OBJECTIVE: To investigate the effect of moxibustion on central insulin resistance related proteins of the rats suffering from diabetic cognitive decline, and analyze the underlying mechanism of moxibustion for cognition improvement. METHODS: Using the intraperitoneal injection of STZ combined with a high-fat diet, the rat model of diabetic cognitive decline were prepared. Twenty successfully-modeled rats were assigned randomly into a model group and a moxibustion group, 10 rats in each one. Besides, a blank group was set up with 10 rats collected. In the moxibustion group, suspending moxibustion was applied to "Baihui" (GV20), "Shenting" (GV24) and "Dazhui" (GV14) at the same time, 20 min in each intervention, once a day, and 6 interventions were delivered weekly and the duration of treatment was consecutive 4 weeks. The random blood glucose was measured using glucometer, and the learning-memory ability was detected by water maze test. HE staining was used to observe the morphology of neurons in the hippocampal tissue, real-time PCR assay was to detect mRNA expression of insulin receptor substrate 1 (IRS1), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) in the hippocampal tissue. The Western blot method was employed to detect the protein expression of IRS1, PI3K, AKT, phosphorylated IRS1 (p-IRS1), phosphorylated PI3K (p-PI3K) and phosphorylated AKT (p-AKT) in the hippocampal tissue, and the ratio of p-IRS1/IRS1, p-PI3K/PI3K and p-AKT/AKT was calculated separately. The immunofluorescence intensity of p-IRS1, p-PI3K, and p-AKT was measured using immunofluorescence. RESULTS: Compared with the blank group, the rats of the model group exhibited higher random blood glucose (P<0.001), longer escape latency (P<0.001), severe pathological damage in the hippocampus, lower mRNA expression of IRS1, PI3K, and AKT (P<0.001), reduced ratio of p-IRS1/IRS1, p-PI3K/PI3K and p-AKT/AKT (P<0.001), and declined immunofluorescence intensity of p-IRS1, p-PI3K, and p-AKT in the hippocampal tissue (P<0.001). In comparison with the model group, for the rats of the moxibustion group, the random blood glucose decreased (P<0.05), the escape latency was shortened (P<0.01), the hippocampal pathological damage was attenuated, the mRNA expression of IRS1, PI3K and AKT increased (P<0.01), the ratio of p-IRS1/IRS1, p-PI3K/PI3K and p-AKT/AKT was elevated (P<0.01, P<0.05), and the immunofluorescence intensity of p-IRS1, p-PI3K, and p-AKT in the hippocampal tissue was strengthened (P<0.01, P<0.05). CONCLUSION In diabetic rats experiencing cognitive decline, moxibustion can enhance the learning-memory ability, which may be attributed to modulating the protein expression of IRS1, PI3K, and AKT, and their phosphorylation, activating insulin signal transduction, and reducing central insulin resistance.
Animals ; Moxibustion ; Insulin Resistance ; Rats ; Male ; Insulin Receptor Substrate Proteins/genetics* ; Rats, Sprague-Dawley ; Humans ; Proto-Oncogene Proteins c-akt/genetics* ; Cognitive Dysfunction/genetics* ; Diabetes Mellitus, Experimental/therapy* ; Hippocampus/metabolism* ; Acupuncture Points ; Phosphatidylinositol 3-Kinases/genetics*

OBJECTIVE: To observe the effect of acupuncture on chondrocyte autophagy in rats of knee osteoarthritis (KOA) and explore its underlying mechanisms. METHODS: Forty male SPF-grade SD rats were randomized into a blank group, a model group, a suspension group, an acupuncture group, and a combined therapy group, 8 rats in each one. Except the blank group, KOA model was prepared by the injection with papain. The suspension exercise therapy (10 min each time, three times daily), acupuncture (at "Yanglingquan" [GB34], "Zusanli" [ST36], and "Dubi" [ST35] on the right side, 30 min each intervention, once daily) and the combined therapy (the suspension exercise therapy combined with acupuncture) were delivered in the suspension group, the acupuncture group and the combined therapy group, respectively. The intervention of each group was performed continuously for 6 days, and 4 consecutive weeks, at the interval of 1 day. Before and after intervention, Lequesne MG score was assessed in the rats. After intervention, HE staining was adopted to observe the cartilaginous tissue morphology of the right knee joints, and Mankin score was evaluated; the serum levels of interleukin (IL)-1β, IL-6, and tumor neurosis factor-α (TNF-α) were measured using ELISA; the real-time PCR was provided to determine the mRNA expression of collagen protein type Ⅱ(COL2), collagen protein type Ⅹ (COL10), phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and autophagy-regulated protein (Beclin-1) in the cartilaginous tissue of the right knee joint; Western blot was employed to detect the protein expression of PI3K, phosphorylated PI3K (p-PI3K), Akt, phosphorylated Akt (p-Akt), mTOR, phosphorylated mTOR (p-mTOR) and Beclin-1 in the cartilaginous tissue of the right knee joint. RESULTS: Compared with the blank group, the rats in the model group showed the higher Lequesne MG score (P<0.01), thinner cartilage of the right knee, reduced chondrocytes and disordered arrangement, and higher Mankin score (P<0.01). Besides, in the model group, the serum levels of IL-1β, IL-6 and TNF-α were elevated (P<0.01), the mRNA expression of COL2 and Beclin-1 and the protein expression of Beclin-1 decreased (P<0.01), the mRNA expression of COL10, PI3K, Akt and mTOR, and the protein expression of p-PI3K, p-Akt and p-mTOR increased (P<0.01) in the cartilaginous tissue of the right knee joint. Compared with the model group, in the suspension group, the acupuncture group and the combined therapy group, the Lequesne MG scores were reduced (P<0.01), the cartilage of the right knee was thickened, the arrangement of chondrocytes was improved, and the Mankin scores were lower (P<0.01). Besides, in these intervention groups, the serum levels of IL-1β, IL-6 and TNF-α were reduced (P<0.01), the mRNA expression of COL2 and Beclin-1 and the protein expression of Beclin-1 increased (P<0.05, P<0.01), the mRNA expression of COL10, PI3K, Akt and mTOR, and the protein expression of p-PI3K, p-Akt and p-mTOR decreased (P<0.05, P<0.01) in the cartilaginous tissue of the right knee joint. When compared with the suspension group and the acupuncture group, in the combined therapy group, the Lequesne MG score was reduced (P<0.01), and the Mankin score was reduced (P<0.05, P<0.01). Besides, the serum levels of IL-1β, IL-6 and TNF-α were reduced (P<0.05), the mRNA expression of COL2 and Beclin-1 and the protein expression of Beclin-1 increased (P<0.05), the mRNA expression of COL10, PI3K, Akt and mTOR, and the protein expression of p-PI3K, p-Akt and p-mTOR decreased (P<0.05, P<0.01) in the cartilaginous tissue of the right knee joint. CONCLUSION Acupuncture can promote cartilage regeneration of knee joint and autophagy in KOA rats, alleviate inflammation, so as to retard cartilage degeneration, which may be possibly associated with the PI3K/Akt/mTOR signaling pathway.
Animals ; Male ; Acupuncture Therapy ; Proto-Oncogene Proteins c-akt/genetics* ; TOR Serine-Threonine Kinases/genetics* ; Osteoarthritis, Knee/physiopathology* ; Phosphatidylinositol 3-Kinases/genetics* ; Rats ; Signal Transduction ; Rats, Sprague-Dawley ; Chondrocytes/metabolism* ; Humans ; Autophagy ; Acupuncture Points

OBJECTIVE: To explore the effect and mechanism of acupuncture at "Weizhong" (BL40) on microcirculation of paravertebral skeletal muscle in rats with chronic blunt injury of lumbar muscle based on the insulin-like growth factor-1 (IGF-1)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway. METHODS: Forty-eight SPF-grade SD rats were randomized into a blank group (8 rats) and a modeling group (40 rats). Chronic blunt injury model was established by weight impact method in the modeling group. Forty rats were successfully modeled, and were randomly divided into a model group, an acupuncture at Weizhong group (Weizhong group), an acupuncture at non-acupoint group (non-acupoint group), an inhibitor group, and an inhibitor+acupuncture at Weizhong group (inhibitor+Weizhong group), 8 rats in each group. In the Weizhong group and the inhibitor+Weizhong group, acupuncture was applied at bilateral "Weizhong" (BL40). In the non-acupoint group, acupuncture was applied at non-acupoints, i.e. points 0.5 cm inward from bilateral "Weizhong" (BL40). The acupuncture intervention was delivered 20 min each time, once a day for continuous 2 weeks. In the inhibitor group and the inhibitor+Weizhong group, intraperitoneal injection of IGF-1 receptor (IGF-1R) inhibitor was given once a day, at a dosage of 2 mg/100 g, for continuous 2 weeks. Before modeling and on the 1st, 7th and 14th days of intervention, the body mass was measured. Before and after modeling, and after intervention, the limb grip strength and paw withdrawal threshold (PWT) were measured. After intervention, the morphology of psoas muscle was observed by HE staining; the ultrastructure of psoas muscle capillaries was observed by electron microscopy; the levels of serum vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) were detected by ELISA; and the protein and mRNA expression of IGF-1, IGF-1R, PI3K, AKT of psoas muscle was detected by Western blot and real-time PCR. RESULTS: Compared with the blank group, in the model group, the body mass on the 7th and 14th days of intervention, the limb grip strength, and the PWT of left and right hind feet were decreased (P<0.001, P<0.01); the skeletal muscle cells showed enlarged intercellular space, loosely arranged and irregularly shaped, the capillaries in the psoas muscle tissues were edematous, and the lumen of the blood vessels was obviously atrophied; the levels of serum VEGF and eNOS were decreased (P<0.001); in psoas muscle, the protein expression of IGF-1 and IGF-1R, as well as the p-PI3K/PI3K, p-AKT/AKT values were decreased (P<0.001), the mRNA expression of IGF-1, IGF-1R, PI3K and AKT was decreased (P<0.001, P<0.05). Compared with the model group, in the Weizhong group, the body weight was increased on the 7th and 14th days of intervention (P<0.001), the limb grip strength and the PWT of the left and right hind feet were increased (P<0.001, P<0.01); the arrangement of the skeletal muscle cells was relatively tight and the intercellular space was reduced, the blood vessels tended to be regular and the structure of the basement membrane was continuous, while the lumens of blood vessels were collapsed locally; the levels of serum VEGF and eNOS were increased (P<0.001); in psoas muscle, the protein expression of IGF-1 and IGF-1R, as well as the p-PI3K/PI3K, p-AKT/AKT values were increased (P<0.001), the mRNA expression of IGF-1, IGF-1R, PI3K and AKT was increased (P<0.001, P<0.01). Compared with the model group, in the inhibitor group, the body mass was decreased on the 7th and 14th days of intervention (P<0.05, P<0.01); the limb grip strength and the PWT of the left hind foot were decreased (P<0.01, P<0.001); the intercellular space of skeletal muscle cells was larger, the nuclei of the cells and erythrocytes were scattered in the intercellular space, the damage of the capillaries in the muscular tissues was serious, the collagen fibers were sparsely distributed and disorganized; the levels of serum VEGF and eNOS were decreased (P<0.001, P<0.01); in psoas muscle, the protein expression of IGF-1 and IGF-1R, as well as the p-PI3K/PI3K and p-AKT/AKT values were decreased (P<0.01, P<0.05, P<0.001), the mRNA expression of IGF-1, IGF-1R, PI3K, and AKT was decreased (P<0.01, P<0.001, P<0.05). Compared with the Weizhong group, in the non-acupoint group and the inhibitor+Weizhong group, the body mass was decreased on the 7th and 14th days of intervention (P<0.001, P<0.01), the limb grip strength was decreased (P<0.001); the morphology of muscle cell was relatively poor, with generally irregular, there was mild collapse and atrophy in the vascular lumen, and mild edema in the endothelial cells; the levels of serum VEGF and eNOS were decreased (P<0.001); in psoas muscle, the protein expression of IGF-1 and IGF-1R, as well as the p-PI3K/PI3K and p-AKT/AKT values were decreased (P<0.01, P<0.001), the mRNA expression of IGF-1, IGF-1R, PI3K, and AKT was decreased (P<0.001, P<0.01, P<0.05). Compared with the Weizhong group, the PWT of the left hind foot was decreased in the non-acupoint group (P<0.001), and PWT of the left and right hind feet was decreased in the inhibitor+Weizhong group (P<0.001). CONCLUSION Acupuncture at "Weizhong" (BL40) promotes lumbar muscle repair in chronic low back pain, its mechanism may be related to the activation of the IGF-1/PI3K/AKT pathway, thereby improving the microcirculation.
Animals ; Insulin-Like Growth Factor I/genetics* ; Acupuncture Therapy ; Rats, Sprague-Dawley ; Rats ; Proto-Oncogene Proteins c-akt/genetics* ; Male ; Humans ; Muscle, Skeletal/metabolism* ; Signal Transduction ; Phosphatidylinositol 3-Kinases/genetics* ; Wounds, Nonpenetrating/metabolism* ; Acupuncture Points

BACKGROUND: G protein-coupled receptor kinase 2 (GRK2) could participate in the regulation of diverse cells via interacting with non-G-protein-coupled receptors. In the present work, we explored how paroxetine, a GRK2 inhibitor, modulates the differentiation and activation of immune cells in rheumatoid arthritis (RA). METHODS: The blood samples of healthy individuals and RA patients were collected between July 2021 and March 2022 from the First Affiliated Hospital of Anhui Medical University. C57BL/6 mice were used to induce the collagen-induced arthritis (CIA) model. Flow cytometry analysis was used to characterize the differentiation and function of dendritic cells (DCs)/T cells. Co-immunoprecipitation was used to explore the specific molecular mechanism. RESULTS: In patients with RA, high expression of GRK2 in peripheral blood lymphocytes, accompanied by the increases of phosphatidylinositol 3 kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR). In animal model, a decrease in regulatory T cells (T regs ), an increase in the cluster of differentiation 8 positive (CD8 + ) T cells, and maturation of DCs were observed. Paroxetine, when used in vitro and in CIA mice, restrained the maturation of DCs and the differentiation of CD8 + T cells, and induced the proportion of T regs . Paroxetine inhibited the secretion of pro-inflammatory cytokines, the expression of C-C motif chemokine receptor 7 in DCs and T cells. Simultaneously, paroxetine upregulated the expression of programmed death ligand 1, and anti-inflammatory cytokines. Additionally, paroxetine inhibited the PI3K-AKT-mTOR metabolic pathway in both DCs and T cells. This was associated with a reduction in mitochondrial membrane potential and changes in the utilization of glucose and lipids, particularly in DCs. Paroxetine reversed PI3K-AKT pathway activation induced by 740 Y-P (a PI3K agonist) through inhibiting the interaction between GRK2 and PI3K in DCs and T cells. CONCLUSION Paroxetine exerts an immunosuppressive effect by targeting GRK2, which subsequently inhibits the metabolism-related PI3K-AKT-mTOR pathway of DCs and T cells in RA.
G-Protein-Coupled Receptor Kinase 2/metabolism* ; Arthritis, Rheumatoid/immunology* ; Animals ; Dendritic Cells/metabolism* ; Paroxetine/therapeutic use* ; Proto-Oncogene Proteins c-akt/metabolism* ; Mice ; Humans ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Male ; Phosphatidylinositol 3-Kinases/metabolism* ; Lymphocyte Activation/drug effects* ; Female ; T-Lymphocytes/metabolism* ; Middle Aged

BACKGROUND: Arsenic trioxide (ATO) is indicated as a broad-spectrum medicine for a variety of diseases, including cancer and cardiac disease. While the role of ATO in hepatic ischemia/reperfusion injury (HIRI) has not been reported. Thus, the purpose of this study was to identify the effects of ATO on HIRI. METHODS: In the present study, we established a 70% hepatic warm I/R injury and partial hepatectomy (30% resection) animal models in vivo and hepatocytes anoxia/reoxygenation (A/R) models in vitro with ATO pretreatment and further assessed liver function by histopathologic changes, enzyme-linked immunosorbent assay, cell counting kit-8, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. Small interfering RNA (siRNA) for extracellular signal-regulated kinase (ERK) 1/2 was transfected to evaluate the role of ERK1/2 pathway during HIRI, followed by ATO pretreatment. The dynamic process of autophagic flux and numbers of autophagosomes were detected by green fluorescent protein-monomeric red fluorescent protein-LC3 (GFP-mRFP-LC3) staining and transmission electron microscopy. RESULTS: A low dose of ATO (0.75 μmol/L in vitro and 1 mg/kg in vivo ) significantly reduced tissue necrosis, inflammatory infiltration, and hepatocyte apoptosis during the process of hepatic I/R. Meanwhile, ATO obviously promoted the ability of cell proliferation and liver regeneration. Mechanistically, in vitro  studies have shown that nontoxic concentrations of ATO can activate both ERK and phosphoinositide 3-kinase-serine/threonine kinase (PI3K-AKT) pathways and further induce autophagy. The hepatoprotective mechanism of ATO, at least in part, relies on the effects of ATO on the activation of autophagy, which is ERK-dependent. CONCLUSION Low, non-toxic doses of ATO can activate ERK/PI3K-AKT pathways and induce ERK-dependent autophagy in hepatocytes, protecting liver against I/R injury and accelerating hepatocyte regeneration after partial hepatectomy.
Animals ; Arsenic Trioxide ; Autophagy/physiology* ; Reperfusion Injury/prevention & control* ; Mice ; Male ; Proto-Oncogene Proteins c-akt/physiology* ; Arsenicals/therapeutic use* ; Oxides/therapeutic use* ; Liver/metabolism* ; Extracellular Signal-Regulated MAP Kinases/metabolism* ; Mice, Inbred C57BL

BACKGROUND: Pancreatic cancer is a lethal malignancy prone to gemcitabine resistance. The single-strand selective monofunctional uracil DNA glycosylase (SMUG1), which is responsible for initiating base excision repair, has been reported to predict the outcomes of different cancer types. However, the function of SMUG1 in pancreatic cancer is still unclear. METHODS: Gene and protein expression of SMUG1 as well as survival outcomes were assessed by bioinformatic analysis and verified in a cohort from Fudan University Shanghai Cancer Center. Subsequently, the effect of SMUG1 on proliferation, cell cycle, and migration abilities of SMUG1 cells were detected in vitro . DNA damage repair, apoptosis, and gemcitabine resistance were also tested. RNA sequencing was performed to determine the differentially expressed genes and signaling pathways, followed by quantitative real-time polymerase chain reaction and Western blotting verification. The cancer-promoting effect of forkhead box Q1 (FOXQ1) and SMUG1 on the ubiquitylation of myelocytomatosis oncogene (c-Myc) was also evaluated. Finally, a xenograft model was established to verify the results. RESULTS: SMUG1 was highly expressed in pancreatic tumor tissues and cells, which also predicted a poor prognosis. Downregulation of SMUG1 inhibited the proliferation, G1 to S transition, migration, and DNA damage repair ability against gemcitabine in pancreatic cancer cells. SMUG1 exerted its function by binding with FOXQ1 to activate the Protein Kinase B (AKT)/p21 and p27 pathway. Moreover, SMUG1 also stabilized the c-Myc protein via AKT signaling in pancreatic cancer cells. CONCLUSIONS SMUG1 promotes proliferation, migration, gemcitabine resistance, and c-Myc protein stability in pancreatic cancer via protein kinase B signaling through binding with FOXQ1. Furthermore, SMUG1 may be a new potential prognostic and gemcitabine resistance predictor in pancreatic ductal adenocarcinoma.
Humans ; Pancreatic Neoplasms/pathology* ; Forkhead Transcription Factors/genetics* ; Signal Transduction/genetics* ; Animals ; Cell Line, Tumor ; Proto-Oncogene Proteins c-akt/metabolism* ; Cell Proliferation/physiology* ; Mice ; Uracil-DNA Glycosidase/genetics* ; Female ; Male ; Gemcitabine ; Mice, Nude ; Apoptosis/physiology* ; Deoxycytidine/analogs & derivatives* ; Cell Movement/genetics*

OBJECTIVES: To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction (BYHWT) on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients (FLS-RA) cultured under a hypoxic condition. METHODS: Forty normal Wistar rats were randomized into two groups (n=20) for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera. FLS-RA were cultured in routine condition or exposed to hypoxia (10% O₂) for 24 h wigh subsequent treatment with IL-1β, followed by treatment with diluted BYHWT-medicated serum (5%, 10% and 20%) or control serum. AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells, and the changes in ROS, ATP level, mitochondrial membrane potential and Ca²⁺ homeostasis were analyzed. The changes in mRNA and protein expressions of BNIP3, PI3K and AKT and mRNA expressions of LC3, Beclin-1 and P62 were detected using RT-qPCR and Western blotting. RESULTS: Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure. The treatment significantly decreased T-AOC concentration, increased ROS production, autophagosome formation and ATPase levels, and lowered mitochondrial membrane potential and Ca²⁺ level in the cells. In IL-1β-induced FLS-RA with hypoxic exposure, treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression, decreased the protein expressions of PI3K and AKT, increased the mRNA expressions of BNIP3 and P62, and lowered the mRNA expressions of PI3K, AKT, LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression. The cells treated with 5% and 10% BYHWT-medicated serum showed no significant changes in LC3 expression. CONCLUSIONS BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.
Drugs, Chinese Herbal/pharmacology* ; Arthritis, Rheumatoid/pathology* ; Animals ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Humans ; Fibroblasts/cytology* ; Rats, Wistar ; Membrane Proteins/metabolism* ; Rats ; Phosphatidylinositol 3-Kinases/metabolism* ; Mitochondria/metabolism* ; Cells, Cultured ; Proto-Oncogene Proteins/metabolism* ; Apoptosis/drug effects* ; Cell Hypoxia ; Synovial Membrane/cytology* ; Male ; Mitochondrial Proteins

OBJECTIVES: To investigate the mechanism mediating the regulatory effect of miR-155-5p on proliferation of human submandibular gland epithelial cells (HSGECs) in primary Sjogren's syndrome (pSS). METHODS: Dual luciferase reporter assay was used to verify the targeting relationship between miR-155-5p and the PI3K/AKT pathway. In a HSGEC model of pSS induced by simulation with TRAIL and INF-γ, the effects of miR-155-inhibitor-NC or miR-155 inhibitor on cell viability, cell cycle, apoptosis and proliferation were evaluated using CKK8 assay, flow cytometry and colony formation assay. ELISA and RT-PCR were used to detect the expressions of inflammatory cytokines and miR-155-5p mRNA in the cells; Western blotting was performed to detect the expressions of proteins in the PI3K/AKT signaling pathway. RESULTS: Dual luciferase assay showed that miR-155-5p targets the PI3K/AKT pathway via PIK3R1 mRNA. The HSGEC model of pSS showed significantly decreased cell viability, cell clone formation ability and expressions IL-10 and IL-4 and increased cell apoptosis, cell percentage in G2 phase, expressions of TNF‑α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-Akt/AKT ratio, and PIK3R1 protein expression. Treatment of the cell models with miR-155 inhibitor significantly increased the cell viability, G1 phase cell percentage, colony formation ability, and expressions of IL-10 and IL-4 levels, and obviously reduced cell apoptosis rate, G2 phase cell percentage, expressions of TNF-α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-AKT/AKT ratio, and PIK3R1 protein expression. CONCLUSIONS In HSGEC model of pSS, inhibition of miR-155-5p can promote cell proliferation and reduced cell apoptosis by targeting PI3K1 mRNA to negatively regulate the overexpression of PI3K/AKT signaling pathway.
Humans ; MicroRNAs/genetics* ; Cell Proliferation ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism* ; Sjogren's Syndrome/pathology* ; Epithelial Cells/cytology* ; Submandibular Gland/cytology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Apoptosis ; Class Ia Phosphatidylinositol 3-Kinase ; Cells, Cultured

OBJECTIVES: To investigate the therapeutic mechanism of Thesium chinense Turcz. (TCT) for antibiotic-associated diarrhea (AAD). METHODS: Network pharmacology, KEGG pathway enrichment analysis and molecular docking were used to identify the shared targets and genes of TCT and AAD, the key signaling pathways and the binding between the active components in TCT and the core protein targets. In a Kunming mouse model of AAD established by intragastric administration of lincomycin hydrochloride, the effects of daily gavage of 1% carboxymethyl cellulose sodium or TCT gel solutions at 1.5 g/kg and 3 g/kg (n=10) on body weight and diarrhea were observed. HE staining, ELISA, 16S rRNA sequencing, and Western blotting were used to examine pathologies, expression levels of IL-6 and TNF-α, changes in gut microbiota, and protein expressions of EGFR, p-EGFR, PI3K, p-PI3K, Akt, and p-Akt in the colon tissues of the mice. RESULTS: We identified a total of 66 active components of TCT and 68 core targets including EGFR, STAT3 and PIK3CA. KEGG pathway enrichment analysis suggested that the therapeutic effects of TCT was mediated primarily through the PI3K/Akt signaling pathway. Molecular docking showed that EGFR had the highest binding affinity with coniferin, and the EGFR-coniferin complex maintained a stable conformation at 10 ns, whose stability was also confirmed by Gibbs free energy analysis. In the mouse models of AAD, treatment with TCT significantly improved colonic tissue morphology, decreased colonic levels of TNF-α and IL-6, increased gut microbiota diversity, and modulated the relative abundances of the key genera including Lactobacillus and Bacteroides. TCT treatment also markedly reduced protein expressions of p-EGFR, p-PI3K and p-Akt in the colon tissues of the mice. CONCLUSIONS TCT can alleviate AAD in mice by modulating gut microbiota composition, regulating the EGFR/PI3K/Akt signaling pathway, and reducing TNF‑α and IL-6 expressions.
Animals ; Gastrointestinal Microbiome/drug effects* ; Signal Transduction/drug effects* ; Mice ; ErbB Receptors/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Diarrhea/drug therapy* ; Phosphatidylinositol 3-Kinases/metabolism* ; Anti-Bacterial Agents/adverse effects* ; Drugs, Chinese Herbal/therapeutic use* ; Molecular Docking Simulation

OBJECTIVES: To explore the molecular mechanism by which miR-124 affects cognitive function of sleep-deprived rats. METHODS: Fifty-four adult male SD rats were randomized into 6 groups (n=9), including a normal control group, a sleep deprivation (SD) model group, and 4 intracerebral microinjection groups in which the rats were subjected to stereotactic injection of miR-124 agomir, miR-124 agomir NC, miR-124 antagomir, or miR-124 antagomir into the lateral ventricle 7 days before SD modeling. The cognitive functions of the rats were evaluated with Morris water maze test, and pathological changes in the hippocampus were observed using HE staining. The expression level of miR-124 in hippocampal tissues of the rats was detected with qRT-PCR, and the expression level of apoptosis-related proteins and signaling pathway proteins were determined using Western blotting. RESULTS: In Morris water maze test, the SD rat models treated with miR-124 agomir showed a significantly shorter escape latency and fewer platform crossings with increased percentage of time and swimming distance in the fourth quadrant as compared with those in SD model group, while the rats treated with miR-124 antagomir exhibited worsened performance in the test. In the SD rat models, treatment with miR-124 agomir obviously lessened pathological changes in the hippocampus, while treatment with miR-124 antagomir significantly worsened the pathological changes. Compared with those in SD model group, the miR-124 agomir-treated rats showed an increased hippocampal expression of miR-124 with upregulated protein expressions of PI3K, p-AKT/AKT, and Bcl-2 and downregulated expressions of Bax and caspase-3 proteins, while rats treated with miR-124 antagomir showed significantly decreased hippocampal expression of miR-124 with lowered expressions of PI3K, p-AKT/AKT and Bcl-2 proteins and increased Bax and caspase-3 protein expressions. CONCLUSIONS High expression of miR-124 alleviates SD-induced cognitive decline and neuronal apoptosis in rats by activating the PI3K/AKT signaling pathway.
Animals ; MicroRNAs/metabolism* ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism* ; Male ; Rats, Sprague-Dawley ; Hippocampus/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Cognition ; Rats ; Sleep Deprivation/metabolism* ; Apoptosis ; Maze Learning