Gut microbiota and its metabolic products in acute respiratory distress syndrome.

The prevalence rate of acute respiratory distress syndrome (ARDS) is estimated at approximately 10% in critically ill patients worldwide, with the mortality rate ranging from 17% to 39%. Currently, ARDS mortality is usually higher in patients with COVID-19, giving another challenge for ARDS treatment. However, the treatment efficacy for ARDS is far from satisfactory. The relationship between the gut microbiota and ARDS has been substantiated by relevant scientific studies. ARDS not only changes the distribution of gut microbiota, but also influences intestinal mucosal barrier through the alteration of gut microbiota. The modulation of gut microbiota can impact the onset and progression of ARDS by triggering dysfunctions in inflammatory response and immune cells, oxidative stress, cell apoptosis, autophagy, pyroptosis, and ferroptosis mechanisms. Meanwhile, ARDS may also influence the distribution of metabolic products of gut microbiota. In this review, we focus on the impact of ARDS on gut microbiota and how the alteration of gut microbiota further influences the immune function, cellular functions and related signaling pathways during ARDS. The roles of gut microbiota-derived metabolites in the development and occurrence of ARDS are also discussed.

Improving myopia awareness via school-based myopia prevention health education among Chinese students.

AIM: To investigate the myopia awareness level, knowledge, attitude, and skills at baseline and to implement and evaluate the efficacy of myopia prevention health education among Chinese students. METHODS: A total of 1000 middle school students from 2 middle schools were invited to participate in the study, and myopia prevention health education was conducted. The students were assessed at baseline, followed by a survey. The efficacy of health education was evaluated using the self-comparison method pre- and post-health education. RESULTS: The study included 957 and 850 pre- and post-health education participants, respectively. The baseline knowledge of all respondents on myopic symptoms (87.5%), myopia is a risk of eyes (72.9%), myopia prevention (91.3%), myopia increases with age (86.7%), performing periodic eye examinations (92.8%), and one first, one foot, and one inch (84.8%) significantly increased after health education (P<0.001 for all). However, the percentage of students who still did not think it necessary to take breaks after 30-40min of continuous near work was 27.0%. The opinion that "myopia can be cured" was still present in 38.3%. CONCLUSION: Implementing school-based myopia prevention health education improves knowledge, attitudes, and skills regarding myopia among Chinese middle school students.

PA-MSHA induces inflamed tumor microenvironment and sensitizes tumor to anti-PD-1 therapy.

A low response rate to immune checkpoint inhibitor (ICI) therapy has impeded its clinical use. As reported previously, an inflamed tumor microenvironment (TME) was directly correlated with patients' response to immune checkpoint blockade (ICB). Thus, restoring the cytotoxic effect of immune cells in the TME is a promising way to improve the efficacy of ICB and overcome primary resistance to immunotherapy. The effect of Pseudomonas aeruginosa mannose-sensitive-hemagglutinin (PA-MSHA) in facilitating T cell activation was determined in vitro and in vivo. Subsets of immune cells were analyzed by flow cytometry. Proteomics was carried out to comprehensively analyze the discriminated cellular kinases and transcription factors. The combinational efficacy of PA-MSHA and αPD-1 therapy was studied in vivo. In this study we demonstrated that PA-MSHA, which is a clinically used immune adjuvant, effectively induced the anti-tumor immune response and suppressed the growth of non-small cell lung cancer (NSCLC) cells. PA-MSHA showed great potential to sensitize refractory "cold" tumors to immunotherapy. It effectively enhanced macrophage M1 polarization and induced T cell activation. In vivo, in combination with αPD-1, PA-MSHA suppressed tumor growth and prolonged the survival time of allograft model mice. These results indicate that PA-MSHA is a potent agent to stimulate immune cells infiltration into the TME and consequently induces inflammation in tumors. The combination of PA-MSHA with αPD-1 is a potential strategy to enhance the clinical response rate to ICI therapy.

Impact of fecal microbiota transplantation on TGF-ß1/Smads/ERK signaling pathway of endotoxic acute lung injury in rats.

Acute lung injury (ALI) is a common clinical disease with high morbidity in both humans and animals. Studies have shown that intestinal microbiota affect the pathology and immune function of respiratory diseases through the "gut-lung axis". The authors investigated the therapeutic effect of fecal microbiota transplantation (FMT) in rats with ALI induced by lipopolysaccharide (LPS). Rats were treated with FMT, and then measured lung wet/dry ratio, PaO2 in artery, proinflammatory marker, and TGF-ß1, Smad3, Smad7, and phosphorylated ERK (p-ERK) protein levels, as well as a histopathologic analysis and high-throughput sequencing of intestinal microbiota. FMT significantly reduced lung wet/dry ratio and TNF-α, IL-1ß, and IL-6 levels, but increased the levels of PaO2 in artery. In addition, FMT significantly decreased the expression of TGF-ß1, Smad3, and p-ERK, while increased the levels of Smad7. Lung histopathological analyses showed that FMT reduced the inflammatory cell infiltration and interstitial lung exudates. High-throughput sequencing of intestinal microbiota analyses showed that FMT reconstructed the structure of intestinal microbiota, and increased the gene abundance of the bacterial community. Therefore, FMT may act on the TGF-ß1/Smads/ERK pathway by regulating intestinal microbiota, inhibiting immune inflammation, reducing the production of inflammatory markers in the body and release, and reducing alveolar epithelial damage and repair, thereby improving the endotoxic ALI in rats induced by LPS.

Interleukin-17A promotes the formation of inflammation in the lung tissues of rats with pulmonary fibrosis.

The aim of the present study was to investigate the effects of interleukin (IL)-17A in a rat model of pulmonary fibrosis. In total, 20 female Wistar rats were randomly divided into a normal saline (NS group) and a bleomycin group (BLM group). The BLM group rats were intratracheally instilled with BLM, while the NS group rats were intratracheally instilled with saline. In each group, half the rats were sacrificed at day 7 and day 28, respectively, following intratracheal instillation. Subsequently, hematoxylin and eosin and Masson's trichrome staining were performed to observe the pathological changes in the lung tissue, while the expression of IL-17A in the lung tissue was detected by immunohistochemistry. In addition, the bronchoalveolar lavage fluid (BALF) was collected and divided into two sections. One section was used for cell counting and classification, and an ELISA was performed to detect the concentration of IL-17A in the BALF. The additional section was used to separate, purify and cultivate alveolar macrophages (AMs). The concentration of IL-17A in the cultivating supernatant was detected by ELISA, and the mRNA expression levels of IL-17A in the AMs were detected using reverse transcription-polymerase chain reaction (RT-PCR). The results revealed that a considerable number of inflammatory cells had infiltrated into the alveolar cavity in the BLM group at day 7, and less alveolitis and more serious fibrosis were observed at day 28, as compared with the NS group. Furthermore, when compared with the NS group, the protein expression levels of IL-17A in the lung tissue were markedly higher in the BLM group at days 7 and 28 (higher at day 7; P<0.05). In addition, the total number of BALF cells in the BLM group was clearly higher at day 7 when compared with the NS group (P<0.05), although a normal level was re-established by day 28. The level of IL-17A in the BALF increased significantly at days 7 and 28 in the BLM group; however, when compared with the level at day 7, the concentration had decreased at day 28. When compared with the NS group, the protein expression levels of IL-17A in the BLM group were notably higher after 12, 24 and 48 h. In addition, the results of the RT-PCR assay revealed that the mRNA expression levels of IL-17A increased significantly at days 7 and 28 in the BLM group when compared with the NS group (P<0.05). Therefore, IL-17A was demonstrated to promote the development of pulmonary inflammation, which may be involved in the development of pulmonary fibrosis.

[B Cell Activating Transcription Factor Regulates Acute Airway Inflammation in Asthmatic Mice].

OBJECTIVE: To investigate the regulatory effect of B cell activating transcription factor (BATF) on acute airway inflammation and its association with retinoic acid orphan nuclear receptors gammat (RORyt) in asthmatic mice. METHODS: 24 female BALB/c mice were randomly and equally divided into three groups (n 8): normal saline (NS) treated, asthma (AS) control and dexamethasone (DEX) treated. AS mice were sensitized and challenged with OVA to establish murine asthma model. Histological changes in lung tissues of the mice were observed by HE staining. Numbers of white blood cell (WBC), polymorphonuclear leukocyte (PMN) and eosinophils (EOS) in the bronchoalveolar lavage fluid (BALF) of the mice were counted. The concentration of interleukin-17 (IL-17) in BALF was measured by ELISA. Quantitative real-time PCR (RT-PCR) was performed to assess the mRNA expressions of BATF, IL-17 and RORγt in the lung tissues. RESULTS: The HE staining showed a higher level of inflammatory cell infiltration around the bronchi of AS mice compared with those treated with NS, predominantly in the forms of EOS, PMN and lymphocytes. The AS and DEX treated mice had higher levels of EOS, PMN, WBC and mRNA expressions of BATF, IL-17 and RORγt in BALF than those treated with NS (P < 0.05). DEX reduced the levels of EOS, PMN, WBC and IL-17 in BALF significantly (P < 0.05). The mRNA expression of BATF in lung tissues of mice was positively correlated with the expression of IL-17, RORγt and the counts of WBC,EOS and PMN in BALF (P < 0.05). CONCLUSION: Asthmatic mice have increased expressions of BATF, IL-17 and RORγt in bronchial and lung tissues. BATF can, through regulating the secretion of Th17 cells, readjust the airway inflammatory. The regulatory function may take effect through synergy with RORγt .

[Effect of Budesonide on Smad4, PDGF-A and PAI-1 in a rat model of pulmonary fibrosis].

AIM: To observe the effect of Budesonide (BUD) on TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue in pulmonary fibrosis rats. METHODS: Forty-five adult female Wistar rats were randomly divided into normal solution (NS) group, BUD group and bleomycin (BLM) group. 9 g/L NaCl solution was instilled into the tratracheaes in NS group, and BLM were used in BUD group and BLM group. NS group and BLM group were inhaled with 9 g/L NaCl solution once everyday at day 0-6, and BUD group were used with BUD. Five rats in every group were killed at 7th, 14th 28th day. The appearances of alveolitis and fibrosis were displayed in HE and Masson staining. The expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue were detected by immunohistochemistry. RESULTS: The extent of the alveolitis in BUD group at 7th, 14th day were significantly lower than that in BLM group(P<0.05). The fibrosis and the expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissues at 7th, 14th, 28th day, BUD group were significantly lower than BLM group(P<0.05). CONCLUSION: After inhaled BUD, the expressions of TGF-ß1, PDGF-A, Smad4 and PAI-1 in lung tissue could be decreased, and the extent of alveolitis and pulmonary fibrosis could be improved in bleomycin-induced pulmonary fibrosis rats.

[Pulmonary fibrosis induces erectile dysfunction in rats].

OBJECTIVE: To study the impact of pulmonary fibrosis on erectile function in rats and its mechanism. METHODS: Forty 12-week-old healthy male SD rats were randomly divided into Groups A (4-week pulmonary fibrosis), B (6-week pulmonary fibrosis), C (4-week control, and D (6-week control). The models of pulmonary fibrosis were established by injection of bleomycin at 5 mg/kg in the trachea, while the controls were injected with normal saline only. At 4 and 6 weeks, all the rats were subjected to determination of the serum testosterone (T) level, arterial blood gas analysis, measurement of intracavernous pressure/mean arterial pressure (ICP/MAP), and examination of NOS activity and cGMP content. The mRNA expressions of eNOS, iNOS and nNOS in the corpus cavernosum penis were detected by real-time PCR, and that of eNOS analyzed by Western blot. RESULTS: The 3 V and 5 V of the ICP/mapx100 in Group C were 16.37 +/- 2.19 and 27.19 +/- 3.18, significantly lower than 30.78 +/- 2.66 and 50.09 +/- 6.97 in Group A (P < 0.05); those in Group D were 10.17 +/- 1.31 and 17.40 +/- 1.74, significantly lower than 31.45 +/- 3.07 and 51.23 +/- 7.23 in Group B (P < 0.05), and so were they in Group D than in C (P < 0.05). PaO2 was significantly lower in Group C than in A ([75.50 +/- 13.87] mmHg vs [103.80 +/- 6.88] mmHg, P < 0.05) , and so was it in Group D than in B ( [83.60 +/- 5.50] mmHg vs [102.70 +/- 5.77] mmHg, P < 0.05). Group C showed a significantly increased serum T level as compared with A ([391.1 +/- 264.7] ng/dl vs [175.9 +/- 53.0] ng/dl, P < 0.05), so did Group D ([745.4 +/- 408.8] ng/dl) versus Group B ([177.8 +/- 52.3] ng/dl) and C (P < 0.05). NOS activity and cGMP content in the corpus cavernosum significantly decreased in Group C ([1.50 +/- 0.14] U/mg prot and [35.69 +/- 3.64] pmol/mg) compared with A ([2.66 +/- 0.39] U/mg prot and [51.10 +/- 7.22] pmol/mg) (P < 0.05), so did they in D ([1.40 +/- 0.20] U/mg prot and [34.55 +/- 4.30] pmol/mg) versus B ([2.75 +/- 0.36] U/mg prot and [52.15 +/- 6.86] pmol/mg) (P < 0.05), but neither showed any significant difference between Groups D and C (P > 0.05). The expression of the eNOS protein was significantly lower in Group C than in A (0.79 +/- 0.01 vs 0.87 +/- 0.01, P < 0.05), so was it in D than in B and C (0.71 +/- 0.02 vs 0.88 +/- 0.01 and 0.79 +/- 0.01, P < 0.05). The expression of eNOS mRNA was significantly higher in Group C than in A (4.46 +/- 0.92 vs 2.61 +/- 0.68, P < 0.05), but did not show any significant difference between D and B (2.79 +/- 0.60 vs 2.69 +/- 0.65, P > 0.05), nor did the expressions of nNOS mRNA and iNOS mRNA between the pulmonary fibrosis groups and the controls (P > 0.05). CONCLUSION: Pulmonary fibrosis may induce erectile dysfunction by suppressing the expression of the eNOS protein and reducing NOS activity and cGMP content in the corpus cavernosum penis of rats.

[Effects of andrographolide on the concentration of cytokines in BALF and the expressions of type I and III collagen mRNA in lung tissue in bleomycin-induced rat pulmonary fibrosis].

AIM: To investigate the effects of andrographolide on the concentration of TNF-α and TGF-ß1 in bronchoalveolar lavage fluid (BALF) and the expressions of type I and III collagen mRNA in Lung tissue in bleomycin (BLM)-induced pulmonary fibrosis in rats. METHODS: 90 healthy SD male rats were randomly divided into 6 groups with 15 rats each group: normal saline (NS) group, BLM group, prednisone (Pred) group and different doses of andrographolide groups (andrographolide group A 62.5 mg/kg, andrographolide group B 125 mg/kg and andrographolide group C 250 mg/kg). BLM was given to BLM group, Pred group and andrographolide group A, B, C by intratracheal instillation, and same volume of NS was given to NS group in the same way. And then NS was given to NS group and BLM group, Pred was given to Pred group and different does of andrographolide were given to andrograoholide group A, B, C by gavage every day. Five rats of each group were killed respectively at day 7, 14, 28 after intratracheal instillation. Alveolitis and fibrosis were observed by HE and Masson staining. Real-time fluorescent quantitative reverse transcription- polymerase chain reaction (FQ RT-PCR) was performed to detect the expressions of type I and III collagen mRNA in lung tissue, and the concentration of TNF-α and TGF-ß1 in BALF was determined by enzyme-linked immunosorbnent assay. Blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were also examined. RESULTS: (1) The AST, ALT, BUN and Cr in every group had no significant diference(P>0.05). (2) Alveolar septal edema, inflammatory cell infiltration and fibrosis were not found in NS group. In the BLM group, lots of inflammatory cells infiltration were observed in the alveolar at day 7; the alveolitis was still existed, but inflammatory cells were significantly reduced, and the number of the fibroblasts and matrix in alveolar septum were obviously increased at day 14, at the same time, alveolar structure was damaged and alveolar septum widened; the inflammation cells infiltration of the alveolar was relieved, pulmonary fibrosis was increased, and parts of alveolar space was disappeared , severe fibrosis was found at day 28. It was similar between andrographolide group A and BLM group in pathomorphology. A lot of inflammatory cells infiltration and local accumulation were observed at day 7 in andrographolide group B, C and Pred group. However, compared with andrographolide group A and BLM group, the fibrosis at day 14, 28 was significantly reduced.(3) The concentration of TGF-ß1, TNF-α in BALF of NS group was significantly lower than that of Pred group, BLM group, andrographolide group A, B, C at each time point(P<0.05). The concentration of TGF-ß1 and TNF-α in BALF of BLM group at day 7, 14, 28 was higher than that of Pred group, andrographolide group B and andrographolide group C (P<0.05). Compared with BLM group, the concentration of TGF-ß1 and TNF-α in BALF of andrographolide group A had no significant difference. (4) The expression of type I and III collagen mRNA in lung tissue of NS group was significantly lower than that of group Pred, BLM, andrographolide group A, B, C at each time point (P<0.05). The expression of type I and III collagen mRNA in lung tissue of BLM group at day 7, 14, 28 was higher than that of Pred group, andrographolide group B and andrographolide group C (P<0.05). Compared with BLM group, the expression of type I and III collagen mRNA in lung tissue of andrographolide group A had no significant difference. CONCLUSION: In BLM-induced rat pulmonary fibrosis, andrographolide could attenuate alveolitis and fibrosis, decrease mRNA expression of collagen I and III in lung tissue and decrease the concentration of TNF-α and TGF-ß1 in BALF. It had no side effects on liver and kidney function.

[Therapeutic effects of aerosolized signal transducer and activator of transcription 1 antisense oligonucleotide administered at different time points on bleomycin-induced pulmonary fibrosis: experiment with rats].

OBJECTIVE: To investigate the curative effects of inhaling signal transducer and activator of transcription 1 (STAT1) antisense oligonucleotide (ASON) on alveolitis and pulmonary fibrosis and the best administration time. METHODS: Twenty-five adult female Wistar rats were randomly divided into 5 equal groups: BLM group, undergoing intra-tracheal perfusion of BLM so as to establish animal models of alveolitis and pulmonary fibrosis and then inhaling aerosolized normal saline (NS); NS group undergoing intra-tracheal perfusion of NS and then inhaling aerosolized NS; ASON 0 d group, undergoing intra-tracheal perfusion of BLM and then inhaling aerosolized STAT1 ASON 3 ml immediately; ASON 7 d group, undergoing intra-tracheal perfusion of BLM and then inhaling STAT1 ASON 3 ml 7 days later; and ASON 14 d group undergoing intra-tracheal perfusion of BLM and then inhaling aerosolized STAT1 ASON 3 ml 14 days later. Aerosolized inhalation was repeated once every other day for 4 times. Twenty-eight days after intra-tracheal perfusion the rats were sacrificed with their lungs taken out to undergo pathological examination. NS was infused into the right lungs to get bronchoalveolar lavage fluid (BALF). ELISA was used to examine the concentrations of transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) in the BALF. RESULTS: The pathology result of the lung tissues showed that compared with the BLM and ASON 14 d groups, the alveolitis and pulmonary fibrosis of the ASON 0 d group were obviously milder. The scores of alveolitis and pulmonary fibrosis of the ASON 0 d group were (1.80 +/- 0.84) and (2.60 +/- 0.55) respectively, both significantly lower than those of the BLM group [(2.40 +/- 0.55) and (4.40 +/- 0.55) respectively] and those of the ASON 7 d group [(2.20 +/- 0.45) and (3.00 +/- 0.71) respectively] (all P < 0.05). The scores of pulmonary fibrosis of the ASON 7 d group was significantly lower than those of the BLM and ASON 14 d groups (both P < 0.05). The concentrations of TGF-beta and TNF-alpha in BALF of the ASON 0 d group were (48.11 +/- 3.46) pg/ml and (1.93 +/- 0.14) ng/ml respectively, both significantly lower than those of the BLM group [(57.67 +/- 2.46) pg/ml and (2.45 +/- 0.25) ng/ml respectively, both P < 0.05]. The concentration of TGF-beta in BALF of the ASON 0 d group was significantly lower than those of the ASON 7 d and ASON 14 d groups [(51.42 +/- 3.57) pg/ml and (55.8 3 +/- 1.79) pg/ml respectively, both P < 0.05]. The concentration of TGF-beta in BALF of the ASON7 d group was significantly lower than those of the BLM and ASON 14 d groups (both P < 0.05). CONCLUSIONS: STAT1 ASON administered in the early stage helps depress the pulmonary fibrosis procedure, and the earlier the drug is administrated the better effect would be obtained. Aerosolized STAT1 ASON can be used as a therapeutic method for pulmonary fibrosis.

[Effect of signal transducer and activator of transcription 1 antisense oligodeoxynucleotides on inflammatory mediators, type I and type III collagen mRNA of rat pulmonary fibrosis].

AIM: To investigate the effect of aerosolized signal transducer and activator of transcription 1 (STAT1) antisense oligodeoxynucleotides (ASON) on the expression of inflammatory mediators in bronchoalveolar lavage fluid (BALF) and typeI and typeIII collagen mRNA of the bleomycin-induced rat pulmonary fibrosis. METHODS: 45 adult female Wistar rats were randomly divided into 3 groups: normal saline (NS) group, bleomycin (BLM) group and ASON group. BLM group and ASON group were intratracheally instilled with bleomycin (BLM) while NS group was instilled with NS. NS group and BLM group were aerosolized with NS while ASON group was aerosolized with STAT1 ASON on day 0, 2, 4 and 6 after intratracheal administration. Then each group was divided into 3 subgroups and the rats were sacrificed on day 7, 14 and 28. The concentration of IFN-gamma, TNF-alpha, TGF-beta1 and PDGF-BB in BALF was detected. The lung tissues were removed and HE and Masson staining was performed to observe the extent of alveolitis and fibrosis. The mRNA levels of typeI and typeIII collagen in the lung tissues were measured. RESULTS: Compared with BLM group, the scores of alveolitis and fibrosis in ASON group were remarkably meliorated (P<0.05). Compared with NS group, the concentration of TNF-alpha, TGF-beta1 and PDGF-BB in BALF in BLM group was significantly increased, but it was lower in ASON group than in BLMA group (P<0.05). The concentration of IFN-gamma in BALF was lower in BLM group than in NS group (P<0.05), but it was higher in ASON group than in BLM group (P<0.05). The mRNA levels of typeI and typeIII collagen at various time points in ASON group were significantly lower than those in BLM group (P<0.05). CONCLUSION: The aerosolized STAT1 ASON has anti-fibrosis effect, which may result from the lessened production of typeI and typeIII collagen through reducing the concentration of cytokines in BALF such as TNF-alpha, PDGF-BB and TGF-beta1 and inhibiting the decline of IFN-gamma in BALF.

The effects of aerosolized STAT1 antisense oligodeoxynucleotides on rat pulmonary fibrosis.

Previous study showed that aerosolized signal transducer and activator of transcription-1 (STAT1) antisense oligodeoxynucleotide (ASON) inhibited the expression of STAT1 and ICAM-1 mRNA and protein in alveolar macrophages (AMs) and decreased the concentrations of TGF-beta, PDGF and TNF-alpha in bronchioalveolar lavage fluid (BALF) in bleomycin (BLM)-induced rat pulmonary fibrosis. Administration of STAT1 ASON ameliorated alveolitis in rat pulmonary fibrosis. However, further investigations are needed to determine whether there is an effect from administration of STAT1 ASON on fibrosis. This study investigated the effect of aerosolized STAT1 ASON on the expressions of inflammatory mediators, hydroxyproline and type I and type III collagen mRNA in BLM-induced rat pulmonary fibrosis. The results showed that STAT1 ASON applied by aerosolization could ameliorate alveolitis and fibrosis, inhibit the expressions of inflammatory mediators, decrease the content of hydroxyproline, and suppress the expressions of type I and type III collagen mRNA in lung tissue in BLM-induced rat pulmonary fibrosis. These results suggest that aerosolized STAT1 ASON might be considered as a promising new strategy in the treatment of pulmonary fibrosis.

[Effect of aerosolized STAT1 antisense oligonucleotides on the expressions of cytokines and collagens in lung tissue of pulmonary fibrosis rats induced by bleomycin].

OBJECTIVE: To investigate the effects of aerosolized signal transducer and activator of transcription 1 (STAT1) antisense oligonucleotides (ASON) on the expressions of TGF-beta(1), PAI-1, collagen Type I and III and hydroxyproline in lung tissue of pulmonary fibrosis induced by bleomycin (BLM) in rats. METHODS: Forty-five adult female Wistar rats were randomly divided into 3 groups: normal saline (NS) group, BLM group and ASON group. The BLM group and the ASON group were intratracheally instilled with BLM while the NS group was instilled with NS. The NS group and the BLM group were aerosolized with NS while the ASON group was aerosolized with STAT1 ASON on day 0, 2, 4, and 6. Then the rats were killed on day 7, 14, and 28. TGF-beta(1) and PAI-1 protein in the tissue of left lung were assessed by immunohistochemistry. The mRNA levels of collagen type I and III of right lung were measured by reverse transcriptase-polymerase chain reaction (RT-PCR). The content of hydroxyproline was also examined. RESULTS: Compared with the BLM group, the scores of alveolitis and fibrosis in the ASON group were remarkably reduced. Both TGF-beta(1) and PAI-1 protein in lung tissue in the BLM group and the ASON group were significantly higher as compared with the NS group. Compared with the BLM group, the mRNA levels of collagen type I and III in the ASON group were decreased. At day 7, 14 and 28, the mRNA levels of collagen type I and III in the ASON group were 1.36 +/- 0.10, 1.19 +/- 0.28, 1.22 +/- 0.24 and 1.20 +/- 0.09, 0.62 +/- 0.09, 0.76 +/- 0.12, respectively, both increased significantly as compared to those in the BLM group (3.29 +/- 0.28, 2.04 +/- 0.25, 1.91 +/- 0.30 and 1.63 +/- 0.15, 1.58 +/- 0.13, 1.12 +/- 0.09, respectively). The content of hydroxyproline (mg/g) in lung tissue in the ASON group at day 7, 14 and 28 was 3.02 +/- 0.13, 3.24 +/- 0.31, and 3.60 +/- 0.16, respectively, which were lower than those of the BLM group (3.76 +/- 0.10, 3.92 +/- 0.30, and 4.62 +/- 0.28, respectively). CONCLUSIONS: Aerosolized STAT1 ASON inhibited alveolitis and fibrosis in BLM-induced pulmonary fibrosis, via inhibition of the expression of PAI-1 and TGF-beta(1), decrease of the mRNA levels of collagen Type I and III, and the reduction of hydroxyproline production.

Aerosolized STAT1 antisense oligodeoxynucleotides decrease the concentrations of inflammatory mediators in bronchoalveolar lavage fluid in bleomycin-induced rat pulmonary fibrosis.

It has been demonstrated that alveolar macrophages (AMs) play a key role in the pathogenesis of pulmonary fibrosis by releasing a variety of cytokines and inflammatory mediators. In addition, abnormal signal transducer and activator of transcription-1 (STAT1) activation in AMs may play a pivotal role in the process of alveolitis and pulmonary fibrosis. In this study, we transfected STAT1 antisense oligodeoxynucleotide (ASON) into rats by aerosolization, and then investigated the effect of STAT1 ASON on inflammatory mediators such as TGF-beta, PDGF and TNF-alpha in bronchoalveolar lavage fluid (BALF) from rats with bleomycin (BLM)-induced rat pulmonary fibrosis. Our results showed that STAT1 ASON by aerosolization could enter into lung tissues and AMs. STAT1 ASON could inhibit mRNA and protein expressions of STAT1 and ICAM-1 in AMs of rat with pulmonary fibrosis, and had no toxic side effect on liver and kidney. Aerosolized STAT1 ASON could ameliorate the alveolitis through inhibiting the secretion of inflammatory mediators in BLM-induced rat pulmonary fibrosis. These results suggest that aerosolized STAT1 ASON might be considered as a promising new strategy in the treatment of pulmonary fibrosis.

[STAT1 antisense oligonucleotides inhibit secretion of TNF-alpha, IL-8 and NO in alveolar macrophages of rats suffering from interstitial pulmonary fibrosis].

AIM: To investigate the effect of signal transducer and activator of transcription 1 (STAT1) antisense oligonucleotides (ASON) on secretion of TNF-alpha, IL-8 and NO by alveolar macrophages (AMs) of rats with bleomycin (BLM)-induced pulmonary fibrosis. METHODS: Five adult female Wistar rats were intratracheally instilled with BLM. After 7 days, the rats were sacrificed under ketamine anaesthesia and bronchoalveolar lavage (BAL) was performed to obtain AMs. AMs were divided into four groups: STAT1 ASON, STAT1 sense oligonucleotides (SON), dexamethasone (DEX) and control groups. Culture medium was collected at 36 hours after adding STAT1 ASON, STAT1 SON and DEX, respectively. The concentrations of TNF-alpha, IL-8 and NO in the culture medium were detected. RESULTS: The concentrations of TNF-alpha, IL-8 and NO in STAT1 ASON group were lower than those in STAT1 SON, DEX and control groups (P<0.05). Moreover, the concentrations of TNF-alpha, IL-8 and NO in DEX group were also lower than those in control and STAT1 SON groups (P<0.05). But compared with control group, the concentrations of TNF-alpha, IL-8 and NO in STAT1 SON group was not significantly different (P<0.05). CONCLUSION: STAT1 ASON can inhibit the secretion of TNF-alpha, IL-8 and NO in AMs. STAT1 may become a target for treating pulmonary fibrosis.

[The effect of STAT1 antisense oligonucleotides on lung fibroblast proliferation and hydroxyproline secretion].

OBJECTIVE: To investigate the effect of signal transducer and activator of transcription 1 (STAT1) antisense oligonucleotides on lung fibroblast proliferation and hydroxyproline secretion. METHODS: Ten adult female Wistar rats were randomly divided into two groups: one group was intratracheally instilled with bleomycin (BLM), while another group with 0.9% NaCl solution (NS). After 7 days, the rats were killed by right ventricle of heart exsanguinations under ketamine anaesthesia, and bronchoalveolar lavage (BAL) was performed to obtain alveolar macrophage (AM). AMs from the BLM group were divided into four groups, treated with STAT1 antisense oligonucleotides, STAT1 sense oligonucleotides, dexamethasone and medium alone (control), respectively. AMs and media were collected after culture for 36 h. The mRNA and protein expression of STAT1 and ICAM-1 in AMs were detected by RT-PCR and Cell-ELISA, respectively. The conditioned media were co-cultured with lung fibroblasts for 30 h, and then the cell proliferation and the concentration of hydroxyproline were examined. RESULTS: (1) The STAT1 mRNA expression by AMs in the STAT1 antisense oligonucleotides group (31.8 +/- 3.5) was lower than those of AMs in the STAT1 sense oligonucleotides group (64.2 +/- 4.3), the dexamethasone group (44.1 +/- 4.6) and the control group (65.5 +/- 4.6) (P < 0.05). Moreover, the STAT1 mRNA expression by AMs in the dexamethasone group was also lower than those of AMs in the STAT1 sense oligonucleotides group and the control group (P < 0.05), but the STAT1 mRNA expression by AMs in the STAT1 sense oligonucleotides group was not different from that of the control group (P > 0.05). The STAT1 mRNA expression by AMs in the NS group (14.9 +/- 3.1) was lower than those of AMs in the STAT1 antisense oligonucleotides group, the STAT1 sense oligonucleotides group, the dexamethasone group and the control group (P < 0.05). (2) The mRNA expression of ICAM-1 showed similar changes to the STAT1 mRNA expression by AMs. (3) The STAT1 protein expression by AMs in the STAT1 antisense oligonucleotides group (4.4 +/- 0.6) or in the NS group (3.7 +/- 0.4) was lower than those of AMs in the STAT1 sense oligonucleotides group (7.7 +/- 0.7), the dexamethasone group (5.9 +/- 0.4) and the control group (7.6 +/- 0.6) (P < 0.05); and the STAT1 protein expression by AMs in the dexamethasone group was also lower than those of AMs in the STAT1 sense oligonucleotides group and the control group (P < 0.05), but the STAT1 protein expression by AMs in the STAT1 sense oligonucleotides group was not different from that of the control group (P > 0.05). (4) The changes of ICAM-1 protein expression, lung fibroblast proliferation and hydroxyproline concentration were consistent with the changes of STAT1 protein expression by AMs. CONCLUSIONS: STAT1 antisense oligonucleotides could inhibit the mRNA and the protein expression of STAT1 and ICAM-1 in AMs. STAT1 antisense oligonucleotides also inhibited lung fibroblast proliferation and hydroxyproline secretion.

[Regulative effect of STAT1 on inflammation of lung tissue in bleomycin-induced rat interstitial pulmonary fibrosis].

OBJECTIVE: To investigate the role of signal transducer and activator of transcription 1 (STAT1) in alveolar macrophage (AM) in bleomycin-induced rat pulmonary fibrosis. METHODS: Fifty adult female Wistar rats were randomly divided into two groups. The rats of BLM group were intratracheally instilled with bleomycin (BLM), and those of the control group with normal saline(NS). The kinetic change of STAT1 activation and the intercellular adhesion molecule-1 (ICAM-1) expression in AM were examined. RESULTS: STAT1 was slightly activated in AM of NS group. After bleomycin treatment, the STAT1 activation of AM significantly increased on day 1, reached the peak value on day 7, and then gradually decreased, yet it remained significantly above the value of the NS group on day 28 (P < 0.05). There were a few positive staining cells for ICAM-1 expression in AM of NS group. After intratracheal instillation of bleomycin, the number of positive staining cells for ICAM-1 expression significantly increased on day 1, reached the peak value on day 7, gradually decreased from then on, but still it was higher than that of the NS group on day 28 (P < 0.05). There was a significant correlation between STAT1 activation and ICAM-1 expression in AM (r = 0.913, P < 0.01); moreover, ICAM-1 expression in AM was significantly correlated with the degree of inflammation in lung tissue (r = 0.947, P < 0.01). CONCLUSION: STAT1 was found abnormally activated in AM in bleomycin-induced rat interstitial pulmonary fibrosis (IPF). The abnormal STAT1 activation may play a role in the pathogenesis of acute alveolitis and pulmonary fibrosis.

STAT1 activation and STAT1-dependent immune-response gene ICAM-1 expression in alveolar macrophages of rats suffered from interstitial pulmonary fibrosis.

AIM: To investigate the role of signal transducer and activator of transcription 1(STAT(1)) in alveolar macrophages (AMs) from rats with bleomycin-induced pulmonary fibrosis. METHODS: Fifty adult female Wistar rats were randomly divided into two groups: One group was intratracheally instilled with bleomycin (BLM), while another group with 9 g/L NaCl solution (NS). The kinetic change of STAT(1) activation and intercellular adhesion molecule-1(ICAM-1) expression in AMs was examined by Western blot and immunohistochemical staining, respectively. RESULTS: (1)STAT(1) was rarely activated in AMs of NS group. After bleomycin treatment, the STAT(1) activation of AMs was significantly increased on day 1, climaxed on day 7, and then gradually decreased, but remaining significantly higher than that of NS group on day 28 (P<0.05). (2)There was only a few cells ICAM-1 higher than that positively stained in AMs of NS group. After intratracheal instillation of bleomycin, the number of ICAM-1 positive stained cells was significantly increased on day 1, reached its peak values on day 7, and then gradually decreased, and still higher than that of NS group on day 28 (P<0.05).(3)There was a significant correlationship between STAT(1) activation and ICAM-1 expression in AMs (r =0.913, P<0.01). Further more, the ICAM-1 expression in AMs was significantly correlated with the severity of inflammation in lung tissues (r =0.947, P<0.01). CONCLUSION: STAT(1) was abnormally activated in AMs of rats with bleomycin-induced interstitial pulmonary fibrosis (IPF). The abnormal STAT(1) activation may play a role in the pathogenesis of acute alveolitis and pulmonary fibrosis.