To explore the effects of apple polyphenols (AP) on lung function and immune imbalance in rats with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) by adjusting the miR-21-5p/SKP2 pathway.

The stable stage of COPD was established by cigarette smoking and endotoxin infusion, and then AECOPD rats were established by nasal infusion of Staphylococcus aureus bacterial solution. They were randomly assigned into the AECOPD group, the AP-25 group, the AP-100 group, the dexamethasone group, the AP-100+NC agomir group, and the AP-100+miR-21-5p agomir group. And rats intervened with normal saline were as the control group. Subsequently, tracheal intubation was performed to measure the peak expiratory and inspiratory flow rates (PEF, PIF), and the ratio of forced expiratory volume to forced vital capacity in 0.3 seconds (FEV0.3/FVC). Serum was collected to measure the levels of tumor necrosis factor -α (TNF-α) and interleukin-6 (IL-6). Bronchoalveolar lavage fluid (BALF) was recovered, and the numbers of neutrophils, lymphocytes and macrophages were determined. Left lung lobe sections were prepared, and lung tissue lesions and scores were observed. The right lung tissue of rats was taken. The proportions of Th17 and Treg cells, the mRNA expression of miR-21-5p and SKP2, and the protein expression of retinoic acid-related orphan receptor γt (RORγt), IL-17A, forkhead box protein P3 (Foxp3), IL-10 and SKP2 in the lung tissue were detected.

The AECOPD group had higher RORγt, IL-17A, miR-21-5p, TNF-α, IL-6, macrophages, neutrophils, lymphocytes, Th17 cell proportion, and Th17/Treg than the control group, and had lower Foxp3, IL-10, SKP2 mRNA and protein expression, Treg cell proportion, PEF, PIF, and FEV0.3/FVC than the control group (P<0.05). The AP-25 group, AP-100 group and dexamethasone group had lower RORγt, IL-17A, miR-21-5p, TNF-α, IL-6, macrophages, neutrophils, lymphocytes, Th17 cell proportion, and Th17/Treg than the AECOPD group, and had higher Foxp3, IL-10, SKP2 mRNA and protein expression, Treg cell proportion, PEF, PIF, and FEV0.3/FVC than the AECOPD group (P<0.05). In addition, miR-21-5p agomir reversed the protective effect of AP on AECOPD rats.

AP improves lung function and immune imbalance in AECOPD rats by inhibiting the miR-21-5p/SKP2 pathway.

To investigate the promoting effect and mechanism of hyperoside (Hyp) on wound healing in diabetic foot ulcer (DFU) rats by inhibiting the nuclear factor-kappa B (NF-κB) pathway and the Nod-like receptor family Pyrin domain-containing 3 (NLRP3) inflammasome.

A total of 50 SD rats were randomly divided into blank group, model group, metformin group, low-dose experimental group and high-dose experimental group, with 10 rats in each group. Fasting blood glucose (FBG), wound healing rate and transcutaneous oxygen partial pressure (TcpO₂) in the tissues surrounding the wounds were measured using a blood glucose meter. Pathological changes in the wound tissues were observed using hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of interleukin (IL)-8, IL-1β, tumor necrosis factor-alpha (TNF-α) and C-reactive protein (CRP). Western blotting was employed to assess the relative expression levels of matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), NF-κB p65, phosphorylated (p)-NF-κB p65 and NLRP3 proteins.

The FBG levels in blank, model, metformin, low-dose experimental and high-dose experimental groups were (5.31±0.91), (22.52±3.18), (11.27±2.35), (13.11±2.67) and (8.76±1.15) mmol·L^-1, respectively; the wound healing rates were (76.91±12.30)%, (9.53±1.01)%, (45.26±6.15)%, (39.94±5.47)% and (57.10±9.26)%, respectively; the TcpO₂ values were (41.09±8.51), (24.35±4.97), (33.27±5.68), (30.84±3.26), and (37.92±4.14) mmHg, respectively; the serum levels of IL-8 were (15.34±0.39), (41.92±3.12), (26.68±2.28), (30.55±3.27) and (17.95±2.70) ng·L^-1, respectively; IL-1β levels were (31.05±4.62), (64.39±7.14), (46.62±5.07), (50.21±3.92) and (39.54±4.25) ng·L^-1, respectively; TNF-α levels were (26.95±4.15), (69.74±7.43), (39.85±4.29), (45.08±4.36) and (35.52±2.95) ng·L^-1, respectively; and CRP levels were (0.69±0.08), (5.11±0.60), (3.23±0.58), (3.68±0.71) and (1.95±0.30) ng·L^-1, respectively; the protein relative expression levels of MMP-9 were 0.23±0.03, 0.99±0.11, 0.62±0.05, 0.74±0.06 and 0.39±0.04, respectively; the MMP-9/TIMP-1 ratios were (22.56±1.23)%, (97.07±5.67)%, (60.18±5.01)%, (70.49±5.98)% and (38.39±4.15)%, respectively; the p-NF-κB p65/NF-κB p65 protein relative expression levels were 0.21±0.02, 1.23±0.10, 0.51±0.04, 0.57±0.06 and 0.33±0.04, respectively; and the NLRP3 protein relative expression levels were 0.25±0.01, 0.93±0.07, 0.72±0.08, 0.80±0.06 and 0.35±0.04, respectively. Statistically significant differences were observed when comparing the model group with the blank group and the low- and high-dose experimental groups with the model group (all P<0.05).

Hyp promotes wound healing in DFU rats by inhibiting NF-κB and NLRP3 pathways, reducing inflammation and reducing MMP-9 expression.

To investigate the effect and underlying mechanism of Sijunzi Decoction in ameliorating insulin resistance (IR) within the pancreatic islets of rats with type 2 diabetes mellitus (T2DM).

A total of 40 Sprague-Dawley (SD) rats were randomly divided into control group (n=10, fed with normal diet) and high-sugar and high-fat group [n=30, fed with high-sugar and high-fat diet coupled with intraperitoneal injection of streptozotocin (STZ) to create a type 2 diabetes mellitus (T2DM) model]. The smoothly modeled SD rats were further randomly assigned into model group, positive control group (0.003 g·mL^-1) and experimental group (0.848 g·mL^-1). The normal group and model group were given distilled water by gavage, while the other two groups were administered the corresponding drugs by gavage for 4 consecutive weeks. Fasting blood glucose (FBG) was measured using a blood glucose meter. Serum levels of fasting insulin (FINS), total cholesterol (TC), total triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in rats of each group were detected with commercial assay kits. The pancreatic index (PI) was calculated based on the detection of pancreatic tissue. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to determine the mRNA expression levels of related proteins, and Western blotting was performed to ascertain the formulation standards of correlated total proteins.

The FBG, FINS, TC, TG, HDL-C, LDL-C and PI in the model group and experimental group were (13.60±0.61) mmol·L^-1 vs. (9.73±0.43) mmol·L^-1, (15.24±0.76) mIU·L^-1 vs. (12.39±1.13) mIU·L^-1, (5.42±0.41) mmol·L^-1 vs. (3.76±0.26) mmol·L^-1, (1.49±0.26) mmol·L^-1 vs. (0.65±0.05) mmol·L^-1, (0.82±0.06) mmol·L^-1 vs. (1.12±0.08) mmol·L^-1, (4.82±0.43) mmol·L^-1 vs. (3.02±0.42) mmol·L^-1, and (0.27±0.09)% vs. (0.34±0.02)%, respectively. The mRNA expression levels of INSR, IRS-1, PI3K, AKT, GLUT4, GSK-3β, and FOXO1 were 0.44±0.16 vs. 0.61±0.16, 0.48±0.13 vs. 0.72±0.16, 0.62±0.30 vs. 0.84±0.12, 0.74±0.20 vs. 0.92±0.02, 0.48±0.22 vs. 0.78±0.24, 1.90±0.16 vs. 1.56±0.25, and 1.83±0.16 vs. 1.60±0.21, respectively. The total protein expression levels of INSR, IRS-1, PI3K, AKT, GLUT4, GSK-3β, and FOXO1 were 0.59±0.29 vs. 0.82±0.18, 0.65±0.28 vs. 0.72±0.16, 0.49±0.14 vs. 0.74±0.15, 0.50±0.17 vs. 0.68±0.12, 0.65±0.28 vs. 0.86±0.14, 2.73±0.19 vs. 2.50±0.22, and 1.61±0.49 vs. 1.21±0.35, respectively. Compared with the model group, all the above indicators in the experimental group showed statistically significant differences (P<0.001, P<0.01, P<0.05).

Sijunzi Decoction can improve pancreatic insulin resistance (IR) in rats with type 2 diabetes mellitus (T2DM) by regulating the expression of 7 signaling factors in the upstream, middle and downstream of the PI3K/AKT signaling pathway.

To investigate the effect of sestrin 2 (SESN2) on JS-1 cell activation through unc-51-like kinase 1 (ULK1) and its mechanism.

The JS-1 cells were divided into seven groups: the control group (cultured under normal conditions), si-NC group (transfected with si-NC), the si-SESN2 group (transfected with si-SESN2), model group (cultured in medium containing 5 μg·L^-1 transforming growth factor-β1 for 24 h), model + oe-SESN2 group (transfected with oe-SESN2 on the basis of the model group), model + si-SESN2 group (transfected with si-SESN2 on the basis of the model group), and model+si-SESN2 + oe-ULK1 group (co-transfected with si-SESN2 and oe-ULK1 on the basis of the model group). The levels of fibrous actin (F-actin) was detected by immunofluorescence. Quantitative real time polymerase chain reaction was used to detect the ULK1 mRNA level. The relevtive expression levels of SESN2, light chain 3 Ⅱ/Ⅰ (LC3Ⅱ/Ⅰ), connective tissue growth factor (CTGF), collagen type I α1 (Col1a1), phospho-c-Jun N-terminal kinase (p-JNK), c-Jun N-terminal kinase (JNK), c-Jun and activating transcription factor 2 (ATF2) were detected by Western Blot. Cell proliferation rate was detected by cell counting kit-8. Transwell assesses the ability of cells to invade.

The relative expression levels of SESN2 protein in the control group, model group, model+oe-SESN2 group, and model+si-SESN2 group were 1.00±0.19, 2.57±0.49, 3.61±0.68, and 1.66±0.31, respectively; the relative fluorescence intensity of F-actin was 1.00±0.18, 4.57±0.92, 6.33±1.19, and 2.81±0.57, respectively; the relative expression levels of ULK1 mRNA were 1.00±0.15, 2.67±0.48, 3.52±0.66, and 1.95±0.37, respectively. The relative expression levels of LC3Ⅱ/Ⅰ protein in the control group, model group, model+oe-SESN2 group, model+si-SESN2 group, and model+si-SESN2+oe-ULK1 group were 1.00±0.20, 2.79±0.53, 3.57±0.68, 1.88±0.35, and 2.51±0.48, respectively; the cell proliferation rates were (100.00±2.23)%, (141.31±4.17)%, (156.52±8.84)%, (109.49±7.35)%, and (127.08±8.16)%, respectively; the number of cell invasions was 66.04±11.45, 137.84±21.73, 215.19±35.28, 75.46±13.51, and 124.27±19.50, respectively; the relative expression levels of CTGF protein were 1.00±0.17, 2.65±0.51, 3.77±0.72, 1.51±0.29, and 2.53±0.51, respectively; the relative expression levels of Col1a1 protein were 1.00±0.15, 2.93±0.57, 3.88±0.75, 1.61±0.31, and 2.72±0.52, respectively; the relative phosphorylation levels of JNK protein were 1.00±0.16, 2.21±0.42, 3.08±0.59, 1.46±0.29, and 2.04±0.37, respectively. Compared with the control group, the above indicators of the model group showed statistically significant differences; compared with the model group, the above indicators of the model+oe-SESN2 group or model+si-SESN2 group showed statistically significant differences; compared with the model+si-SESN2 group, the above indicators of the model+si-SESN2+oe-ULK1 group all showed statistically significant differences (P<0.05, P<0.01, P<0.001).

SESN2 activates ULK1, promotes JS-1 cell activation, promotes cell proliferation and invasion, and regulates cell homeostasis, possibly through the activation of JNK signaling pathway.

To evaluate the uncertainty for the determination of 10,11-dihydro-10-hydroxycarbazepine (MHD) in human plasma by ultra performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

An isotope-labeled internal standard was employed. Chromatographic separation was achieved within 3 min on a BEH C₁₈ column (2.1 mm×50.0 mm, 1.7 μm) using a gradient elution at a flow rate of 0.4 mL·min^-1, with a mobile phase consisting of methanol containing 0.1% formic acid and water containing 5% methanol plus 0.1% formic acid. Plasma sample concentrations were determined by UHPLC-MS/MS with electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode. The method was validated for selectivity, calibration curve, lower limit of quantification, precision, recovery, matrix effect, and stability. The influencing factors in the quantification of MHD were analyzed and evaluated to calculate the individual uncertainties, combined uncertainty, and finally the expanded uncertainty.

MHD demonstrated good linearity within the range of 0.03 to 6.00 mg·L^-1. The accuracy (bias from the theoretical concentration) for within-run and between-run analyses was within ±15%, and the precision (coefficient of variation) was within 13.32%. Plasma samples showed good stability under the following conditions: 9 hours at 25 ℃, 5 hours at 4 ℃, three freeze-thaw cycles, and storage at -80 ℃ for 18 days. Based on the calculated uncertainty values for each contributing factor, repeatability, matrix effect, and recovery represented the major sources of uncertainty for the low-concentration quality control (QC) sample, with values of 1.96×10^-2, 5.49×10^-2, and 7.25×10^-2, respectively. For the high-concentration QC sample, matrix effect and recovery contributed substantially to the overall uncertainty, with values of 4.92×10^-2 and 2.01×10^-2, respectively. The expanded uncertainties (P=95%, k=2) for MHD at low (0.09 mg·L^-1) and high (4.50 mg·L^-1) concentrations in human plasma were 1.62×10² mg·L^-1 and 0.48 mg·L^-1, respectively.

The measurement uncertainty of MHD concentration in human plasma determined by UHPLC-MS/MS is primarily contributed by repeatability, matrix effect and recovery at low concentration levels, while dominated by matrix effect and recovery at high concentrations

To investigate the mechanism of professor Fu Deyu’s core prescription, Danyu Hemai Granules (DYHMG), in treating hypertension using network pharmacology, and to conduct preliminary validation through molecular docking technology.

Active components and targets of Danyu Hemai Granules were screened with traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP), traditional Chinese medicine integrated database (TCMID), and HERB database. Hypertension-related targets were retrieved from GeneCards and OMIM. Intersection targets were identified, and protein-protein interaction (PPI) networks were constructed via STRING. The "Herb-Active Component-Target-Disease" network was visualized using Cytoscape to identify core targets. GO and KEGG pathway enrichment analyses were performed with Metascape. Finally, molecular docking validation was performed between key active components and core targets.

A total of 127 active ingredients and 312 targets of DYHMG, and 5 251 hypertension-related targets were identified, resulting in 229 common targets. Analysis suggested DYHMG exerts its antihypertensive effects primarily through active ingredients such as quercetin, kaempferol, luteolin, beta-sitosterol, stigmasterol, and wogonin. These ingredients acted on core targets including signal transducer and activator of transcription 3 (STAT3), RAC-alpha serine/threonine-protein kinase (AKT1), and estrogen receptor alpha (ESR1). Key pathways involved were lipid and atherosclerosis, the HIF-1 signaling pathway, insulin resistance, calcium signaling pathway, cAMP signaling pathway, MAPK signaling pathway, necroptosis, the renin-angiotensin system (RAS), and efferocytosis pathways.

Danyu Hemai Granules exhibits the characteristics of multi-component, multi-target, and multi-pathway in the treatment of hypertension. This study can provide ideas for the in-depth research on Danyu Hemai Granules in the treatment of hypertension and offer a scientific basis for its rational clinical application.