Pi-Pa-Run-Fei-Tang alleviates lung injury by modulating IL-6/JAK2/STAT3/IL-17 and PI3K/AKT/NF-κB signaling pathway and balancing Th17 and Treg in murine model of OVA-induced asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Pi-Pa-Run-Fei-Tang (PPRFT) is an empirical TCM prescription for treating asthma. However, the underlying mechanisms of PPRFT in asthma treatment have yet to be elucidated. Recent advances have revealed that some natural components could ameliorate asthma injury by affecting host metabolism. Untargeted metabolomics can be used to better understand the biological mechanisms underlying asthma development and identify early biomarkers that can help advance treatment. AIM OF THE STUDY: The aim of this study was to verification the efficacy of PPRFT in the treatment of asthma and to preliminarily explore its mechanism. MATERIALS AND METHODS: A mouse asthma model was built by OVA induction. Inflammatory cell in BALF was counted. The level of IL-6, IL-1ß, and TNF-α in BALF were measured. The levels of IgE in the serum and EPO, NO, SOD, GSH-Px, and MDA in the lung tissue were measured. Furthermore, pathological damage to the lung tissues was detected to evaluate the protective effects of PPRFT. The serum metabolomic profiles of PPRFT in asthmatic mice were determined by GC-MS. The regulatory effects on mechanism pathways of PPRFT in asthmatic mice were explored via immunohistochemical staining and western blotting analysis. RESULTS: PPRFT displayed lung-protective effects through decreasing oxidative stress, airway inflammation, and lung tissue damage in OVA-induced mice, which was demonstrated by decreasing inflammatory cell levels, IL-6, IL-1ß, and TNF-α levels in BALF, and IgE levels in serum, decreasing EPO, NO, and MDA levels in lung tissue, elevating SOD and GSH-Px levels in lung tissue and lung histopathological changes. In addition, PPRFT could regulate the imbalance in Th17/Treg cell ratios, suppress RORγt, and increase the expression of IL-10 and Foxp3 in the lung. Moreover, PPRFT treatment led to decreased expression of IL-6, p-JAK2/Jak2, p-STAT3/STAT3, IL-17, NF-κB, p-AKT/AKT, and p-PI3K/PI3K. Serum metabolomics analysis revealed that 35 metabolites were significantly different among different groups. Pathway enrichment analysis indicated that 31 pathways were involved. Moreover, correlation analysis and metabolic pathway analysis identified three key metabolic pathways: galactose metabolism; tricarboxylic acid cycle; and glycine, serine, and threonine metabolism. CONCLUSION: This research indicated that PPRFT treatment not only attenuates the clinical symptoms of asthma but is also involved in regulating serum metabolism. The anti-asthmatic activity of PPRFT may be associated with the regulatory effects of IL-6/JAK2/STAT3/IL-17 and PI3K/AKT/NF-κB mechanistic pathways.

[Effect of oblique needling at Ashi-point on expression of MyoD1/Pax7 in mice with quadriceps femoris injury].

OBJECTIVE: To observe the effect of oblique needling at Ashi-point on behavior, and cell morphology, myogenic differentiation antigen (MyoD1) and paired box transcription factor Pax7 (Pax7) of quadriceps femoris tissue in quadriceps femoris injured mice. METHODS: A total of 24 C57BL/6 male mice were randomly divided into control, model, shallow insertion and deep insertion groups, with 6 mice in each group. The quadriceps femoris injury model was established by single intramuscular injection of 0.5% bupivacaine (BPVC). Twenty-four hours after modeling, mice of the two acupuncture groups were received oblique needling on the surface or through the muscle belly of quadriceps femoris for once, the oblique needling was lifted and inserted 3 times. The climbing pole test was conducted 24 h after modeling and 24 h after EA. Histopathological changes of quadriceps femoris was observed by H.E. staining. The expressions of MyoD1 and Pax7 were detected by immunohistochemistry. RESULTS: Compared with the control group, the score of climbing pole test was lower (P<0.01), and the expressions of MyoD1 and Pax7 significantly increased (P<0.01) in the model group. After the intervention and compared with the model group, the score of climbing pole test was higher (P<0.01), and the expressions of MyoD1 and Pax7 obviously increased (P<0.01) in the two acupuncture groups. The therapeutic effect of deep insertion group was apparently superior to that of shallow insertion group in up-regulating the climbing pole test score and expressions of MyoD1 and Pax7 (P<0.05, P<0.01). H.E. stain showed large areas of inflammatory infiltration, muscle cells swelling, atrophy, rupture, degeneration and necrosis, different cell sizes and morphologies, enlarged intervals, nuclear aggregation, deep nuclear staining, nuclear pyknosis, and hemorrhage in the model group, which was relatively milder in both needling groups. CONCLUSION: Oblique needling at Ashi-point can effectively promote the benign repair of injured quadriceps muscle and promote the recovery of exercise ability in mice, which may be associated with its effect in up-regulating the expression of MyoD1 and Pax7 protein. The role of deep insertion is superior to that of shallow insertion.

Evaluation of thrombin inhibitory activity of catechins by online capillary electrophoresis-based immobilized enzyme microreactor and molecular docking.

An online capillary electrophoresis (CE)-based thrombin (THR) immobilized enzyme microreactor (IMER) method was established to screen THR inhibitors in this study. S-2366 was used as chromogenic substrate for determination of THR activity and other kinetic constants. After continuously run for 50 times, the prepared IMER could still remain 89% of the initial immobilized enzyme activity. The Michaelis-Menten constant (Km) of immobilized THR was measured as 0.514 mmol/L and the half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) of argatroban on THR were determined as 78.07 and 26.53 nmol/L, respectively, which indicated that CE-based THR IMER was successfully established and could be applied to screen THR inhibitors. Then the prepared IMER was used to investigate the inhibitory potency on THR of four main catechins in green tea including epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG). The results showed that ECG and EGCG had good THR inhibition activity and their inhibition rates at concentration of 200 µmol/L were 53.2 ±â€¯3.8% and 55.8 ±â€¯2.6%, respectively, which was in consistent with the results of microplate reader assay. Additionally, molecular docking results showed that the benzopyran groups of ECG and EGCG were inserted into the THR active pocket and interacted with residues LYS60F, TRP60D, TRY60A, IEU99, GLY216, HIS57 and SER195, but EC and EGC did not. Therefore, the developed CE-based THR IMER is reliable method for measuring THR inhibitory activity of natural inhibitors.