Mechanic study based on untargeted metabolomics of Pi-pa-run-fei-tang on pepper combined with ammonia induced chronic cough model mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Pi-pa-run-fei-tang (PPRFT), a traditional Chinese medicine formula with long-standing history, demonstrated beneficial effect on chronic cough. However, the mechanism underlying efficacy unclear. In current research, we explored the impact and molecular mechanism of chronic cough mouse stimulating with capsaicin combined with ammonia. AIM OF THE STUDY: To investigate the metabolic modulating effects, and potential mechanisms underlying the therapeutic effect of PPRFT in chronic cough. MATERIALS AND METHODS: Chronic cough mouse models were created by stimulating mice by capsaicin combined with ammonia. Number of coughs and cough latency within 2 min were recorded. With lung tissue and serum samples collected for histopathology, metabolomics, RT-qPCR, immunohistochemistry, and WB analysis. Lymphocytes were isolated and flow cytometric assays were conducted to evaluate the differentiation between Th17 and Treg cell among CD4+ cells. RESULTS: Results indicated that PPRFT obviously reduced the number of coughs, prolonged cough latency, reduced inflammatory cell infiltration and lung tissues damage, and decreased the serum level of IL-6, IL-1ß, TNF-α, and IL-17 while increasing IL-10 levels. Notably, PPRFT suppressed Th17 cell divergence and promoted Treg cell divergence. Furthermore, serum metabolomic assays showed that 46 metabolites differed significantly between group, with 35 pathways involved. Moreover, mRNA levels of IL-6, NF-κB, IL-17, RORγT, JAK2, STAT3, PI3K and AKT in lung tissues remarkably reduced and mRNA levels of IL-10 and FOXP3 were elevated after PPRFT pretreatment. Additionally, PPRFT treatments decreased the protein levels of IL-6, NF-κB, IL-17, RORγT, p-JAK2, p-STAT3, p-PI3K, and p-AKT and increased the protein levels of IL-10 and FOXP3, but no significantly effects to the levels on JAK2, STAT3, PI3K, and AKT in the lungs. CONCLUSION: Conclusively, our result suggested the effect with PPRFT on chronic cough may be mediated through IL-6/JAK2/STAT3 and PI3K/AKT/NF-κB pathway, which regulate the differentiation between Th17 and Treg cell. This beneficial effect of PPRFT in capsaicin and ammonia-stimulated chronic cough mice indicates its potential application in treating chronic cough.

Effect of Panax quinquefolius extract on Mycobacterium abscessus biofilm formation.

Mycobacterium abscessus (M. abscessus) can exist either as planktonic bacteria or as a biofilm. Biofilm formation is one of the important causes of conversion to resistance to antibiotics of bacteria that were previously sensitive when in their planktonic form, resulting in infections difficult to manage. Panax quinquefolius and its active ingredient ginsenosides have the potential ability in fighting pathogenic infections. In this study, the P. quinquefolius extract (PQE) showed good antibacterial/bactericidal activity against the M. abscessus planktonic cells. The extract reduced the biomass, thickness, and number of M. abscessus in the biofilm and altered its morphological characteristics as well as the spatial distribution of dead/alive bacteria. Moreover, the ginsenoside CK monomer had a similar inhibitory effect on M. abscessus planktonic bacteria and biofilm formation. Therefore, PQE and its monomer CK might be potential novel antimicrobial agents for the clinical prevention and treatment of M. abscessus, including biofilms in chronic infections.

Clinical Potential of Hypoxia Inducible Factors Prolyl Hydroxylase Inhibitors in Treating Nonanemic Diseases.

Hypoxia inducible factors (HIFs) and their regulatory hydroxylases the prolyl hydroxylase domain enzymes (PHDs) are the key mediators of the cellular response to hypoxia. HIFs are normally hydroxylated by PHDs and degraded, while under hypoxia, PHDs are suppressed, allowing HIF-α to accumulate and transactivate multiple target genes, including erythropoiesis, and genes participate in angiogenesis, iron metabolism, glycolysis, glucose transport, cell proliferation, survival, and so on. Aiming at stimulating HIFs, a group of small molecules antagonizing HIF-PHDs have been developed. Of these HIF-PHDs inhibitors (HIF-PHIs), roxadustat (FG-4592), daprodustat (GSK-1278863), vadadustat (AKB-6548), molidustat (BAY 85-3934) and enarodustat (JTZ-951) are approved for clinical usage or have progressed into clinical trials for chronic kidney disease (CKD) anemia treatment, based on their activation effect on erythropoiesis and iron metabolism. Since HIFs are involved in many physiological and pathological conditions, efforts have been made to extend the potential usage of HIF-PHIs beyond anemia. This paper reviewed the progress of preclinical and clinical research on clinically available HIF-PHIs in pathological conditions other than CKD anemia.

Melatonin induces the rejuvenation of long-term ex vivo expanded periodontal ligament stem cells by modulating the autophagic process.

BACKGROUND: Stem cells that have undergone long-term ex vivo expansion are most likely functionally compromised (namely cellular senescence) in terms of their stem cell properties and therapeutic potential. Due to its ability to attenuate cellular senescence, melatonin (MLT) has been proposed as an adjuvant in long-term cell expansion protocols, but the mechanism underlying MLT-induced cell rejuvenation remains largely unknown. METHODS: Human periodontal ligament stem cells (PDLSCs) were isolated and cultured ex vivo for up to 15 passages, and cells from passages 2, 7, and 15 (P2, P7, and P15) were used to investigate cellular senescence and autophagy change in response to long-term expansion and indeed the following MLT treatment. Next, we examined whether MLT could induce cell rejuvenation by restoring the autophagic processes of damaged cells and explored the underlying signaling pathways. In this context, cellular senescence was indicated by senescence-associated ß-galactosidase (SA-ß-gal) activity and by the expression of senescence-related proteins, including p53, p21, p16, and γ-H2AX. In parallel, cell autophagic processes were evaluated by examining autophagic vesicles (by transmission electronic microscopy), autophagic flux (by assessing mRFP-GFP-LC3-transfected cells), and autophagy-associated proteins (by Western blot assay of Atg7, Beclin-1, LC3-II, and p62). RESULTS: We found that long-term in vitro passaging led to cell senescence along with impaired autophagy. As expected, MLT supplementation not only restored cells to a younger state but also restored autophagy in senescent cells. Additionally, we demonstrated that autophagy inhibitors could block MLT-induced cell rejuvenation. When the underlying signaling pathways involved were investigated, we found that the MLT receptor (MT) mediated MLT-related autophagy restoration by regulating the PI3K/AKT/mTOR signaling pathway. CONCLUSIONS: The present study suggests that MLT may attenuate long-term expansion-caused cellular senescence by restoring autophagy, most likely via the PI3K/AKT/mTOR signaling pathway in an MT-dependent manner. This is the first report identifying the involvement of MT-dependent PI3K/AKT/mTOR signaling in MLT-induced autophagy alteration, indicating a potential of autophagy-restoring agents such as MLT to be used in the development of optimized clinical-scale cell production protocols.

Oral Exposure to ZnO Nanoparticles Disrupt the Structure of Bone in Young Rats via the OPG/RANK/RANKL/IGF-1 Pathway.

PURPOSE: To evaluate the effects of ZnO NPs on bone growth in rats and explore the possible mechanisms of action. MATERIALS AND METHODS: Three-week-old male rats received ultrapure water or 68, 203, and 610 mg/kg zinc oxide nanoparticles (ZnO NPs) for 28 days, orally. RESULTS: The high-dosage groups caused significant differences in weight growth rate, body length, and tibia length (P<0.05), all decreasing with increased ZnO NP dosage. There were no significant differences in body mass index (BMI) (P>0.05). The zinc concentration in liver and bone tissue increased significantly with increased ZnO NP dosage (P<0.05). Clearly increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were observed in the 610 mg/kg ZnO NP group (P>0.05), whereas alkaline phosphatase (ALP) increased in the 610 mg/kg ZnO NP group (P<0.05). Significant differences in insulin-like growth factor type 1 (IGF-1) levels and a decrease in calcium (Ca) levels were observed in 203 and 610 mg/kg ZnO NP groups (P<0.05). Phosphorus (P) levels increased and the Ca/P ratio decreased in the 610 mg/kg ZnO NP group (P<0.05). Micro-computed tomography (micro-CT) of the tibia demonstrated signs of osteoporosis, such as decreased bone density, little trabecular bone structure and reduced cortical bone thickness. Micro-CT data further demonstrated significantly decreased bone mineral density (BMD), trabecular number (Tb.N), and relative bone volume (BV/TV) with increasing dosage of ZnO NPs. Osteoprotegerin (OPG) expression and the ratio of OPG to receptor activator of nuclear factor-κB ligand (RANKL) were statistically lower in the 610 mg/kg ZnO NP group (P<0.05), whereas RANKL expression did not change significantly (P>0.05). CONCLUSION: We infer that ZnO NPs affect bone growth in young rats directly or indirectly by altering IGF-1 levels. Overall, the results indicate that ZnO NPs promote osteoclast activity and increase bone loss through the OPG/RANK/RANKL/IGF-1 pathway.

Triterpenes possessing an unprecedented skeleton isolated from hydrolyzate of total saponins from Gynostemma pentaphyllum.

Five novel triterpenes were isolated from hydrolyzate of total saponins from Gynostemma pentaphyllum and identified as gypensapogenin A (1), gypensapogenin B (2), gypensapogenin C (3), 3-O-ß-d-glucopyranosyl-gypensapogenin D (4) and gypensapogenin D (5), two of which (1 and 2) possess unprecedented ring A. The cyclization of the side chains-3 formed five-membered cyclic ketone rings similar to ring E in 1. The 21-oic acid-21, 23-lactone was present in the side chains of 4 and 5. We also proposed the possible formation mechanisms of compounds 1-3. Compounds 1-5 were evaluated for cytotoxic activities in three cell lines including A549, U87 and Hep3B and compound 3 showed significant activities toward A549 and U87 human cancer cells (with IC 50 values at 0.11 and 0.58 µm respectively).