Scorpio and centipede ameliorate asthma by inhibiting the crosstalk between ferroptosis and inflammation in airway epithelial cells.

Objectives: We previously revealed that scorpio and centipede (SC) improve the inflammatory response in asthma, whereas it is unclear whether ferroptosis is involved in this process. Materials and Methods: The asthmatic mouse model was established and lung tissues were collected for histopathological examination. The levels of tumor necrosis factor-α (TNF-α), interleukin- (IL-)1ß, Fe2+, malondialdehyde (MDA), glutathione peroxidase 4 (GPX4), ferritin heavy chain 1(FTH1), and reactive oxygen species (ROS) were assessed in asthmatic mice and mouse airway epithelial cells. Results: Our results showed that ferroptosis was induced in asthmatic mice, as evidenced by the reduction of FTH1 and GPX4 expression and the increase of MDA and Fe2+ levels (all P<0.05). Ferrostatin-1 repressed inflammation and ferroptosis of asthmatic mice. Additionally, SC significantly inhibited the levels of TNF-α, IL-1ß, MDA, and Fe2+, while enhancing FTH1 and GPX4 expression. However, SC plus erastin showed the reverse results. Moreover, ferroptosis remarkably increased in asthmatic airway epithelial cells, while SC suppressed ferroptosis of the cells (all P<0.05). Conclusion: SC ameliorated asthma by inhibiting the crosstalk between ferroptosis and inflammation in airway epithelial cells.

Elucidation of the mechanisms and molecular targets of KeChuanLiuWei-Mixture for treatment of severe asthma based on network pharmacology.

KeChuanLiuWei-Mixture (KCLW) is widely used as a Chinese medicine prescription to treat severe asthma. However, the underlying therapeutic mechanism of KCLW remains unclear. In this study, a network pharmacology method was used to identify the chemical constituents of KCLW by the TCMSP database and ultra-performance liquid chromatography coupled with time-of-flight mass spectrometry. Differential expression identification, protein-protein interaction (PPI) network and functional enrichment analysis were used to screen key targets of KCLW for severe asthma. Our results confirmed that quercetin, luteolin, kaempferol, and wogonin are the most critical active ingredients in KCLW. Moreover, the 16 relevant severe asthma-related targets of KCLW were obtained by overlapping the PPI networks of the KCLW putative targets and severe asthma-related genes, among which the most important targets were IL-6, NOS2, VEGFA, CXCL2, and PLAT. Functionally, the 16-targets and their interacting differentially expressed genes were primarily related to biological functions and pathways related to immunity and inflammation, such as inflammatory response, T cell differentiation, Nrf2/HO-1 signaling pathway, TGF-ß/Smad signaling pathway, and NF-κB signaling pathway. KCLW inhibited inflammation in PDGF-BB-induced airway smooth muscle cells. In summary, this study demonstrates the active substance and potential therapeutic mechanism of KCLW in severe asthma, and offers a clinical direction for KCLW against severe asthma.

A retrospective study of Pupingqinghua prescription versus Lianhuaqingwen in Chinese participants infected with SARS-CoV-2 Omicron variants.

Background: Coronavirus disease (COVID-19) seriously endangers global public health. Pupingqinghua prescription (PPQH) is an herbal formula from traditional Chinese medicine used for treatment of SARS-CoV-2 infection. This study aims to evaluate the clinical efficacy and safety of PPQH in Chinese participants infected with the SARS-CoV-2 Omicron variant. Methods: A total of 873 SARS-CoV-2 (Omicron)-infected patients were included. Among them, the patients were divided into the PPQH group (653 cases) and LHQW group (220 cases) according to different medications. The effectiveness indicators (hematological indicators, Ct values of novel Coronavirus nucleic acid tests, and viral load-shedding time) and safety indicators (liver and kidney function and adverse events) were analyzed. Results: There was no significant difference in baseline characteristics between the PPQH group and the LHQW group, except the gender; After the treatment, the levels of IL-5, IL-6, IL-10, NK cells, and INF-α of the patients in the PPQH group showed a downward trend (p < 0.05); The viral load shedding time was 5.0 (5.0, 7.0) in the PPQH group and 5.0 (4.0, 7.0) in the LHQW group; both PPQH and LHQW can shorten the duration of symptoms of fever, cough, and sore throat. The re-positive rate of COVID-19 test was 1.5 % in the PPQH group and 2.3 % in the LHQW group. In terms of safety, the levels of γ-GTT decreased significantly (p < 0.01); gastrointestinal reaction was the primary adverse reaction, and the reaction rate was 4.7 % in the PPQH group and 9.5 % in the LHQW group. Conclusion: PPQH can shorten the length of hospital stay and improve clinical symptoms of patients with SARS-COV-2 (Omicron), and it also has a good safety profile.

Scorpion and centipede alleviates severe asthma through M2 macrophage-derived exosomal miR-30b-5p.

Asthma is one of the most common chronic inflammatory diseases. Although the scorpion and centipede (SC) significantly ameliorates asthma and changes exosomal miRNAs, the molecular mechanism is still obscure. Here, we show that SC improves inflammation in asthmatic mice and increases M2 macrophage-derived exosomes (M2Φ-Exos) by promoting M2 macrophage polarization. The M2Φ-Exos remarkably inhibits airway epithelial cell pyroptosis by reducing the expression of NLRP3, caspase-1, and LI-1ß and mitochondrial swelling. Furthermore, miR-30b-5p is up-regulated in M2Φ-Exos compared with M1Φ-Exos. Overexpression of miR-30b-5p in M2Φ-Exos prevents airway epithelial cell pyroptosis, while down-regulation of miR-30b-5p promotes pyroptosis. We also uncover that pyroptosis is increased in asthmatic mice, while SC blocks pyroptosis. Moreover, miR-30b-5p overexpressed M2Φ-Exos further enhances the ameliorative effect of SC, which significantly down-regulates IRF7 expression. Our results collectively reveal that M2Φ-Exos induced by SC could carry miR-30b-5p to mitigate severe asthma by inhibiting airway epithelial cell pyroptosis. Most importantly, our findings may provide a potential clinical application of M2Φ-Exos for treating severe asthma.

Evaluating the Effects of Heat-Clearing Traditional Chinese Medicine in Stable Bronchiectasis by a Series of N-of-1 Trials.

PURPOSE: The purpose of this study is to study the effects of heat-clearing Traditional Chinese Medicine (TCM) in the stable stage of bronchiectasis via N-of-1 trials. METHODS: The N-of-1 trials in this study were randomized and double-blinded with crossover comparisons consisting of three pairs. Each pair was of two 4-week periods. Each patient took the individualized decoction in the experimental period and the individualized decoction was removed of heat-clearing drugs, mainly including heat-clearing and detoxifying drugs, in the control period for three weeks. After three weeks, the patients stopped taking the decoction for one week. The primary outcome was from patients' self-reporting symptoms scores on a 1-7-point Likert scale. Mixed-effects models were used to conduct statistical analysis on these N-of-1 trials. RESULTS: Of the 21 patients enrolled, 15 completed three pairs of N-of-1 trials (71.43%). (1) Seen from the individual level, no statistical difference between the experimental decoction and the control (P > 0.05) was observed. However, 5 patients found better decoctions according to the clinical criteria. (2) As revealed by the group data of all the N-of-1 trials, the control was better than the individualized decoction in terms of symptom scores on the Likert scale (1.94 ± 0.69 versus 2.08 ± 0.68, P = 0.04, mean difference, and 95% CI: 0.19 (0.01, 0.37)) and on CAT scores (13.66 ± 6.57 versus 13.95 ± 6.97, P = 0.04, mean difference, and 95% CI: 0.86 (0.042, 1.67)), but such differences were not clinically significant. The other outcomes, such as Likert scale score of respiratory symptoms and 24-hour sputum volume, showed no statistical difference. CONCLUSION: The experimental design of this study can make the TCM individualized treatment fully play its role and can detect the individualized tendencies according to the severity of phlegm and heat in some subjects. With the intermittent use or reduced use of heat-clearing drugs, most of the subjects, at the group level, enrolled in the series of N-of-1 trials may improve the symptoms and quality of life while saving the cost of TCM and reducing the potential side effects of heat-clearing TCM. This trial is registered with clinicaltrials.goc (NCT03147443).

A Series of N-of-1 Trials for Traditional Chinese Medicine Using a Bayesian Method: Study Rationale and Protocol.

Background. Our previous studies showed that N-of-1 trials could reflect the individualized characteristics of traditional Chinese medicine (TCM) syndrome differentiation with good feasibility, but the sensitivity was low. Therefore, this study will use hierarchical Bayesian statistical method to improve the sensitivity and applicability of N-of-1 trials of TCM. Methods/Design. This is a randomized, double-blind, placebo-controlled, three-pair crossover trial for a single subject, including 4-8 weeks of run-in period and 24 weeks of formal trial. In this study, we will recruit a total of 30 participants who are in the stable stage of bronchiectasis. The trial will be divided into three pairs (cycles), and one cycle contains two observation periods. The medications will be taken for three weeks and stopped for one week in the last week of each observation period. The order of syndrome differentiation decoction and placebo will be randomly determined. Patient self-reported symptom score (on a 7-point Likert scale) is the primary outcome. Discussion. Some confounding variables (such as TCM syndrome type and potential carryover effect of TCM) will be introduced into hierarchical Bayesian statistical method to improve the sensitivity and applicability of N-of-1 trials of TCM, and the use of prior available information (e.g., "borrowing from strength" of previous trial results) within the analysis may improve the sensitivity of the results of a series of N-of-1 trials, from both the individual and population level to study the efficacy of TCM syndrome differentiation. It is the exploration of improving the objective evaluation method of the clinical efficacy of TCM and may provide reference value for clinical trials of TCM in other chronic diseases. This trial is registered with ClinicalTrials.gov (ID: NCT04601792).

Small RNA Sequencing Reveals Exosomal miRNAs Involved in the Treatment of Asthma by Scorpio and Centipede.

Asthma is a common respiratory disease with inflammation in the lungs. Exosomes and microRNAs (miRNAs) play crucial role in inflammation, whereas the role of exosomal miRNA in asthma remains unknown. Here, we aimed to identify the key exosomal miRNAs and their underlying mechanisms involved in scorpio and centipede (SC) treatment in asthma. Eighteen mice were randomly divided into three groups: control group, asthma group, and SC treatment group. Effect of SC was assessed by hematoxylin-eosin staining and real-time PCR. Exosomes from asthma and SC treatment groups were analyzed by small RNA-seq. Results revealed SC significantly alleviated the pathogenesis of asthma and suppressed the release of inflammatory cytokines. A total of 328 exosomal miRNAs were differentially expressed between the exosomes from asthma and SC-treated mice, including 118 up- and 210 downregulated in SC-treated mice. The altered exosomal miRNAs were primarily involved in the function of transcription, apoptotic process, and cell adhesion; and pathway of calcium, Wnt, and MAPK signaling. Real-time PCR verified exosomal miR-147 was downregulated, while miR-98-5p and miR-10a-5p were upregulated in SC-treated mice compared to asthma mice. Moreover, the target genes of miR-147-3p, miR-98-5p, and miR-10a-5p were mainly enriched in Wnt and MAPK inflammatory signaling. miR-10a-5p promoted the proliferation of mouse lung epithelial cells and downregulated the expression of Nfat5 and Map2k6. These data suggest SC-induced exosomal miRNAs might mediate the inflammatory signaling and might be involved in the SC treatment in asthma. The exosomal miRNAs might be promising candidates for the treatment of asthma.

Investigation into the Individualized Treatment of Traditional Chinese Medicine through a Series of N-of-1 Trials.

PURPOSE: To compare the efficacy of individualized herbal decoction with standard decoction for patients with stable bronchiectasis through N-of-1 trials. METHODS: We conducted a single center N-of-1 trials in 17 patients with stable bronchiectasis. Each N-of-1 trial contains three cycles. Each cycle is divided into two 4-week intervention including individualized decoction and fixed decoction (control). The primary outcome was patient self-reported symptoms scores on a 1-7 point Likert scale. Secondary outcomes were 24-hour sputum volume and CAT scores. RESULTS: Among 14 completed trials, five showed that the individualized decoction was statistically better than the control decoction on symptom scores (P < 0.05) but was not clinically significant. The group data of all the trials showed that individualized decoction was superior to control decoction on symptom scores (2.13 ± 0.58 versus 2.30 ± 0.65, P = 0.002, mean difference and 95% CI: 0.18 (0.10, 0.25)), 24 h sputum volume (P = 0.009), and CAT scores (9.69 ± 4.89 versus 11.64 ± 5.59, P = 0.013, mean difference and 95% CI: 1.95 (1.04, 2.86)) but not clinically significant. CONCLUSION: Optimizing the combined analysis of individual and group data and the improvement of statistical models may make contribution in establishing a method of evaluating clinical efficacy in line with the characteristics of traditional Chinese medicine individual diagnosis and treatment.

α-Asarone suppresses the proliferation and migration of ASMCs through targeting the lncRNA-PVT1/miR-203a/E2F3 signal pathway in RSV-infected rats.

Asthma is a chronic inflammatory pulmonary disease and respiratory syncytial virus (RSV) infection is a common cause of lower respiratory tract illness in infants and young children. α-Asarone presents many pharmacological effects and has been demonstrated to be useful in treating asthma. However, the functional mechanism of α-asarone in RSV-infected asthma has not been investigated. Long non-coding RNAs (lncRNAs) have been reported to play critical roles in many biological processes. Although many lncRNAs have been characterized, few were reported in asthma, especially in RSV-induced asthma. Currently, a novel post-transcriptional regulation has been proposed in which lncRNAs function as competing endogenous RNAs (ceRNAs) to competitively sponge miRNAs, thereby regulating the target genes. In the present study, we established an RSV-infected Sprague-Dawley rat model and demonstrated that lncRNA-PVT1 is involved in the mechanism of α-asarone in treating RSV-induced asthma, and lncRNA-PVT1 regulates the expression of E2F3 by functioning as a ceRNA which competitively sponges miR-203a.

Effect of "yang-warming and kidney essence-replenishing" herbal paste on cold-related asthma exacerbation.

OBJECTIVE: To observe the effect of "Yang-warming and kidney essence-replenishing" herbal paste on cold-related asthma exacerbation. METHODS: One hundred and fifty one patients with moderate to severe persistent asthma were randomly divided into a treatment group (n = 74) and a control group (n = 69). Both groups were given basic treatment according to the Global Initiative for Asthma scheme. The treatment group was also treated with herbal paste in the winter. The frequency of asthma exacerbation, cold and cold-related asthma exacerbation, scores of the asthma control test (ACT), and kidney-deficiency syndrome during the one-year follow-up in each group were recorded. RESULTS: Compared with the control group, the onset frequencies of catching cold, asthma exacerbation, cold-related asthma exacerbation, and kidney-deficiency syndrome score were significantly reduced. There was no significant difference in scores of ACT between the two groups. CONCLUSION: "Yang-warming and kidney essence-replenishing" herbal paste could reduce cold-related asthma exacerbation.

[Effects of medicinal extract for tonifying kidney to relieve asthma on glucocorticoid receptor expression in lung tissues of rats with bronchial asthma].

OBJECTIVE: To observe the effects of medicinal extract for tonifying kidney to relieve asthma on glucocorticoid receptor (GR) expression in rats with asthma, and to explore its mechanism in treating asthma. METHODS: Sixty SD rats were randomly divided into normal control group, untreated group, dexamethasone group, and medicinal extract-prevented, medicinal extract-treated, and medicinal extract-prevented and -treated groups, with ten rats in each group. Asthma was induced by intraperitoneal injection of ovalbumin (OVA) and forced inhalation of atomized OVA. Expression of GR in lung tissues was detected by immunohistochemical method. Pathological changes of the lung tissues were observed by HE straining. RESULTS: Expression of GR was lower in the untreated group than in the normal control group (P<0.05). Expressions of GR in medicinal extract groups were up-regulated as compared with those in the untreated group and dexamethasone group (P<0.05, P<0.01), and there were no significant differences as compared with the normal control group (P>0.05). CONCLUSION: Medicinal extract for tonifying kidney to relieve asthma can increase the expression of GR in lung tissues of asthmatic rats, which may be one of its mechanisms in preventing and treating asthma.

[Effects of Kechuanluo oral liquid on eosinophil apoptosis and its regulation factors in lung tissues of asthmatic mice].

OBJECTIVE: To explore the mechanism of Kechuanluo oral liquid, a compound Chinese herbal medicine, in inhibiting allergic airway inflammation by observing the effects of Kechuanluo on eosinophil (EOS) apoptosis and its regulation factors in asthmatic mice. METHODS: Fifty-six BALB/c mice were randomly divided into normal control group (n=16), untreated group (n=16), Western medicine group (n=12) and Kechuanluo group (n=12). Except for the mice in normal control group, asthma was induced in BALB/c mice by using ovalbumin (OVA) and potassium aluminium sulfate. The mice were intragastrically administered with normal saline, Kechuanluo (30 ml/kg daily) and prednisolone tablets (10 mg/kg daily) respectively for two weeks. At 0 hour, the 3rd, 7th and 14th day after the end of OVA sensitization, the EOSs of lung tissues were counted by improved-Giemsa staining method; immunohistochemical method and image analysis were used to detect the expressions of Fas, FasL and Bcl-XL in the EOSs in the four groups; and apoptotic rates of the EOSs in the lung tissues of asthmatic mice were detected by terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling (TUNEL) technique. RESULTS: Compared with the untreated group, airway inflammations of the mice in the Kechualuo group and Western medicine group were lessened and the EOS counts decreased on the 3rd, 7th and 14th day after the last OVA sensitization (P<0.05). On the 3rd day, the EOSs apoptotic rates, the expression areas of Fas in the EOSs and FasL in the lung tissues were significantly higher in the Kechuanluo group than those in the untreated group (P<0.01), furthermore, the EOS apoptotic rate reached the peak level. Inversely, the EOS count and the expression area of Bcl-XL in the EOSs were obviously lower in the Kechuanluo group (P<0.05). On the 7th and 14th day, the expression areas of Fas and Bcl-XL in the EOSs were significantly decreased in the Kechuanluo group (P<0.01), and on the 14th day, the EOS apoptotic rate and the expression area of FasL in the lung tissues were obviously lower either. The effects exhibited in the Western medicine group were similar to those in the Kechuanluo group. CONCLUSION: There is airway inflammation with eosinophilic infiltration in asthmatic mice, accompanied with suppressed apoptosis and delayed apoptosis. Kechuanluo can induce and accelerate EOS apoptosis in early inflammation by inhibiting eosinophilic inflammation, improving Fas expression in EOSs and FasL expression in the lung tissues, and reducing Bcl-XL expression in EOSs.

[Function of MMP/TIMP on airway remodeling of bronchial asthma and treating effects of Zhichuan capsule].

OBJECTIVE: To explore the mechanism of the bronchial asthma and to study the treating effects of Zhichuan Capsule on the airway remodeling of asthmatic model rats. METHODS: The rat model was established by being sensitized and activated with different density of ovalbumin through prolonged and repeated exposure for 8 weeks. The rats were randomly divided into model group, Zhichuan Capsule treated group, dexameson treated group, and Zhichuan Capsule and dexameson treated group. Another group of normal rats were taken as control. General histological changes were observed by hematoxylin and eosin stained sections. Being standardized by internal perimeter (Pi), the wall thickness (d), internal area (Ai), outer area (Ao) and wall area (WA) of the airway were quantified by computer-assisted image analysis system. The express of MMP-9, TIMP-1, Col I, Col III and ColV in the airway were examined by immunocytochemical methods. During the course of airway remodeling, the dynamic changes of model rats were observed at different time points (2, 4, 6 and 8 weeks after the activating). Statistical comparison was performed by ANOVA followed by Fisher LSD test. RESULTS: (1) Histologic examination showed eosinophil infiltration within the airway walls, epithelial damage, excessive mucus in the lumen and edema in the submucosa of the airways in model rats, and that the collagen deposition increased accompanied by increasing of TIMP-1. In the model rats, MMP-9 increased at the time point of 2 weeks, but it decreased in the late stage (8 weeks after activating) of airway remodeling. And the level of TIMP-1 was far higher than MMP-9 at the time point of 8 weeks. (2) Zhichuan Capsule could down-regulate the level of TIMP-1 in the airway wall, as well as the thickness of airway wall and the collagen deposition. And there were progressing effects when it was used together with dexameson. CONCLUSION: (1) The early increase of MMP-9 is a key point to start remodeling; and the increase of TIMP-1 in the late stage, which inhibits collagenase activity, may play an important role in developing airway fibrosis. Imbalance between MMP-9 and TIMP-1 is a marker of airway remodeling. (2) Zhichuan Capsule can decrease the deposition of collagen and suppress the airway remodeling by inhibiting the TIMP-1 expression.

[Morphological changes following airway remodeling in asthmatic rats].

OBJECTIVE: To explore the establishment of an asthmatic model with airway remodeling in rats by observing the morphological changes of the airway in different stages. METHODS: Animals were divided into two groups: asthmatic group and control group. The subjects were observed at 5 phases: before activation and 2, 4, 6, 8 weeks after activation. General histological changes were observed using hematoxylin and eosin stained sections. Being standardized by internal perimeter (Pi), the wall thickness (d), internal area (Ai), outer area (Ao) and wall area (WA) of the airway were quantified by computer-assisted image analysis system. And the membrane airways were divided into 3 grades (large, media and small airways) to be analyzed. RESULTS: The changes of small airway appeared earlier and greater than the others. The changes of media airway appeared later than the small one. The changes of large airway appeared as later as the 4th week after activation. There were no differences before and after activation in control group (P>0.05). CONCLUSION: (1) Prolonged, repeated and low density Ag activation could establish asthmatic model with airway remodeling. (2) The morphological changes of the airways increased following airway remodeling, and the greatest increase occurred at small airway. (3)The morphological changes in the model rats were similar to that in human beings.

GM-CSF and IFN-gamma-induced expression of human leucocyte antigen class II molecules on basophils of umbilical cord blood.

AIM: To determine whether basophils expressed human leucocyte antigen (HLA) class II molecules. METHODS: Basophils in umbilical cord blood were separated and purified with methods of density gradient centrifugation and immunomagnetic microbeads. The isolated basophils were cultured in RPMI-1640 plus 10 % fetal calf serum at 37 in a humidified atmosphere with 5 % CO2 and stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interferon (IFN)-gamma for 20-60 h. The expression of HLA class II molecules on basophils was measured with fluorescence activated cell sorter (FACS). RESULTS: The purity of isolated basophils was > or = 83.5 %. After treated with GM-CSF 10 microg/L or IFN-gamma 100 kU/L, the expression of HLA class II molecules became detectable on membranous surface and at a higher level at 20 h, the percentage of expression was 10.2 %+/-2.1 % and 11.3 %+/-1.0 %, respectively. CONCLUSION: Basophils possessed the potency of HLA class II molecular expression.