of the study drug, all of whom completed at least one PRO assessment. Statistically significant difference of overall score changes from baseline favoured furmonertinib in physical functioning (between-group difference 2.14 [95% CI 0.25-4.04], p = 0.027), nausea/vomiting (-1.56 [95% CI -2.62 to -0.49], p = 0.004), appetite loss (-2.24 [95% CI -4.26 to -0.23], p = 0.029), diarrhoea (-3.36 [95% CI -5.19 to -1.54], p < 0.001), alopecia (-2.62 [95% CI -4.54 to -0.71], p = 0.007), and pain in other parts (-4.55 [95% CI -7.37 to -1.74], p = 0.002), but not reached clinical relevance. Time to deterioration in physical functioning (hazard ratio 0.63 [95% CI 0.42-0.94], p = 0.021), cognitive functioning (0.73 [95% CI 0.54-0.98], p = 0.034), nausea/vomiting (0.64 [95% CI 0.41-0.99], p = 0.042), appetite loss (0.63 [95% CI 0.43-0.92], p = 0.016), diarrhoea (0.63 [95% CI 0.46-0.85], p = 0.002), dyspnoea (0.72 [95% CI 0.53-0.98], p = 0.034), cough (0.67 [95% CI 0.44-1.00], p = 0.049), dysphagia (0.54 [95% CI 0.35-0.83], p = 0.004), and alopecia (0.62 [95% CI 0.42-0.90], p = 0.012) was longer with furmonertinib versus gefitinib. Interpretation: In patients with locally advanced or metastatic EGFR mutation-positive NSCLC, furmonertinib showed improved scores and delayed deterioration in several functioning and symptoms compared to gefitinib. Funding: Shanghai Allist Pharmaceutical Technology Co., Ltd and the National Science and Technology Major Project for Key New Drug Development (2017ZX09304015).

[Expression and significance of immunoglobulin free light chains in serum and lung tissues from patients with chronic obstructive pulmonary disease].

OBJECTIVE: To study the association of free immunoglobulin light chain (FLC) with clinical manifestations and lung inflammation in smokers with normal lung function and chronic obstructive pulmonary disease (COPD) patients. METHODS: Thirty-two patients with peripheral lung cancer undergoing surgical resection were enrolled from the Department of Thoracic Surgery,Affiliated Hospital of Xuzhou Medical College. They were divided into non-smoking with normal lung function group (non-smoking group, 10 cases), smoking with normal lung function group (smoking group, 12 cases) and smoking with stable COPD group (COPD group, 10 cases). Their preoperative fasting serum and lung tissues away from cancer were used in the study.Enzyme-linked immunesorbent assays (ELISA) were used to detect the levels of FLC-λ and FLC-κ in serum and lung tissue homogenates. The expression of FLC-λ and FLC-κ in the airway epithelium, alveolar wall and blood vessel wall was detected by immunohistochemistry. The correlation between FLC levels and pulmonary functions were analyzed. RESULTS: The serum levels of FLC-λ and FLC-κ in COPD group and smoking group were (35 ± 11),(38 ± 12) and (26 ± 9),(26 ± 8) mg/L, respectively. They were all significantly increased compared with the non-smoking group [(16 ± 7),(16 ± 5) mg/L]. The differences were all statistically significant (q = 3.590-7.482, P < 0.01), and those of the COPD group were significantly higher than those of the smoking group (q = 3.209-4.198, P < 0.05 and P < 0.01). The concentrations of FLC-λ and FLC-κ in lung tissue homogenates of the COPD group and the smoking group were (1.29 ± 0.31),(1.32 ± 0.30) and (0.86 ± 0.42),(0.85 ± 0.37) µg/mg, respectively. They were all significantly increased compared with those of the non-smoking group [(0.45 ± 0.18),(0.42 ± 0.13) µg/mg],(q = 4.178- 9.795, P < 0.05 and P < 0.01). The levels of FLC-λ and FLC-κ in the lung tissue homogenates from the COPD group were significantly higher than those from the smoking group (q = 4.269-4.349, all P < 0.05). The expression of FLC-λ and FLC-κ was detected in airway epithelium, alveolar wall and blood vessel wall. The levels of FLC-λ and FLC-κ in serum and lung tissue homogenates showed a negative correlation with FEV1 percentage of predicted value (r = -0.476 to -0.591, all P < 0.01). CONCLUSIONS: Expressions of FLC were increased in the serum and the lung tissues of COPD patients and smokers with normal lung function, and closely correlated with airflow limitation. The results suggest that FLC plays a proinflammatory role in the pathogenesis of COPD.

A Double-Blind Randomized Placebo-Controlled Phase 3 Trial of Tobramycin Inhalation Solution in Adults With Bronchiectasis With Pseudomonas aeruginosa Infection.

BACKGROUND: Few large-scale studies have demonstrated the efficacy of tobramycin nebulization in bronchiectasis. We evaluated the efficacy and safety of nebulized tobramycin inhalation solution (TIS) in adults with bronchiectasis with Pseudomonas aeruginosa infection. RESEARCH QUESTION: Can TIS effectively reduce sputum P aeruginosa density and improve the bronchiectasis-specific quality of life in patients with bronchiectasis with P aeruginosa infection? STUDY DESIGN AND METHODS: This was a phase 3, 16-week, multicenter, randomized, double-blind, placebo-controlled trial. Eligible adults with bronchiectasis were recruited from October 2018 to July 2021. On the basis of usual care, patients nebulized TIS (300 mg/5 mL twice daily) or normal saline (5 mL twice daily) via vibrating-mesh nebulizer. Treatment consisted of two cycles, each consisting of 28 days on-treatment and 28 days off-treatment. The coprimary end points included changes from baseline in P aeruginosa density and Quality-of-Life Bronchiectasis Respiratory Symptoms score on day 29. RESULTS: The modified intention-to-treat population consisted of 167 patients in the tobramycin group and 172 patients in the placebo group. Compared with placebo, TIS resulted in a significantly greater reduction in P aeruginosa density (adjusted mean difference, 1.74 log10 colony-forming units/g; 95% CI, 1.12-2.35; P < .001) and greater improvement in Quality-of-Life Bronchiectasis Respiratory Symptoms score (adjusted mean difference, 7.91; 95% CI, 5.72-10.11; P < .001) on day 29. Similar findings were observed on day 85. TIS resulted in a significant reduction in 24-h sputum volume and sputum purulence score on days 29, 57, and 85. More patients became culture negative for P aeruginosa in the tobramycin group than in the placebo group on day 29 (29.3% vs 10.6%). The incidence of adverse events and serious adverse events were comparable between the two groups. INTERPRETATION: TIS is an effective treatment option and has an acceptable safety profile in patients with bronchiectasis with P aeruginosa infection. TRIAL REGISTRATION: ClinicalTrials.gov; No. NCT03715322; URL: www. CLINICALTRIALS: gov.

Anti-inflammatory effects of moxifloxacin on rat airway smooth muscle cells exposed to allergen: Inhibition of extracellular-signal-regulated kinase and nuclear factor-κB activation and of interleukin-8 and eotaxin synthesis.

BACKGROUND AND OBJECTIVE: Moxifloxacin (MXF) has been shown to possess immunomodulatory properties in addition to its antimicrobial effects. We investigated the effects of MXF on cytokine secretion and signal transduction mechanisms in naive control and allergen-exposed airway smooth muscle cell (ASMC) stimulated with tumour necrosis factor (TNF)-α. METHODS: An animal model was established. ASMC was derived from rat airway tissue and cultured in vitro, then incubated with 10 ng/mL of TNF-α. Interleukin (IL)-8 and eotaxin secretion were measured by enzyme-linked immunosorbent assay, and activation of extracellular-signal-regulated kinase (ERK)1/2 and nuclear factor (NF)-κB p65 was measured by western blotting, with or without the addition of MXF (20 µg/mL) and/or dexamethasone (DXM) (10(-6) M). RESULTS: Baseline IL-8 and eotaxin secretion did not differ between control and allergen-exposed cells. Stimulation with TNF-α increased IL-8 and eotaxin secretion, with increased IL-8 secretion by allergen-exposed compared with naive control ASMC, post-TNF-α stimulation (P = 0.001). Baseline phosphorylation of ERK1/2 (p-ERK1/2) and NF-κB p65 was higher in allergen-exposed than in control ASMC. TNF-α increased p-ERK1/2 and NF-κB p65 levels, with higher levels in allergen-exposed ASMC, post-TNF-α stimulation (P < 0.001). MXF and the combination of MXF with DXM suppressed the secretion of IL-8 and eotaxin, but DXM alone did not affect IL-8, post-TNF-α stimulation (P > 0.05). MXF, DXM and the combination of MXF with DXM inhibited TNF-α-stimulated p-ERK1/2 and NF-κB p65 levels by 34, 40 and 62%, and 33, 38 and 64%, respectively. CONCLUSIONS: MXF suppressed the secretion of pro-inflammatory cytokines by allergen-exposed rat ASMC, partly by inhibiting NF-κB and ERK activation. DXM may have additional or synergistic effects with MXF.

[The effects of leptin on apoptosis of airway smooth muscle cells via the PI3K/Akt signaling pathway].

OBJECTIVE: To observe the effects of leptin on the expression of Akt, Pho-Akt, Bcl-2, Bax, caspase-3 and the apoptosis of airway smooth muscle cells (ASMCs), and to explore the possible mechanisms. METHODS: ASMCs were derived from rat airway tissue and cultured in vitro. The cells were randomly divided into 5 groups including a control group, leptin at concentrations of 50, 100, 200 µg/L groups (group Lep50, Lep100, Lep200), and PI3K specific antagonist with Lep200 group. Then the cells of different groups were incubated for 24 h. An apoptosis detection kit was used for annexin V and PI staining. The expression of Akt, phosphorylation Akt, Bcl-2, Bax, caspase-3 were measured by Western blot. RESULTS: The apoptosis rates of ASMCs in group Lep50, Lep100 and Lep200 were (3.97 ± 0.39)%, (1.88 ± 0.72)% and (0.77 ± 0.11)%, respectively, all significantly lower than that in the control group (7.38 ± 0.49)% (F = 89.57, P < 0.05). Furthermore, the concentration of leptin was negatively related to the apoptosis rate (r = -0.711, P < 0.05). The apoptosis rates of PI3K specific antagonist with Lep200 group (3.29 ± 0.36)% was higher than that of group Lep200 (0.77 ± 0.11)% (F = 89.57, P < 0.01). After the intervention of leptin, the expression of Bcl-2 was upregulated and positively correlated with leptin concentration (r = 0.939, P < 0.05); Bax was downregulated and negatively related to the leptin concentration (r = -0.908, P < 0.05); while the Bcl-2/Bax ratio was raised after leptin treatment (F = 20.56, P < 0.05). Leptin inhibited the activation of caspase-3 in the negative way. (r = -0.961, P < 0.05). The results also showed that leptin significantly increased phosphorylation of Akt that positively related to leptin concentration (r = 0.958, P < 0.05). Compared with group Lep200, the expression of Pho-Akt and Bcl-2 in PI3K specific antagonist with Lep200 group were downregulated (F = 32.93, 19.48, respectively, P < 0.05), while the expression of Bax and caspase-3 was increased (F = 10.10, 29.86, respectively, P < 0.05); the Bcl-2/Bax ratio was lower in group Lep200 as compared to the PI3K specific antagonist with Lep200 group (F = 20.56, P < 0.05). CONCLUSION: Leptin can significantly inhibit ASMC apoptosis partially via the PI3K/Akt signaling pathway.

Furmonertinib (AST2818) versus gefitinib as first-line therapy for Chinese patients with locally advanced or metastatic EGFR mutation-positive non-small-cell lung cancer (FURLONG): a multicentre, double-blind, randomised phase 3 study.

BACKGROUND: Furmonertinib (AST2818) is an irreversible, selective, third-generation EGFR tyrosine-kinase inhibitor. We aimed to investigate the efficacy and safety of furmonertinib versus the first-generation EGFR tyrosine-kinase inhibitor gefitinib as first-line treatment in patients with EGFR mutation-positive locally advanced or metastatic non-small-cell lung cancer (NSCLC). METHODS: The FURLONG study is a multicentre, double-blind, randomised, phase 3 study done in 55 hospitals across mainland China. We enrolled patients who were aged 18 years or older and had histologically confirmed, locally advanced or metastatic, stage IIIB, IIIC, or IV unresectable NSCLC with EGFR exon 19 deletions or exon 21 Leu858Arg mutation on tissue biopsy confirmed by a central laboratory. Eligible patients were stratified according to EGFR mutation (exon 19 deletions or exon 21 Leu858Arg) and CNS metastases (with or without) and randomly assigned (1:1) to receive either oral furmonertinib (80 mg/day) or oral gefitinib (250 mg/day) in 21-day cycles until disease progression, the occurrence of intolerable toxicities, withdrawal of consent, or other discontinuation reasons judged by the investigators. Investigators, clinicians, participants, independent review centre (IRC) members, the sponsor, and those analysing the data were all masked to treatment allocation. The primary endpoint was IRC-assessed progression-free survival and, along with safety, was analysed in the full analysis set, which comprised all randomly assigned patients who had received at least one dose of study drug. This study is registered with ClinicalTrials.gov, NCT03787992, and is ongoing for survival follow-up. FINDINGS: Between May 30, 2019, and Dec 5, 2019, 750 patients were screened, of whom 358 were randomly assigned to receive either furmonertinib and gefitinib-matching placebo (n=178) or gefitinib and furmonertinib-matching placebo (n=180). 178 patients randomly assigned to furmonertinib and 179 patients randomly assigned to gefitinib were treated and were included in the full analysis set. Median follow-up was 21·0 months (IQR 18·0-23·5) in the furmonertinib group and 21·0 months (18·0-23·5) in the gefitinib group. Median IRC-assessed progression-free survival was 20·8 months (95% CI 17·8-23·5) in the furmonertinib group and 11·1 months (9·7-12·5) in the gefitinib group (hazard ratio 0·44, 95% CI 0·34-0·58; p<0·0001). Treatment-related adverse events of a grade 3 or more occurred in 20 (11%) of 178 patients in the furmonertinib group and in 32 (18%) of 179 patients in the gefitinib group. Treatment-related serious adverse events were reported in ten (6%) patients in the furmonertinib group and in 11 (6%) patients in the gefitinib group. Ten (6%) patients in the furmonertinib group and three (2%) patients in the gefitinib group died due to adverse events, which were all judged to be possibly unrelated to study treatment by the investigators. INTERPRETATION: Furmonertinib showed superior efficacy compared with gefitinib as first-line therapy in Chinese patients with EGFR mutation-positive NSCLC, along with an acceptable safety profile without new signals. Furmonertinib is a new potential treatment option for this population. FUNDING: Shanghai Allist Pharmaceuticals and the China National Major Project for New Drug Innovation. TRANSLATION: For the Chinese translation of the abstract see Supplementary Materials section.

Central Nervous System Efficacy of Furmonertinib (AST2818) Versus Gefitinib as First-Line Treatment for EGFR-Mutated NSCLC: Results From the FURLONG Study.

INTRODUCTION: Furmonertinib (AST2818) is a pan-EGFR tyrosine kinase inhibitor with central nervous system (CNS) antitumor activity. We report the CNS efficacy of furmonertinib compared with gefitinib in untreated EGFR-sensitizing mutation-positive NSCLC from the FURLONG study. METHODS: FURLONG was a randomized, double-blind, phase 3 study conducted in 55 hospitals in the People's Republic of China. Patients 1:1 randomly received furmonertinib 80 mg once daily or gefitinib 250 mg once daily treatment. At screening, all the patients underwent brain imaging examination. Patients with asymptomatic steady CNS metastases at baseline constituted this preplanned CNS subgroup analysis. RESULTS: A total of 358 patients were enrolled in the FURLONG study. In the 133 (37%) patients who had measurable or nonmeasurable CNS lesions, CNS progression-free survival was 20.8 months (95% confidence interval [CI]: 15.2-25.3) in the furmonertinib group and 9.8 months (95% CI: 7.2-18.0) in the gefitinib group (hazard ratio = 0.40 [95% CI: 0.23-0.71], p = 0.0011). In the 60 patients (17%) who had measurable CNS lesions, CNS objective response rate was 91% (95% CI: 72-99) with furmonertinib and 65% (95% CI: 48-80) with gefitinib (OR = 6.82 [95% CI: 1.23-37.67], p = 0.0277). The least-square mean of CNS depth of response was 62% (95% CI: 51-72) in the furmonertinib group and 39% (95% CI: 30-47) in the gefitinib group, the mean difference was 23% (95% CI: 10-37, p = 0.0011). CONCLUSIONS: Furmonertinib first-line treatment was found to have superior efficacy in CNS progression-free survival, CNS objective response rate, and CNS depth of response compared with gefitinib in patients with EGFR-mutated NSCLC with CNS metastases.

[The effect of moxifloxacin on apoptosis of airway smooth muscle cells and mitochondria membrane potential].

OBJECTIVE: To observe the effects of moxifloxacin at various concentrations on the expression of Caspase-3, the alteration of mitochondria membrane potential (ΔΨm) and the apoptosis of airway smooth muscle cells (ASMCs), and to explore the possible mechanisms. METHODS: ASMCs were derived from rat airway tissues and cultured in vitro. The cells were randomly divided into 5 groups including a control group and 4 groups to which moxifloxacin was added at different concentrations (40, 80, 120, 200 mg/L, groups M40, M80, M120 and M200 respectively). Then the cells of different groups were incubated for 48 h. An apoptosis detection kit was used for annexin V and PI staining, and JC-1 probe was employed to measure mitochondrial depolarization in ASMCs, and the protein of Caspase-3 was measured by Western blot. RESULTS: The apoptosis rates of ASMCs in groups M40, M80, M120 and M200 were (2.95 ± 0.21)%, (7.39 ± 0.63)%, (13.39 ± 0.40)% and (21.20 ± 1.42)%, respectively, all of which were higher than that in the control group (0.94 ± 0.05)%, F = 399.77, P < 0.01. Furthermore, the concentration of moxifloxacin was positively related to the apoptosis rate (r = 0.974, P < 0.01). Compared to the control group (the ratio of orange-red fluorescence to green fluorescence was 10.02 ± 0.20), there was a shift from mitochondrial orange-red fluorescence to green fluorescence among groups with the concentrations of moxifloxacin increasing (6.54 ± 0.15, 4.48 ± 0.14, 2.25 ± 0.10 and 1.99 ± 0.12); the difference was significant (F = 1565.12, P < 0.01), and there was a dose-dependent response (r = -0.946, P < 0.01). The results of Western blot indicated that the expression of Caspase-3 increased with the concentrations of moxifloxacin increasing (0.45 ± 0.05, 0.59 ± 0.04, 0.69 ± 0.06 and 0.84 ± 0.04, respectively), and there was a very low expression of Caspase-3 in the control group (0.31 ± 0.03). The expression of Caspase-3 showed a positive correlation with the concentration of moxifloxacin (r = 0.979, P < 0.01). The apoptosis rate of ASMCs in the different groups had a remarkable correlation with the ΔΨm and Caspase-3 (r = -0.887, P < 0.01; r = 0.955, P < 0.01). There was also a remarkable negative correlation between ΔΨm and Caspase-3 (r = -0.951, P < 0.01). CONCLUSION: Moxifloxacin was shown to promote ASMC apoptosis by altering ΔΨm.

Hypoxia-inducible factor-1α promotes proliferation of airway smooth muscle cells through miRNA-103-mediated signaling pathway under hypoxia.

The hypoxia-inducible factor-1α (HIF-1α) activated during asthma development plays a causative role in the abnormal proliferation of airway smooth muscle (ASM) cells and consequential airway remodeling. Although the underlying mechanisms of HIF-1α activity have not been fully revealed, HIF-1α-regulated miRNA signaling is considered important for disrupted differentiation and proliferation of local cells in various tissues under inflammation. We aimed to identify the key miRNA signaling involved in HIF-1α regulation of the proliferation of ASM cells. This study was based on primary ASM cells isolated from adult male rats. Three percent O2 and 21% O2 were set as hypoxic and normoxic condition for ASM cell treatment, respectively. Knockdown of HIF-1α was performed through transfection of pSUPER-shHIF-1α plasmid. Overexpression and knockdown of miRNA-103 were performed through transfection of miRNA-103 mimic or inhibitor, respectively. Levels of HIF-1α, PTEN, and PCNA were determined with Western blot and RT-qPCR. Hypoxia increased HIF-1α and miRNA-103 expression and proliferation in ASM cells. Knockdown of HIF-1α suppressed hypoxia-induced upregulation of proliferation and miRNA-103 expression in ASM cells. Knockdown of miRNA-103 displayed similar effects as knockdown of HIF-1α in ASM cells under hypoxia, while overexpression of miRNA-103 played the opposite role. Additionally, increased or decreased expression of PTEN was also detected when HIF-1α/miRNA-103 was knocked down under hypoxia or miRNA-103 was overexpressed under normoxia, respectively. Our results suggest that HIF-1α promotes the proliferation of ASM cells via upregulating miRNA-103 expression under hypoxia, and PTEN is involved in the miRNA-103-mediated signaling pathway.

Bevacizumab biosimilar LY01008 compared with bevacizumab (Avastin) as first-line treatment for Chinese patients with unresectable, metastatic, or recurrent non-squamous non-small-cell lung cancer: A multicenter, randomized, double-blinded, phase III trial.

BACKGROUND: Previous studies have demonstrated the preclinical pharmacological and toxicological consistency, and clinical pharmacokinetic equivalence of bevacizumab biosimilar LY01008 with reference bevacizumab (Avastin). This randomized controlled trial aimed to compare the efficacy and safety of LY01008 with Avastin in first-line treatment of Chinese patients with advanced or recurrent non-squamous non-small cell lung cancer (NSCLC). METHODS: Stage IIIB-IV NSCLC patients with evaluable lesions, good physical status, and adequate organ functions from 67 centers across China were randomized in a ratio of 1:1 to receive LY01008 or Avastin 15 mg/kg intravenously in combination with paclitaxel/carboplatin (combined treatment) for 4-6 cycles, followed by maintenance monotherapy with LY01008 until disease progression, intolerable toxicity, or death. The primary endpoint was objective response rate (ORR) in accordance with Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 confirmed by independent radiological review committees (IRRC). Secondary endpoints included disease control rate (DCR), duration of response (DoR), progression-free survival (PFS), overall survival (OS), and safety. This study was registered in ClinicalTrials.gov (NCT03533127). RESULTS: Between December 15th , 2017, and May 15th , 2019, a total of 649 patients were randomized to the LY01008 (n = 324) or Avastin (n = 325) group. As of September 25th , 2019 for primary endpoint analysis, 589 patients received ORR evaluation, with a median number of combined treatment cycles of 5 (range 1-6) and median duration of treatment of 3.0 (range 0.0-5.1) months. ORR of response-evaluable patients in the LY01008 and Avastin groups were 48.5% and 53.0%, respectively. The stratified ORR ratio was 0.91 (90% CI 0.80-1.04, within the prespecified equivalence margin of 0.75-1.33). Up to May 15th , 2020, with a median follow-up of 13.6 (range 0.8-28.4) months, no notable differences in DCR, median DoR, median PFS, median OS, and 1-year OS rate were observed between the LY01008 and Avastin groups. There were no clinically meaningful differences in safety and immunogenicity across treatment groups. CONCLUSIONS: LY01008 demonstrated similarity to Avastin in terms of efficacy and safety in Chinese patients with advanced or recurrent non-squamous NSCLC. LY01008 combined with paclitaxel/carboplatin is expected to become a new treatment option for unresectable, metastatic, or recurrent non-squamous NSCLC patients in the first-line setting.

The profibrotic effects of chronic microaspiration of bile acids on lungs of rats at different stages.

This study aimed to explore the profibrotic effects of chronic microaspiration of two major bile acids, including chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA), on lungs of rats at different stages, as well as the underlying mechanisms in vivo. A rat model was induced by weekly intratracheal instillation of DCA and CDCA. Our results showed that chronic microaspiration of bile acids resulted in alveolar structure disorder, and inflammatory cells infiltration in the pulmonary interstitium at the early stage. Subsequently, numerous fibroblasts were proliferated, and collagen deposition was profoundly increased over the interstitium of the airways and vessels. Compared with control group, the expression of α-smooth muscle actin, type I collagen, hydroxyproline, transforming growth factor-ß1 (TGF-ß1), and matrix metalloproteinase-9 in the lung tissues were remarkably elevated at the 2nd week, reached the highest level at the 6th week, and maintained high at the 8th week in both DCA- and CDCA-treated groups (P < 0.05). Furthermore, chronic microaspiration of bile acids led to higher levels of glutathione and malondialdehyde, while lower level of superoxide dismutase in lung tissues compared with controls (P < 0.05), thereby resulting in the oxidant/antioxidant enzyme imbalance in the formation of fibrosis. In addition, we also found a consistent growth in the expression of farnesoid X receptor (FXR) in both DCA- and CDCA-treated groups. Our findings suggested that chronic microaspiration of bile acids could initiate the process of pulmonary fibrosis from the early phase and promote its progression in a time-dependent manner, which likely involved the TGF-ß1, oxidative stress, and FXR-related pathways.

Optimization of Nebulized Budesonide in the Treatment of Acute Exacerbation of Chronic Obstructive Pulmonary Disease.

Background: Clinical studies have suggested nebulized budesonide (NB) as an alternative to systemic corticosteroids for patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD). However, the optimal budesonide dose for AECOPD remains unclear. Objectives: To compare the efficacy and safety of different doses of NB in the management of AECOPD. Patients and Methods: A total of 321 AECOPD patients with moderate-to-severe exacerbation were randomly divided into three groups and treated with NB. The low dose group (L) was given 4 mg/day (n=95, 1 mg Q6h), while high-dose group 1 (H1, n=111, 2 mg Q6h) and high-dose group 2 (H2, n=115, 4 mg Q12h) were given 8 mg/day. Patients also received routine treatment including oxygen therapy, expectorant, nebulization bronchodilators, antibiotics, and fluid rehydration. The COPD assessment test (CAT), lung function, and artery blood gas were evaluated before and after 3 hrs and 5 days of treatment. In addition, hospital stay, frequency of acute exacerbations within 3 months of discharge, and adverse events during treatment were compared. Results: H1 and H2 showed improved spirograms and CAT score faster than L. In H2, forced expiratory volume in 1 s (FEV1%) at 3 hrs and FEV1%, forced expiratory flow after 50% of the forced vital capacity has been exhaled (FEF50%), mean forced expiratory flow between 25% and 75% of forced vital capacity (FEF25-75%) and CAT score at 5 days were significantly improved compared to L. FEV1% improved most in H2, moderately in H1, and least in L, with significant differences between groups at 5 days. No differences between groups were observed in adverse effects, hospital stay, and frequency of exacerbations within 3 months of discharge. Conclusion: Compared to the conventional dose (4 mg/day), a high dose (8 mg/day) of NB improved pulmonary function and symptoms more effectively in the early treatment of AECOPD, especially when given as 4 mg twice daily.

[Effects of leptin on airway inflammation and the expression of Th1/Th2 cytokines in asthmatic rats].

OBJECTIVE: To investigate the effects of leptin on airway inflammation and the expression of Th1/Th2 cytokines. METHODS: The obesity and acute asthma models were established in 40 female SD rats, which were randomly divided into a normal weight control group (group A), a normal weight asthmatic group (group B), a normal weight intervention group (group C), an obese control group (group D) and an obese asthmatic group (group E). The airway resistance and airway responsiveness were calculated by transpulmonary pressure and gas flow rate. The numbers of leukocytes, eosinophils (EOS) and neutrophils (N) in bronchoalveolar lavage fluid (BALF) were counted. The concentrations of interleukin-4 (IL-4), interferon-gamma (IFN-gamma) and leptin in serum and BALF were determined by ELISA. The protein and mRNA expression of leptin was measured by Western blot and RT-PCR respectively. RESULTS: The airway resistance in group C and E [(0.890 +/- 0.106) cm H2Oxml(-1)xs(-1), (1.024 +/- 0.096) cm H2Oxml(-1)xs(-1), (1.129 +/- 0.107) cm H2Oxml(-1)xs(-1), (0.946 +/- 0.104) cm H2Oxml(-1)xs(-1), (1.124 +/- 0.095) cm H2Oxml(-1)xs(-1), (1.135 +/- 0.105) cm H2Oxml(-1)xs(-1), respectively.] was increased significantly compared to group B [(0.638 +/- 0.128) cm H2Oxml(-1)xs(-1), (0.745 +/- 0.073) cm H2Oxml(-1)xs(-1), (0.773 +/- 0.090) cm H2Oxml(-1)xs(-1)] (q = 7.128, 8.712, 8.318, 11.300, 11.258, 11.447, all P < 0.05). The numbers of leukocyte and neutrophils in group C and E [(91 +/- 9) x 10(4)/ml, (108 +/- 21) x 10(4)/ml, (12.4 +/- 4.0) x 10(4)/ml, (14.2 +/- 5.9) x 10(4)/ml, respectively.] were increased significantly compared to group B [(79 +/- 7) x 10(4)/ml, (2.4 +/- 1.1) x 10(4)/ml] (q = 2.923, 7.063, 8.629, 10.182, all P < 0.05). The concentrations of IFN-gamma were [(42.3 +/- 3.5) ng/L, (45.1 +/- 4.8) ng/L, (19.2 +/- 1.8) ng/L, (20.3 +/- 1.5) ng/L] in group C and E respectively, which were significantly higher than those of group B [(16.5 +/- 1.4) ng/L, (9.3 +/- 1.0) ng/L] (q = 21.607, 23.952, 16.919, 18.799, all P < 0.05). The protein and mRNA expression of leptin in lung tissue in group C and E [(0.40 +/- 0.07) ng/L, (0.44 +/- 0.05) ng/L, (0.34 +/- 0.06) ng/L, (0.38 +/- 0.04) ng/L, respectively.] were remarkably higher than those of group B [(0.31 +/- 0.03) ng/L, (0.21 +/- 0.04) ng/L] (q = 4.648, 6.713, 8.222, 10.752, all P < 0.05). CONCLUSION: Leptin could aggravate airway inflammation featured by infiltration of neutrophils and enhancement of Th1 type inflammation.

[Effects of fasudil on the expression of Rho kinase-1 and airway inflammation in a mouse model of asthma.].

OBJECTIVE: To explore the role of Rho kinase-1 (ROCK-1) in airway inflammation of asthma by observing the effects of fasudil, a specific inhibitor of ROCK-1, on the expression of Rho kinase-1 and airway inflammation in a mouse model of asthma. METHODS: Twenty-four female BALB/c mice were randomly divided into 3 groups (n = 8 each): a control group, an asthmatic group and a treatment group. Mice in the asthmatic and the treatment groups were sensitized by intraperitoneal injection of OVA (25 microg) precipitated with 1 mg of alum in 200 microl of saline on days 1 and 15, and subsequently challenged by nebulization of 2% OVA on days 22-26. Mice in the control group were sensitized with Al(OH)3 saline and challenged with saline instead of OVA. Mice of the treatment group were injected intraperitoneally with fasudil (10 mg/kg) 1 h before each OVA challenge. All the mice were killed 24 h after the final challenge, and bronchoalveolar lavage fluid (BALF) was collected for counting total inflammatory cells and eosinophils (EOS). Cytokines and chemokines in BALF were measured by ELISA. The lung tissue slides were examined histologically. The protein and mRNA expression of ROCK-1 were measured by immunohistochemistry and RT-PCR respectively. RESULTS: (1) OVA challenge in mice of the asthmatic group caused a marked increase in the number of the total cells and eosinophils in BALF (q = 25.909, 35.002, respectively, all P < 0.01). When fasudil was applied, both the total cell counts and the eosinophil numbers were significantly decreased. The total cell number was decreased from (1.45 +/- 0.12) x 10(9)/L to (0.89 +/- 0.09) x 10(9)/L (q = 16.676, P < 0.01), and the number of eosinophils was decreased from (0.52 +/- 0.06) x 10(9)/L to (0.20 +/- 0.04) x 10(9)/L (q = 21.537, P < 0.01). (2) Compared with the control group, OVA challenge in mice of the asthmatic group induced eotaxin, IL-5 and IL-13 release into BALF (q = 18.246, 23.009, 25.826, respectively, all P < 0.01). The eotaxin, IL-5 and IL-13 levels in BALF after OVA challenge were (45 +/- 8) ng/L, (157 +/- 23) ng/L and (429 +/- 46) ng/L, respectively. Application of fasudil resulted in inhibition of the augmented levels of eotaxin, IL-5 and IL-13 in BALF, decreased to (20 +/- 5) ng/L, (57 +/- 14) ng/L and (254 +/- 28) ng/L, respectively (q = 13.119, 17.503, 8.449, respectively, all P < 0.01). (3) Mice in the control group showed no detectable inflammatory response in the lung, whereas OVA-challenged mice induced infiltration of inflammatory cells around airways and blood vessels. The majority of the infiltrated inflammatory cells were eosinophils. Application of fasudil significantly reduced the infiltration of inflammatory cells in the peribronchial areas compared with the asthmatic mice. (4) The expression levels of ROCK-1 mRNA and protein in mice of the asthmatic group (0.67 +/- 0.05 and 1.09 +/- 0.06) were much higher than those of the control group (0.26 +/- 0.05 and 0.87 +/- 0.09) (q = 25.614, 8.156, all P < 0.01). When fasudil was administered, the expression levels of ROCK-1 mRNA and protein were significantly attenuated to 0.35 +/- 0.04 and 0.98 +/- 0.08, compared with those of the asthmatic group (q = 20.379, 4.135, all P < 0.01). (5) The expression level of ROCK-1 mRNA was positively correlated with the number of eosinophils and the levels of eotaxin, IL-5 and IL-13 in BALF (r = 0.709, 0.600, 0.613, 0.650, all P < 0.01). CONCLUSION: Airway inflammation of bronchial asthma was improved by inhibiting expression and activity of ROCK-1 by fasudil, suggesting that ROCK-1 may be involved in asthmatic airway inflammation induced by OVA challenge.

Tobacco smoking aggravates airway inflammation by upregulating endothelin-2 and activating the c-Jun amino terminal kinase pathway in asthma.

BACKGROUND: Asthma is closely associated with tobacco smoking (TS) and is more difficult to effectively treat after exposure to TS. OBJECTIVE: To observe the effects of TS on the expression of endothelin-2 (ET-2) and airway inflammation in asthmatic rats and to explore the related mechanisms. METHODS: We established an animal model of asthma with ovalbumin (OVA)/Al(OH)3 and subjected different animal groups to TS and/or dexamethasone/bosentan. The differences in the inflammatory cell infiltration, the pathological changes to the bronchial wall and the bronchial smooth muscle thickness, and the expression of ET-2, c-Jun amino terminal kinase (JNK1/2), malondialdehyde (MDA), and glutathione peroxidase (GSH) in the lung tissue and of interleukin (IL)-7 in bronchoalveolar lavage fluid (BALF) were assessed. RESULTS: Exposure to TS or OVA caused an obvious increase in the inflammatory cells in the BALF over what was observed in the control group. In asthma models, the expression of ET-1, JNK1/2, MDA, and GSH in the lung tissues, as well as that of IL-17 in the BALF, was increased. After treatment with dexamethasone/bosentan, the expression of IL-17, JNK1/2, MDA, and GSH decreased compared to the smoking group; airway inflammation and the staining intensity in the lung tissue were also reduced. CONCLUSION: TS exposure can clearly exacerbate airway inflammation in asthmatic rats, while bosentan can alleviate airway inflammation through regulation of the ET-2/JNK1/2 signalling pathway.

Downregulation of BarH-like homeobox 2 promotes cell proliferation, migration and aerobic glycolysis through Wnt/ß-catenin signaling, and predicts a poor prognosis in non-small cell lung carcinoma.

BACKGROUND: Human BarH-like homeobox 2 (Barx2), a homeodomain factor of the Bar family, plays a critical role in cell adhesion and cytoskeleton remodeling, and has been reported in an increasing array of tumor types except non-small cell lung carcinoma (NSCLC). The purpose of the current study was to characterize the expression of Barx2 and assess the clinical significance of Barx2 in NSCLC. METHODS: Quantitative real-time polymerase chain reaction, immunohistochemistry and western blot analysis were used to examine mRNA and protein expression, respectively. The relationships between Barx2 expression and clinicopathological variables were analyzed. Cell Counting Kit-8 and plate colony formation assay were used to detect cell proliferation. Transwell assay was used to examine cell migration ability. Glucose uptake, lactate, adenosine triphosphate, and lactate dehydrogenase assays were used to detect aerobic glycolysis. RESULTS: Barx2 is downregulated in NSCLC tissues compared with para-carcinoma. Furthermore, Barx2 expression shows a negative correlation with advanced TNM stage and a high level of Ki-67. Survival analysis reveals that Barx2 level is an independent prognostic factor for NSCLC patients. The Barx2 (low) Ki-67 (high) group had the worst prognosis. Furthermore, the data indicate that downregulation of Barx2 expression promotes cell proliferation, migration, and aerobic glycolysis, including increased lactate dehydrogenase activity, glucose utilization, lactate production, and decreased intracellular adenosine triphospahte level. Furthermore, Barx2 acts as a negative regulator of the canonical Wnt/ß-catenin pathway. Reactivation of Wnt/ß-catenin pathway by LiCl can reverse the inhibiting effect of Barx2. CONCLUSIONS: These findings reveal that Barx2 serving as a tumor suppressor gene could decrease cell proliferation, migration, and aerobic glycolysis through inhibiting the Wnt/ß-catenin signaling pathway, and predicts a good prognosis in NSCLC.