Qingfei Tongluo Formula Mitigates Mycoplasma pneumoniae Infection via the PERK Signaling Pathway.

Mycoplasma pneumoniae pneumonia (MPP) is usually found in school-aged children and relapses easily because of antibiotic resistance. The Qingfei Tongluo formula (QTF) is a clinically used traditional Chinese medicine to treat MPP. Our previous study demonstrated that QTF exhibited ameliorative effects on the experimental MPP mice model. In this study, the function and underlying QTF mechanism in MPP was attempted to be further explored. Mycoplasma pneumoniae (MP) was applied to infect A549 cells and BALB/c mice to mimic MPP in vitro and in vivo. Cytokine release and reactive oxygen species (ROS) production were analyzed using enzyme-linked immunosorbent assay (ELISA) assay and flow cytometry. Western blot analysis was used to detect the protein involved in ER stress. MP infection was found to enhance cytokine release and ER stress in vitro and in vivo, and this effect could be alleviated by QTF. Moreover, protein kinase RNA-like endoplasmic reticulum kinase (PERK) knockdown alleviated MP infection-induced cytokine release, ROS production, and ER stress in A549 cells while the PERK overexpression exhibited the opposite effects. In conclusion, QTF alleviated MP infection-induced cytokine release, ROS production, and ER stress via PERK signaling pathway inhibition.

Geraniol relieves mycoplasma pneumonia infection-induced lung injury in mice through the regulation of ERK/JNK and NF-κB signaling pathways.

BACKGROUND: Pneumonia is a serious pediatric lung injury disease caused by Mycoplasma pneumoniae (M. pneumoniae) with increasing global prevalence every year. The WHO has reported that nearly 19% of children die due to pneumonia worldwide. OBJECTIVE: The present research was conducted to discover the ameliorative properties of geraniol against M. pneumoniae-provoked pneumonia in mice through the modulation of inflammatory responses. METHODOLOGY: The pneumonia was provoked in the male Swiss albino mice via infecting animals with 100 µl of M. pneumoniae for 2 days and supplemented concurrently with 20 mg/kg of geraniol for 3 days. 100 mg/kg of azithromycin was used as a standard drug. The nitric oxide (NO) level and MPO activity were measured using kits. The SOD activity, GSH, and MDA levels were studied using standard methods. The polymerase chain reaction (PCR) study was performed to examine the M. pneumoniae DNA load. The inflammatory cytokines status was assessed by assay kits. The ERK1/2, JNK1/2, and NF-κB expressions were studied by reverse-transcription (RT-PCR). The lung tissues were analyzed microscopically to investigate the histological alterations. RESULTS: Geraniol treatment effectively reduced lung weight, NO level, and MPO activity in the pneumonia mice. The total cells and M. pneumoniae DNA load were also decreased by the geraniol. The SOD activity and GSH level were improved and MDA was decreased by the geraniol treatment. The IL-1, IL-6, IL-8, TNF-α, and TGF status were appreciably depleted by the geraniol in the pneumonia mice. Geraniol also suppressed the ERK1/2 and NF-κB expressions in the lung tissues. Histological findings also suggest the therapeutic roles of geraniol against pneumonia in mice. CONCLUSION: In summary, our results proved the beneficial roles of geraniol against the M. pneumoniae-provoked pneumonia. Geraniol could be a hopeful therapeutic agent to treat pneumonia in the future.

Proteomic and mechanistic study of Qingxuan Tongluo formula and curcumin in the treatment of Mycoplasma pneumoniae pneumonia.

OBJECTIVE: Mycoplasma pneumoniae (MP) is the only pathogen in the Mycoplasma family that can cause respiratory symptoms, including acute upper respiratory tract infection and bronchitis, which are often attributed to Mycoplasma pneumoniae pneumonia (MPP). MPP is one of the diseases that commonly affects the pediatric respiratory system, but its pathogenesis is unclear. This study investigated the therapeutic effects and mechanisms of Qingxuan Tongluo formula and its main component, curcumin, on MPP. METHODS: A mouse model of MPP was obtained by nasal drip of the MP strain. The effects of Qingxuan Tongluo formula and curcumin on the treatment of MPP were studied. The proteomic profiles of the alveolar lavage fluid of mice in the model group, Qingxuan Tongluo formula group and curcumin group were evaluated by LC-MS/MS. ELISA and immunohistochemistry were used to verify the possible presence of MP infection biomarkers and drug target proteins. RESULTS: Compared with the mice in the model group, the MPP mice in the Qingxuan Tongluo formula group had significantly reduced fever and cough and prolonged the cough incubation period. Moreover, the pulmonary pathology of the MPP mice was significantly improved, and the lung histopathological score was decreased. After treatment with Qingxuan Tongluo formula and curcumin, the functional and pathway abnormalities caused by MP were mainly inhibited. Levels of HSP90AA1, GRP94, ENO1 and PLG expression were verified by ELISA and immunohistochemistry. CONCLUSION: Qingxuan Tongluo formula significantly reduced fevers and cough and prolonged the cough incubation period of MPP mice. Qingxuan Tongluo formula and curcumin significantly improved the pathological changes in lung tissue caused by MP infection. Proteomics analyses indicated that Qingxuan Tongluo formula and curcumin may have therapeutic effects on MPP by regulating energy metabolism, relieving oxidative stress and activating the fibrinolytic system. ENO1 and PLG were found to be potential drug targets.

Polydatin suppresses the development of lung inflammation and fibrosis by inhibiting activation of the NACHT domain-, leucine-rich repeat-, and pyd-containing protein 3 inflammasome and the nuclear factor-κB pathway after Mycoplasma pneumoniae infection.

Mycoplasma pneumoniae (MP) can infect both the upper and lower respiratory tracts. Polydatin (PD), a traditional Chinese medicine, is known to have anti-inflammation and antifibrosis properties. However, the protective effects of PD against MP pneumonia (MPP) remain unclear. So, the aim of this study was to describe the therapeutic effects and underlying mechanisms of PD against MPP. BALB/c mice were assigned to three groups: a normal control group, MP infection group, or PD-treated MP infection group. BEAS-2B cells transfected with or without NACHT domain-, leucine-rich repeat-, and pyd-containing protein 3 (NLRP3) were used to confirm the protective mechanisms of PD. Immunohistochemical analysis, Western blot analysis, enzyme-linked immunosorbent assay, and flow cytometry were used in this study. The results showed that PD treatment suppressed MP-induced lung injury in mice by suppressing the expression of inflammatory factors and inhibiting the development of pulmonary fibrosis. Meanwhile, PD treatment inhibited activation of the NLRP3 inflammasome and nuclear factor κB (NF-κB) pathway. Overexpression of NLRP3 reversed the protective effect of PD against MP-induced injury of BEAS-2B cells. Taken together, these results indicate that PD treatment suppressed the inflammatory response and the development of pulmonary fibrosis by inhibiting the NLRP3 inflammasome and NF-κB pathway after MP infection.

[Serum metabonomics in mice infected with mycoplasma pneumoniae by UPLC-Q-TOF-MS].

Ultra high performance liquid chromatography coupled with tandem quadrupole time of flight mass spectrometry(UPLC-Q-TOF-MS) was applied to metabonomics study in BALB/c mice infected with mycoplasma pneumoniae(MP) to analyze the changes in serum endogenous metabolites, identify potential biomarkers associated with mycoplasma pneumoniae pneumonia(MPP), analyze the metabolic pathway and explore the pathogenic mechanism of MPP. The BALB/c mice were inoculated with MP by repeated intranasal infectious routes to establish MPP models, and the results of the lung tissue biopsy, IgM and mycoplasma nucleic acid content determination showed that the models of MP in BALB/c mice were successfully established. UPLC-Q-TOF-MS was used to analyze the serum metabolic profiling of BALB/c mice infected with MP, and then principal component analysis(PCA) was combined with orthogonal partial least squares discriminant analysis(OPLS-DA) for data processing. The results showed that there were significant differences in serum metabolic profile between the MP infected mice and the normal mice. Forty-seven potential biomarkers such as ornithine, cortisol, vitamin A and tryptophan were screened out by database searching and MS information matching. These potential biomarkers related to 17 metabolic pathways including retinol metabolism, arginine and proline metabolism, steroid hormone synthesis and so on. The metabonomic research method for serum of mice infected with mycoplasma pneumoniae based on UPLC-Q-TOF-MS was established in this study. The metabolic changes of endogenous small molecules in mice infected with MP were reflected in the overall level, laying the foundation for the selection and evaluation of MPP drugs.

Comparison of the ameliorative effects of Qingfei Tongluo formula and azithromycin on Mycoplasma pneumoniae pneumonia.

Mycoplasma pneumoniae pneumonia (MPP) is a common disease in children. Qingfei Tongluo formula (QTF) has been used for the treatment of MPP clinically, but the therapeutic effect remains unclear compared to conventional treatments with Western medicines. Therefore, the aim of this study was to assess changes in the expression levels of relevant factors associated with microcirculation after MPP and to compare the therapeutic effect of QTF with that of azithromycin (AZM) on experimental mice with MPP. A total of 174 children admitted with clinical diagnoses of pneumonia (80 MPP and 94 non-MPP) were used to identify differences in the expression patterns of factors in the microcirculation using an enzyme-linked immunosorbent assay. A BALB/c mouse model of MPP infection was established to determine the therapeutic effect of QTF. The results showed that the expression level of thrombomodulin (TM), vascular endothelial growth factor (VEGF), d-dimer (D-D), interleukin (IL)-6, and IL-10 were upregulated after MPP both clinically in children and in the mouse model. After 3 days of therapy, the amount of total MPP DNA decreased, especially in the mid- and high-dose QTF treatment groups. The expression levels of VEGF, IL-6, and IL-10 also decreased in response to treatment with QTF or AZM. However, there was no influence on D-D levels. QTF treatment also decreased TM expression. In conclusion, QTF treatment inhibited the progression of MPP, reduced vascular permeability, and improved pulmonary microcirculation more effectively than conventional treatment with Western medicine.

Ameliorative effects of Qingfei Tongluo formula on experimental mycoplasmal pneumonia in mice.

Mycoplasma pneumoniae pneumonia (MPP) is a common disease in children. Qingfei Tongluo formula (QTF) has been used for the treatment of MPP clinically, but the chemical constituents and mechanism involved remain unclear. This study aimed to analyze the main chemical constituents and to explore the possible mechanism of action associated with QTF treatment of MPP. Liquid chromatography-mass spectrometry was employed to identify the compounds contained in the QTF extract. A BALB/c mouse model of MP infection was established. After treatment with QTF (0.85 and 1.70 g/kg) for 3 days, hematoxylin and eosin staining was performed in lung tissues for histological examination. Inflammatory cytokines were detected by ELISA. Western blot analysis was used for detecting phosphorylated proteins involved in MAPK and nuclear factor (NF)-κB signaling pathways. In the mouse model, a large amount of pulmonary interstitial infiltration of lymphocytes and plasmacytes were seen as well as bronchus and vasodilation congestion. Following QTF treatment, inflammation was alleviated significantly compared with the model group. Inflammatory cytokines [interleukin (IL)-6, transforming growth factor-ß1, IL-8, IL-1ß and tumor necrosis factor-α] in bronchoalveolar lavage fluid were decreased dramatically. In addition, we found that QTF inhibited activation of phosphorylation of JNK, ERK and NF-κB. In conclusion, QTF alleviates MPP inflammation possibly via inhibitory activation of MAPK/NF-κB pathways, which can act as a new agent for MPP treatment.

[Effect of Huchang Qingfei concentrated pellets on E-cadherin expression in lung tissue of mice infected with Mycoplasma pneumoniae].

OBJECTIVE: To investigate the effects of Huchang Qingfei concentrated pellets on the expression of E-cadherin (E-cd) in the lung tissue from mice infected with Mycoplasma pneumoniae (MP). METHOD: A mice model of Mycoplasmal pneumonia (MPP) was developed by repeatedly intranasal infectious route. Transmission electronic microscope (TEM) and immunohistochemistry stain were performed to observe the pathological changes and expression of E-cd in lung tissues. RESULT: Under TEM it was found that the cellular membrane was ruptured, mitochondria was denatured, crista was broken in the pulmonary cells of the model group; the all above parameters in Huchang medicated group were improved obviously. The immunohistochemistry test showed that strong positive brown stain of E-cd expression was found in the pulmonary epithelial cell membrane and bronchial periphery in the model group, however, in the medicated group, the E-cd expression level in the cellular membrane was decreased and the expression ratio was dropped significantly as compared with the model controls. CONCLUSION: Huchang Qingfei concentrated pellets can inhibit the overexpression of E-cd in the lung tissue of mice with MP-infection, which may be helpful for prevention and treatment of pulmonary injury caused by MPP.

[Effect of selenium status on the morbidity of interstitial pneumonia in rats infected with mycoplasma].

To investigate the effect of selenium(Se) status on morbidity and process of interstitial pneumonia, we used the Wistar rats as the animal model infected with Mycoplasma Pneumonia (MP). The rats were maintained on the based diets with different doses of Se, infected with MP and exposed to sodium selenite(1.5-2.0 ml/day, containing Se 2 micrograms/ml) or 0.9% NaCl (1.5-2.0 ml/d) by gavage for different time. The experimental rats were divided into five groups, group A, feeding with normal control diet and without infected MP, group B with adequate Se diet and infected MP, group C with Se deficiency diet infected MP and added Se at the same time, group D with Se deficiency diet and added Se after suffered with MP, and group E with Se deficiency diet and infected MP. The results showed that the MP morbidity of the rats fed with high Se diet was significantly lower than that of the rats fed with low Se diet. The pathological change was significant in the low Se group and relatively mild in the high Se group. After infected with MP, the supplementation of sodium selenite to the low Se rats might decrease the MP morbidity and shorten the disease course compared with the group without supplementing Se. The incidence of myocarditis in the infected group with normal Se level diet or in the infected group with Se supplement was lower than that in the group without Se supplementation. The concentrations of Se in plasma and glutathione peroxidase in plasma and white blood cells decreased to a certain level after infection. The present study indicated a positive influence of Se supplement on the morbidity, disease course, and state of interstitial pneumonia in rats.