Network pharmacology analysis and experimental validation of Xiao-Qing-Long-Tang's therapeutic effects against neutrophilic asthma.

BACKGROUND: Xiao-Qing-Long-Tang (XQLT), a classical Chinese herbal medicine formula, has been extensively used for allergic asthma treatment. However, there is limited research on its anti-inflammatory effects and mechanisms specifically in neutrophilic asthma (NA). PURPOSE: This study aims to investigate the potential therapeutic effects of XQLT against NA using a combination of network pharmacology and experimental validation. STUDY DESIGN: By utilizing traditional Chinese medicine and disease databases, we constructed an XQLT-asthma network to identify potential targets of XQLT for NA. In the experimental phase, we utilized an ovalbumin (OVA)/lipopolysaccharide (LPS)-induced model for neutrophilic asthma and examined the therapeutic effects of XQLT. RESULTS: Our research identified 174 bioactive components within XQLT and obtained 140 target genes of XQLT against asthma. Functional enrichment analysis revealed that these target genes were primarily associated with inflammation and cytokines. In the experimental validation, mice induced with OVA-LPS showcased eosinophilic and neutrophilic cell infiltration in peri-bronchial areas, elevated levels of IL-4 and IL-17 in both serum and lung, increased percentages of Th2 and Th17 cells in the spleen, as well as elevated levels of CD11b+ and CD103+ dendritic cells (DCs) within the lung. Treatment with XQLT effectively reduced IL-4 and IL-17 levels, decreased the percentages of Th2, Th17, CD11b+, and CD103+ DCs, and improved inflammatory cell infiltrations in lung tissues. These findings serve as a foundation for the potential clinical application of XQLT in neutrophilic asthma.

miR-34a-5p up-regulates the IL-1ß/COX2/PGE2 inflammation pathway and induces the release of CGRP via inhibition of SIRT1 in rat trigeminal ganglion neurons.

Migraine is a debilitating neurological condition, with a global prevalence rate of 10.68% in men and 18.79% in women. Elucidation of the molecular mechanisms underlying migraines is of great importance for improving the quality of life of patients. The release of the neuropeptide calcitonin gene-related peptide (CGRP) from trigeminal nerve terminals is involved in the pathogenesis of migraine. Recent studies have shown that up-regulation of miR-34a-5p expression is associated with acute migraine attacks. Here, we investigated whether alteration of the expression of miR-34a-5p induces the release of the vasoactive peptide CGRP. We isolated primary rat trigeminal ganglion neurons and performed gain- and loss-of-function assays to alter the expression level of miR-34a-5p. Down-regulation of miR-34a-5p inhibited the expression of interleukin-1ß (IL-1ß)/cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2), decreased IL-1ß, PGE2 and CGRP release, and up-regulated the expression of silencing information regulator 1 (SIRT1) in trigeminal ganglion, whereas overexpression of miR-34a-5p enhanced the expression of IL-1ß/COX2/PGE2, increased the release of IL-1ß, PGE2 and CGRP, and decreased the expression of SIRT1 in trigeminal ganglion. In addition, overexpression of miR-34a-5p induced apoptosis in primary rat trigeminal neurons. In summary, these findings suggest that miR-34a-5p up-regulates the IL-1ß/COX2/PGE2 inflammation pathway, induces apoptosis and enhances release of CGRP via inhibition of SIRT1 expression in trigeminal ganglion neurons; thus, miR-34a-5p may have potential as a therapeutic target for the treatment of migraine.

[Effects of electroacupuncture on miR-34a-5p/SIRT1 signaling in the trigeminal ganglion of rats with migraine].

OBJECTIVE: To investigate the effect of electroacupuncture(EA) on miR-34a-5p, silent mating type information regulation 2 homolog-1 (SIRT1) and nuclear factor-κB subunit p65 (NF-κB p65) in the trigeminal ganglion of rats with migraine, so as to explore the mechanisms of EA underlying prevention of migraine. METHODS: Male SD rats were randomly divi-ded into normal, sham operation, model, EA, and EA plus EX527(a SIRT1 inhibitor) groups, with 10 rats in each group. The rat model of migraine was established by electrical stimulation of the trigeminal ganglion. Before modeling, EA was applied at "Waiguan"(TE5) and "Fengchi"(GB20) for 20 min each time, once a day for 5 consecutive days, and intraperitoneal injection of EX527 (5 mg/kg) every day simultaneously. Serum prostaglandin E2 (PGE2) and calcitonin gene-related peptide (CGRP) concentrations were measured by enzyme-linked immunosorbent assay. The levels of miR-34a-5p, SIRT1 and interleukin-1ß (IL-1ß) mRNA，and protein expression of SIRT1, IL-1ß, NF-κB p65, NF-κB Ac-p65 and cyclooxygenase-2 （COX2） in trigeminal ganglia were detected by real-time quantitative PCR and Western blot, separately. RESULTS: The serum concentrations of PGE2 and CGRP， the expression of miR-34a-5p, IL-1ß mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expression in the trigeminal ganglion were remarkably increased (P<0.05), while the SIRT1 mRNA and protein decreased (P<0.05) in the model group in contrast to the normal group. Following EA intervention， the serum PGE2 and CGRP concentrations, miR-34a-5p expression, IL-1ß mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expression were significantly down-regulated (P<0.05), and SIRT1 mRNA and protein significantly up-regulated (P<0.05). Compared with the EA group, the serum concentrations of PGE2 and CGRP, IL-1ß mRNA and protein, NF-κB p65, NF-κB Ac-p65 and COX2 protein expressions increased (P<0.05), and SIRT1 protein decreased (P<0.05) in the EA plus EX527 group. CONCLUSION: In migraine rats, EA can inhibit miR-34a-5p expression in the trigeminal ganglion, increase SIRT1 expression, down-regulate IL-1ß/COX2 inflammation signals, reduce PGE2 synthesis, and thus redue CGRP released from the peripheral terminals, which may be one of the mechanisms of EA in preventing migraine.

5-Aza-2'-deoxycytidine protects against emphysema in mice via suppressing p16Ink4a expression in lung tissue.

BACKGROUND: There is a growing realization that COPD, or at least emphysema, involves several processes presenting in aging and cellular senescence. Endothelial progenitor cells (EPCs) contribute to neovascularization and play an important role in the development of COPD. The gene for p16Ink4a is a major dominant senescence one. The aim of the present study was to observe changes in lung function, histomorphology of lung tissue, and expression of p16Ink4a in lung tissue and bone marrow-derived EPCs in emphysematous mice induced by cigarette-smoke extract (CSE), and further to search for a potential candidate agent protecting against emphysema induced by CSE. MATERIALS AND METHODS: An animal emphysema model was induced by intraperitoneal injection of CSE. 5-Aza-2'-deoxycytidine (5-Aza-CdR) was administered to the emphysematous mice. Lung function and histomorphology of lung tissue were measured. The p16Ink4a protein and mRNA in EPCs and lung tissues were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction, respectively. RESULTS: CSE induced emphysema with increased p16Ink4a expression in lung tissue and bone marrow-derived EPCs. 5-Aza-CdR partly protected against emphysema, especially in the lung-morphology profile, and partly protest against the overexpression of p16Ink4a in EPCs and lung tissue induced by CSE. CONCLUSION: 5-Aza-CdR partly protected against emphysema in mice via suppressing p16Ink4a expression in EPCs and lung tissue.

Decitabine enhances stem cell antigen-1 expression in cigarette smoke extract-induced emphysema in animal model.

Stem cell antigen-1 (Sca-1) is a mouse glycosyl phosphatidylinositol-anchored protein and a cell surface marker found on hematopoietic stem cells (HSCs). Despite decades of study, its biological functions remain little known. Sca-1 is a typical marker of bone marrow-derived HSCs, it is also expressed by a mixture of tissue-resident stem, progenitor cells in nonhematopoietic organs. Endothelial progenitor cell (EPC) is a subtype of HSC and contributes to endothelial repair by homing in on locations of injury. Abnormal genetic methylation has been detected in smoking-related diseases. The present study aimed to investigate the lung function and histomorphology, the expression of Sca-1 gene in lung tissues, and bone marrow-derived EPCs in cigarette smoke extract (CSE)-induced emphysema mice, and to further determine whether Decitabine (Dec), the most widely used inhibitor of DNA methylation, could protect against the damages caused by CSE. The results of the present study demonstrated that Dec could partly protect against CSE-induced emphysema in mice, enhance Sca-1 expression in lung tissue, and bone marrow-derived EPCs. The results suggested that the depletion of the progenitor cell pool and DNA methylation of Sca-1 gene may be involved in the progression of emphysema in mice.

Comparison between cigarette smoke-induced emphysema and cigarette smoke extract-induced emphysema.

BACKGROUND AND OBJECTIVE: Emphysema is the main pathological feature of COPD and also is the focus of the related research. Although several emphysema animal models have been established, exact comparison of findings is seldom. The present study aimed to compare cigarette smoke (CS) exposure-induced emphysema model and intraperitoneal injection of cigarette smoke extract (CSE)-induced emphysema model to evaluate the effectiveness of the two different modeling methods. METHODS: Six-week-old male C57BL/6 J mice were used and randomly divided into two groups: CS exposure and intraperitoneal injection of CSE. Each group was subdivided into two subgroups: control and CS or CSE. Lung function, mean linear intercept (MLI), destructive index (DI), apoptotic index (AI), total and differential cells count in broncholavolar lavage fluid (BALF), SOD and IL-6 concentration in serum were measured. RESULTS: Compared with their respective controls, lung function was significantly decreased in CS and CSE groups (P < 0.01); MLI, DI, and AI of lung tissue were significantly higher in CS and CSE groups (P < 0.01); total number of leukocytes, the number and percentage of neutrophils (NEUs), and the number of macrophages (MAC) in BALF were significantly higher in CS and CSE groups (P < 0.01); SOD concentration in serum was significantly decreased in CS and CSE groups (P < 0.01); IL-6 concentration in serum was significantly increased in in CS and CSE groups (P < 0.01). There was no significant difference between CS group and CSE group in any of the parameters described above. CONCLUSIONS: Both CS exposure and intraperitoneal injection of CSE could induce emphysema and the effectiveness of the two different modeling methods were equal.

Dual effects of cigarette smoke extract on proliferation of endothelial progenitor cells and the protective effect of 5-aza-2'-deoxycytidine on EPCs against the damage caused by CSE.

UNLABELLED: Cigarette smoke is a major public health problem associated with multitude of diseases, including pulmonary and vascular diseases. Endothelial progenitor cells (EPCs) contribute to neovascularization and play an important role in the development of these diseases. The effect of CSE on EPCs is seldom studied. The aim of the current study is to observe the effect of CSE on biological behavior of EPCs and, further, to search for potential candidate agent in protection of proliferation of EPCs against the damage caused by CSE exposure in vitro. METHODS: The proliferations of EPCs isolated from bone marrow of C57BL/6J mice were assessed by MTT after incubating the EPCs with a series of concentrations of CSE (1.0%, 2.5%, 5.0%, and 10.0%) for different times (3, 6, and 24 hours) as well as with 1.0% CSE in presence of 5-AZA-CdR for 24 hours. RESULTS: The proliferations of EPCs were significantly enhanced after 3 hours of exposure to concentrations of 1.0% and 2.5% CSE but depressed when exposed to concentrations of 5.0% and 10.0% CSE. Furthermore, the 5-AZA-CdR in concentrations of 2.0 µmol/L and 5.0 µmol/L partly protected against the depression of proliferation of EPCs caused by CSE exposure. CONCLUSIONS: The CSE showed dual effects on proliferation of EPCs isolated from mice. The 5-AZA-CdR partly protected the proliferation of EPCs against the damage caused by CSE exposure in vitro, suggesting that DNA methylation may be involved in the dysfunction of EPCs induced by CSE.

[Study on influencing factors in efficiency of introducing gene into human keratinocyte (KC)].

OBJECTIVE: To observe the effect of plasmids in different size and gene transfection protocol on efficiency of introducing gene into human KC. METHODS: Four plasmids in different size, inclu-ding pSUPER-enhanced green fluorescent protein (EGFP), pEGFP-N2, pHSER-green fluorescent protein (GFP) and ploxP-EGFP, were transfected into immortal human KC line (HaCaT) and human embryo kid-ney cell line (293FT) separately following transfection protocols of liposome (LTP), cation polymerizer (CPTP), electroporation combined with nucleus transfection agent (ETP) and lentivirus. 293FT was used as control. GFP expression was observed under inverted fluorescence microscope. The transfection efficiency (TE) was calculated. RESULTS: (1) The four plasmids could be introduced into HaCaT (TE, 1.0%-3.3%) and 293FT (TE, 80.0%-84.7% ) following LTP. (2) The four plasmids could also be introduced into HaCaT (TE, 1.0%-3.7% ) and 293FT (TE, 81.3%-86.7% ) following CPTP. (3) Two shorter plasmids (pSUPER-EGFP and pEGFP-N2) could be introduced into HaCaT by ETP with higher TE than the othr two longer plasmids (pHSER-GFP and ploxP-EGFP), which were 22.3% and 19.0% vs. 4.0% and 3.3%, respectively. (4) pHSER-GFP packaged by lentivirus could be introduced into HaCaT with the TE reaching 97.0%, which surpassed the above three protocols. CONCLUSIONS: It is difficult to introduce exogenous gene into human KC by LTP or CPTP; TE of lentivirus transfection protocol apparently surpasses

[Clone screening and interference efficiency of seed cells with CCL20 gene knockdown for tissue-engineered skin].

OBJECTIVE: To screen stable cell clones of CCL20 gene knockdown and assess their interference effects, recombinant lentivirus vectors with CCL20 gene specific shRNA were applied to infect human immortal keratinocyte line (HaCaT). METHODS: The three pHSER-CCL20-shRNA-GFP vectors (pHCG-1 and pHCG-2 were CCL20 gene specific, and pHCG-3 was used as mismatch control) have been previously constructed. The virus packaging cell line 293FT was transfected with these vectors by using CaCl2 methods to produce lentiviral particles. After the viral titers of these three harvested cell supernatants were determined by flow cytometry, HaCaT cells were transfected by these viruses and screened under the pressure of G418. The CCL20 mRNA from HaCaT cell clones and the CCL20 protein levels in the supernatants of HaCaT cell clones were detected by Real-time RT-PCR and ELISA, respectively. RESULTS: The titers of three lentiviruses were 7.08 x 10(5) transduced units (TU)/ml, 1.88 x 10(5) TU /ml and 2.08 x 10(5) TU/ml, respectively. Two HaCaT cell clones from each lentiviral vectors were obtained after G418 screening for 5 - 8 weeks. Four CCL20 gene specific clones showed stable interference effect in both Real-time RT-PCR and ELISA. The mRNA expression and protein level of CCL20 gene specific clones were down regulated significantly. CONCLUSIONS: The four human immortal keratinocyte clones with long term CCL20 gene knockdown have been screened by recombinant lentivirus vectors with CCL20 gene specific shRNA. These clones might be served as seed cells for novel tissue-engineered skin with lower rejection.