Tumor cell-intrinsic programmed death-ligand 1 (PD-L1) signals mediate tumor initiation, progression and metastasis, but their effects in ameloblastoma (AM) have not been reported. In this comprehensive study, we observed marked upregulation of PD-L1 in AM tissues and revealed the robust correlation between elevated PD-L1 expression and increased tumor growth and recurrence rates. Notably, we found that PD-L1 overexpression markedly increased self-renewal capacity and promoted tumorigenic processes and invasion in hTERT⁺-AM cells, whereas genetic ablation of PD-L1 exerted opposing inhibitory effects. By performing high-resolution single-cell profiling and thorough immunohistochemical analyses in AM patients, we delineated the intricate cellular landscape and elucidated the mechanisms underlying the aggressive phenotype and unfavorable prognosis of these tumors. Our findings revealed that hTERT⁺-AM cells with upregulated PD-L1 expression exhibit increased proliferative potential and stem-like attributes and undergo partial epithelial‒mesenchymal transition. This phenotypic shift is induced by the activation of the PI3K-AKT-mTOR signaling axis; thus, this study revealed a crucial regulatory mechanism that fuels tumor growth and recurrence. Importantly, targeted inhibition of the PD-L1-PI3K-AKT-mTOR signaling axis significantly suppressed the growth of AM patient-derived tumor organoids, highlighting the potential of PD-L1 blockade as a promising therapeutic approach for AM.
Ameloblastoma/metabolism* ; Humans ; B7-H1 Antigen/metabolism* ; Neoplasm Recurrence, Local/pathology* ; Signal Transduction ; Cell Proliferation ; Up-Regulation ; TOR Serine-Threonine Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Telomerase/metabolism* ; Jaw Neoplasms/metabolism* ; Epithelial-Mesenchymal Transition ; Animals ; Cell Line, Tumor ; Female ; Male

Psoriasis is a chronic inflammatory skin disease, and its pathogenesis is largely modulated by abnormal angiogenesis. Previous research has indicated that AlkB homolog 5 (ALKBH5), an important demethylase affecting N⁶-methyladenosine (m⁶A) modification, plays a role in regulating angiogenesis in cardiovascular and eye diseases. Our present study found that ALKBH5 was upregulated and co-localized with cluster of differentiation 31 (CD31) in the skin of IMQ group compared with control group. ALKBH5-deficient mice decreased IMQ-induced psoriatic dermatitis and exhibited histological improvements, including decreased epidermal thickness, hyperkeratosis, numbers of dermal capillary vessels and inflammatory cell infiltration. ALKBH5-KO mice alleviated angiogenesis in psoriatic lesions by downregulating the protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway. Additionally, the expression of ALKBH5 was significantly upregulated in IL-17A-induced human umbilical vein endothelial cells (HUVECs), which further promoted the expression of angiogenesis-related cytokines and endothelial cell proliferation. Cell proliferation and angiogenesis were suppressed in ALKBH5 knockdown group, whereas ALKBH5 overexpression promoted these processes. The regulation of angiogenesis in HUVECs by ALKBH5 was facilitated through the AKT-mTOR pathway. Collectively, ALKBH5 plays a pivotal role in psoriatic dermatitis and angiogenesis, which may offer a new potential targets for treating psoriasis.
Animals ; Psoriasis/chemically induced* ; Mice ; Humans ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells/metabolism* ; AlkB Homolog 5, RNA Demethylase/genetics* ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Cell Proliferation ; Mice, Knockout ; Disease Models, Animal ; Signal Transduction ; Male ; Skin/blood supply* ; Mice, Inbred C57BL ; Angiogenesis

OBJECTIVE: To investigate the effects of calcitonin gene-related peptide (CGRP) on autophagy in hypoxic/reoxygenated (H/R) cardiomyocytes and its relationship with the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway. METHODS: The rat cardiomyocyte cell line H9c2 was routinely cultured in vitro and passaged for experiments when the cells grew to 80% fusion. (1) CGRP dosage screening experiment: the cells were divided into blank control group, H/R group and different dosages of CGRP pretreatment groups. H9c2 cells were placed in a closed hypoxia chamber for 2 hours and then reoxygenated in a conventional incubator for 12 hours to prepare the H/R model. The CGRP pretreatment groups were pretreated with 0.01, 0.1, 0.5, 1, 5, and 10 μmol/L CGRP before the modeling process. The blank control group was not given any treatment. Cell counting kit-8 (CCK-8) was used to detect the cell survival rate, and the most suitable drug dosage was screened out. (2) Intervention experiment: H9c2 cells were divided into blank control group, H/R group, CGRP+H/R group, and CGRP+PI3K target inhibitor ly294002 (LY)+H/R group. H/R group was prepared as cellular H/R model. CGRP (1 μmol/L) alone or in combination with LY (10 μmol/L) was administered to CGRP+H/R group and CGRP+LY+H/R group, respectively, prior to the preparation of cellular H/R model. The blank control group was cultured routinely without treatment. The cell survival rate was detected by CCK-8. The level of lactate dehydrogenase (LDH) release was detected by colorimetric assay. The expressions of autophagy-related proteins [autophagy effector protein Beclin-1, microtubule-associated protein 1 light chain 3-II (LC3-II), autophagy protein p62] and PI3K/Akt/mTOR signaling pathway proteins [phosphorylated Akt (p-Akt), phosphorylated mTOR (p-mTOR)] were detected by Western blotting. RESULTS: (1) Results of CGRP dosage screening experiment: compared with the blank control group, the cell survival rate of the H/R group decreased significantly; and after giving 0.1, 0.5, 1, 5 μmol/L CGRP for pretreatment, the cell survival rate increased significantly, and intervention effect of 1 μmol/L CGRP was the best, and the difference was statistically significant when compared with that of the H/R group [(74.23±6.18)% vs. (23.43±4.09)%, P < 0.01], so it was used as the intervention dosage for the subsequent experiment. (2) Intervention experiment results: compared with the blank control group, the cell survival rate in the H/R group was significantly reduced, the level of LDH release was significantly increased, the protein expressions of Beclin-1 and LC3-II were significantly increased, and the protein expressions of p62, p-Akt and p-mTOR were significantly reduced, indicating that the death of cardiomyocytes occurred after the treatment of H/R and was accompanied by the elevation of autophagy level, and this process was associated with the activation of PI3K/Akt/mTOR signaling pathway. Compared with the H/R group, CGRP pretreatment increased cell survival rate [(76.02±2.43)% vs. (46.15±3.29)%, P < 0.01], decreased the level of LDH release (U/L: 169.83±11.65 vs. 590.17±34.50, P < 0.01), and down-regulated the protein expressions of Beclin-1 and LC3-II [Beclin-1 protein (Beclin-1/β-actin): 1.27±0.15 vs. 1.93±0.19, LC3-II protein (LC3-II/LC3-I): 1.27±0.13 vs. 1.98±0.18, both P < 0.01], up-regulated the protein expressions of p62, p-Akt, p-mTOR [p62 protein (p62/β-actin): 0.96±0.02 vs. 0.63±0.05, p-Akt protein (p-Akt/Akt): 0.76±0.04 vs. 0.48±0.02, p-mTOR protein (p-mTOR/mTOR): 1.13±0.09 vs. 0.68±0.15, all P < 0.05], suggesting that CGRP was able to reduce the H/R-induced cardiomyocyte injury, and this process was accompanied by a decrease in the level of cellular autophagy and activation of the PI3K/Akt/mTOR signaling pathway. Compared with the CGRP+H/R group, the cell survival rate was significantly lower than that in the CGRP+LY+H/R group [(56.95±6.63)% vs. (76.02±2.43)%, P < 0.01], LDH release level was significantly higher (U/L: 436.00±27.44 vs. 169.83±11.65, P < 0.01), and the protein expressions of Beclin-1 and LC3-II were significantly up-regulated [Beclin-1 protein (Beclin-1/β-actin): 1.63±0.12 vs. 1.27±0.15, LC3-II protein (LC3-II/LC3-I): 1.61±0.13 vs. 1.27±0.13, both P < 0.01], and significantly down-regulated p62, p-Akt, and p-mTOR protein expressions [p62 protein (p62/β-actin): 0.57±0.09 vs. 0.96±0.02, p-Akt protein (p-Akt/Akt): 0.45±0.01 vs. 0.76±0.04, p-mTOR protein (p-mTOR/mTOR): 0.66±0.06 vs. 1.13±0.09, all P < 0.05], suggesting that PI3K-targeted inhibitor was able to reverse the protective effect of CGRP on H/R cells. CONCLUSIONS CGRP pretreatment attenuated H/R-induced cardiomyocyte injury, increased cell survival rate, and reduced cellular LDH release. This effect may be achieved through inhibiting the activation of PI3K/Akt/mTOR signaling pathway.
Animals ; Myocytes, Cardiac/drug effects* ; Signal Transduction/drug effects* ; Rats ; TOR Serine-Threonine Kinases/metabolism* ; Calcitonin Gene-Related Peptide/pharmacology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Cell Line ; Cell Hypoxia ; Phosphatidylinositol 3-Kinases/metabolism*

OBJECTIVE: Glucocorticoid-induced osteoporosis (GIOP) is a common complication of prolonged glucocorticoid therapy. Chlorogenic acid (CGA), a polyphenol with antioxidant properties that is extracted from traditional Chinese medicines such as Eucommiae Cortex, has potential anti-osteoporotic activity. This study aimed to investigate the possible effects of CGA on GIOP in mice and murine long bone osteocyte Y4 (MLO-Y4) cells and explore the underlying molecular mechanisms. METHODS: The protective effects of CGA were initially evaluated in the GIOP mouse model induced by dexamethasone (Dex). The micro-computed tomography, hematoxylin-eosin staining, silver nitrate staining, and serum detection were used to assess the efficacy of CGA for improving bone formation in vivo. Then, network pharmacology analysis was used to predict the potential targets and molecular mechanisms underlying the therapeutic efficacy of CGA against GIOP. After that, 2',7'-dichlorofluorescein diacetate staining, flow cytometry, real-time quantitative reverse transcription polymerase chain reaction, and Western blotting were used to verify the mechanisms of CGA against GIOP in vitro. RESULTS: Animal experiments showed that CGA treatment effectively attenuated Dex-induced decreases in bone mass and strength and improved disrupted osteocyte morphology in mice. The protein-protein interaction analysis highlighted erb-b2 receptor tyrosine kinase (ERBB2), which is also known as human epidermal growth factor receptor 2 (HER2), caspase-3, kinase insert domain receptor, matrix metallopeptidase 9, matrix metallopeptidase 2, proto-oncogene tyrosine-protein kinase Src, and epidermal growth factor receptor as core targets. The Kyoto Encyclopedia of Genes and Genomes analysis revealed several significantly enriched pathways (P < 0.05), including the ERBB, phosphoinositide 3 kinase-AKT serine/threonine kinase 1 (AKT), and mechanistic target of rapamycin kinase (mTOR) pathways. Cellular experiments verified that CGA enhanced bone formation and promoted autophagy while inhibiting apoptosis in MLO-Y4 cells exposed to Dex, which was associated with the upregulated expression of HER2 and activation of the HER2/AKT/mTOR signaling pathway. CONCLUSION CGA exerted anti-osteoporotic effects against GIOP, partially through targeting osteocytes and modulating the HER2/AKT/mTOR signaling pathway. Please cite this article as: Xie AN, Zhang SZY, Zhang Y, Cao JL, Wang CL, Wang LB, Wu HJ, Zhang J, Dai WW. Chlorogenic acid mitigates glucocorticoid-induced osteoporosis via modulation of HER2/AKT/mTOR signaling pathway. J Integr Med. 2025; 23(6):670-682.
Animals ; Chlorogenic Acid/therapeutic use* ; Osteoporosis/metabolism* ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Mice ; Glucocorticoids/adverse effects* ; Receptor, ErbB-2/metabolism* ; Proto-Oncogene Mas ; Dexamethasone/adverse effects* ; Osteocytes/drug effects* ; Osteogenesis/drug effects* ; Male ; Cell Line ; Mice, Inbred C57BL ; Humans

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

The present study aimed to investigate the effects of eccentric treadmill exercise on adaptive hypertrophy of skeletal muscle in rats. Thirty-two 3-month-old Sprague Dawley (SD) rats were selected and randomly assigned to one of the four groups based on their body weights: 2-week quiet control group (2C), 2-week downhill running exercise group (2E), 4-week quiet control group (4C), and 4-week downhill running exercise group (4E). The downhill running protocol for rats in the exercise groups involved slope of -16°, running speed of 16 m/min, training duration of 90 min, and 5 training sessions per week. Twenty-four hours after the final session of training, all the four groups of rats underwent an exhaustion treadmill exercise. After resting for 48 h, all the rats were euthanized and their gastrocnemius muscles were harvested for analysis. HE staining was used to measure the cross-sectional area (CSA) and diameter of muscle fibers. Transmission electron microscope was used to observe the ultrastructural changes in muscle fibers. Purithromycin surface labeling translation method was used to measure protein synthesis rate. Immunofluorescence double labeling was used to detect the colocalization levels of lysosomal-associated membrane protein 2 (Lamp2)-leucyl-tRNA synthetase (LARS) and Lamp2-mammalian target of rapamycin (mTOR). Western blot was used to measure the protein expression levels of myosin heavy chain (MHC) IIb and LARS, as well as the phosphorylation levels of mTOR, p70 ribosomal protein S6 kinase (p70S6K), and eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1). The results showed that, compared with the 2C group rats, the 2E group rats showed significant increases in wet weight of gastrocnemius muscle, wet weight/body weight ratio, running distance, running time, pre- and post-exercise blood lactate levels, myofibrillar protein content, colocalization levels of Lamp2-LARS and Lamp2-mTOR, and LARS protein expression. Besides these above changes, compared with the 4C group, the 4E group further exhibited significantly increased fiber CSA, fiber diameter, protein synthesis rate, and phosphorylation levels of mTOR, p70S6K, and 4E-BP1. Compared with the quiet control groups, the exercise groups exhibited ultrastructural damage of rat gastrocnemius muscle, which was more pronounced in the 4E group. These findings suggest that eccentric treadmill exercise may promote mTOR translocation to lysosomal membrane, activating mTOR signaling via up-regulating LARS expression. This, in turn, increases protein synthesis rate through the mTOR-p70S6K-4E-BP1 signaling pathway, promoting protein deposition and inducing adaptive skeletal muscle hypertrophy. Although the ultrastructural changes of skeletal muscle are more pronounced, the relatively long training cycles during short-term exercise periods have a more significant effect on promoting gastrocnemius muscle protein synthesis and adaptive hypertrophy.
Animals ; Rats, Sprague-Dawley ; Physical Conditioning, Animal/physiology* ; Rats ; Muscle, Skeletal/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Male ; Hypertrophy ; Adaptation, Physiological/physiology* ; Adaptor Proteins, Signal Transducing/metabolism* ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism* ; Intracellular Signaling Peptides and Proteins

This study explores the mechanism of Guben Jiannao Liquid on Alzheimer's disease(AD) by regulating autophagy based on the liver kinase B1(LKB1)/adenosine monophosphate-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR) pathway. Male SD rats were randomly divided into the blank group, model group, low-dose and high-dose groups of Guben Jiannao Liquid, and rapamycin group, with 10 rats in each group. Except for the blank group, all other groups of rats were injected bilaterally in the hippocampus with β-amyloid(Aβ)_(1-42) to establish the AD model. The low-dose(6.21 g·kg~(-1)) and high-dose(12.42 g·kg~(-1)) groups of Guben Jiannao Liquid and rapamycin group(1 mg·kg~(-1)) were given the corresponding drugs by gavage, and the blank and model groups were given an equal volume of saline by gavage for four weeks. Morris water maze was used to test the learning and memory ability of rats in each group; hematoxylin-eosin(HE) and Nissl staining were used to observe the morphological and quantitative changes of neurons and Nissl bodies in the CA1 region of rat hippocampus; immunohistochemistry was utilized to detect Aβ-positive cell expression in the CA1 region of rat hippocampus; transmission electron microscopy was employed to observe ultrastructural changes in rat hippocampal tissue, and Western blot was used to examine the protein expression levels of LKB1, p-AMPK/AMPK, p-mTOR/mTOR, Beclin1, p62, and LC3-Ⅱ in the hippocampal tissue of the rats. The results showed that compared with those in the blank group, rats in the model group had elevated evasion latency and decreased number of platform transversal and residence time in the platform quadrant. The number of neurons in the hippocampal area was reduced, and the morphology was impaired. The average integral optical density value of Aβ-positive cells was elevated; the expression levels of LKB1, p-AMPK/AMPK, Beclin1, and LC3-Ⅱ were decreased, and the expression levels of p-mTOR/mTOR and p62 were increased. Compared with those in the model group, rats in the low-dose and high-dose groups of Guben Jiannao Liquid had shorter evasion latency, higher number of platform transversal, longer residence time in the platform quadrant, increased number of neurons, decreased expression of Aβ-positive cells and average integral optical density values, and increased number of autophagic lysosomes in hippocampal tissue. The expression levels of LKB1, Beclin1, and LC3-Ⅱ were elevated in the hippocampus of rats in the low-dose group of Guben Jiannao Liquid. The expression levels of LKB1, p-AMPK/AMPK, Beclin1, and LC3-Ⅱ were elevated in the hippocampal tissue of rats in the high-dose group of Guben Jiannao Liquid, and the expression levels of p-mTOR/mTOR and p62 were decreased. The findings suggest that Guben Jiannao Liquid can improve cognitive impairment in AD rats, and its mechanism of action may be related to the activation of the LKB1/AMPK/mTOR signaling pathway and the up-regulation of autophagy level.
Animals ; Alzheimer Disease/physiopathology* ; Male ; TOR Serine-Threonine Kinases/genetics* ; Autophagy/drug effects* ; Rats, Sprague-Dawley ; Protein Serine-Threonine Kinases/genetics* ; AMP-Activated Protein Kinases/genetics* ; Rats ; Drugs, Chinese Herbal/administration & dosage* ; Signal Transduction/drug effects* ; AMP-Activated Protein Kinase Kinases ; Humans ; Hippocampus/metabolism*

This study investigated the effects and action mechanism of tannins from Galla chinensis cream(TGCC) on the skin wound of rat tail. Male Sprague Dawley(SD) rats were randomly divided into a control group, model group, model+low-dose TGCC(50 mg per rat) group, model+high-dose TGCC group(100 mg per rat), and model+TGC+FAK inhibitor(Y15) cream(100 mg+10 mg per rat) group, with 10 rats in each group. After the rat tail skin injury model was successfully constructed, in the treatment group, corresponding drugs were applied to the wound surface, while in the control and model groups, the same amount of cream base as the TGCC group was applied by the same method. Then, sterile gauze was wrapped around the wound edge, and these operations were performed three times a day for 28 consecutive days. The wound healing status at the third, seventh, eleventh, fourteenth, twenty-first, and twenty-eighth days was recorded, and the wound healing rate and healing time were calculated. On the day after the last dose of medication, rat serum and tail skin wound tissue were collected for analyzing the activities of serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), creatinine(CREA), urea, reactive oxygen species(ROS), interferon gamma(IFN-γ), interleukin(IL)-1β, IL-6, IL-4, IL-10, tumor necrosis factor(TNF)-α, as well as catalase(CAT), glutathione(GSH), lactate dehydrogenase(LDH), malondialdehyde(MDA), myeloperoxidase(MPO), superoxide dismutase(SOD), total antioxidant capacity(T-AOC), platelet endothelial cell adhesion molecule-1(CD31), and leukocyte differentiation antigen 34(CD34) in the wound tissue of rat tail skin. Hematoxylin-eosin, Masson, and sirius red staining were used to observe the morphological changes in the wound tissue of rat tail skin. The thickness of the epidermis, the number of fibroblasts and blood vessels, and the contents of collagen fibers, typeⅠ collagen(COLⅠ), and COLⅢ were calculated. The mRNA expressions of keratin 10(KRT10), KRT14, vascular endothelial growth factor(VEGF), fibroblast growth factor(FGF), epidermal growth factor(EGF), CD31, CD34, matrix metallopeptidase-2(MMP-2), MMP-9, COLⅠ, COLⅢ, desmin, fibroblast specific protein 1(FSP1), IFN-γ, IL-1β, TNF-α, IL-4, IL-6, and IL-10 in skin wound tissue were determined by quantitative real-time polymerase chain reaction(PCR). Western blot was utilized to detect the protein expressions of KRT10, KRT14, VEGF, FGF, EGF, MMP-2, MMP-9, COLⅠ, COLⅢ, desmin, FSP1, focal adhesion kinase(FAK), phosphorylated focal adhesion kinase(p-FAK), phosphatidylin-ositol-3-kinase(PI3K), phosphorylated phosphatidylin-ositol-3-kinase(p-PI3K), protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), mammalian target of rapamycin(mTOR), and phosphorylated mammalian target of rapamycin(p-mTOR). The results manifest that TGCC can dramatically elevate the healing rate of rat tail wounds and shorten wound healing time. Besides, it can reduce serum ROS levels, the contents of MDA, MPO, and LDH in the rat skin wound tissue, as well as the serum IFN-γ, IL-1β, IL-6, and TNF-α levels and the mRNA expression levels of IFN-γ, IL-1β, IL-6, and TNF-α in the skin wound tissue. It can elevate the activities of CAT, GSH, SOD, and T-AOC in wound tissue, the IL-4 and IL-10 contents in serum, and the mRNA expressions of IL-4 and IL-10 in the wound tissue. In addition, TGGC can inhibit inflammatory cell infiltration and increase the epidermal thickness, counts of fibroblasts and blood vessels, and contents of collagen fibers, COLⅠ, and COLⅢ. Besides, TGCC can elevate the mRNA and protein expressions of epidermal differentiation markers(KRT10 and KRT14), endothelial cell markers(CD31 and CD34), angiogenesis and fibroblast proliferation, differentiation markers(VEGF, FGF, EGF, COLⅠ, COLⅢ, desmin, and FSP1), reduce the mRNA and protein expressions of gelatinases(MMP-2 and MMP-9), and increase protein expressions of p-FAK, p-PI3K, p-Akt, p-mTOR, as well as ratios of p-FAK/FAK, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR. These results suggest that TGCC can significantly facilitate skin wound healing, and its mechanism may be related to the activation of the FAK/PI3K/Akt/mTOR signaling pathway, inhibition of inflammatory cell infiltration in skin wound tissue, elevation of epidermal thickness, counts of fibroblasts and vessels, and contents of collagen fiber, COLⅠ, and COLⅢ, and reduction of MMP-2 and MMP-9 expressions, thus accelerating wound healing.
Animals ; Male ; Wound Healing/drug effects* ; Rats ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; TOR Serine-Threonine Kinases/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Skin/metabolism* ; Proto-Oncogene Proteins c-akt/genetics* ; Tannins/pharmacology* ; Humans ; Drugs, Chinese Herbal/administration & dosage* ; Focal Adhesion Kinase 1/genetics*

To investigate the mechanism of action of Syngnathus extract in treating knee osteoarthritis of rats, forty-eight male SD rats were randomly divided into the blank group, model group, positive drug group, as well as low-dose, medium-dose, and high-dose groups of Syngnathus extract. The rat model of knee osteoarthritis was constructed by intra-articular injection of sodium iodoacetate. After successful modeling, celecoxib(18 mg·kg~(-1)·d~(-1)) and Syngnathus extract(0.4, 0.8, and 1.6 g·kg~(-1)·d~(-1)) were given in different groups by gavage intervention for two weeks. Hematoxylin-eosin(HE) staining was used to observe the histopathological changes of cartilage in knee joints, and enzyme-linked immunosorbent assay(ELISA) was used to detect the expression level of inflammatory factors in serum. Real-time fluorescence quantitative PCR, Western blot, and immunohistochemistry were used to detect the levels of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target protein of rapamycin(mTOR) pathway-related mRNA and protein expression. The results showed that, comparied with the blank group, the cartilage surface of the knee joints of rats in the model group was uneven, with disorganized levels and defective cartilage tissue. The serum levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) and the mRNA levels of PI3K, Akt, and mTOR in cartilage tissue, as well as the protein expression levels of phosphorylated PI3K(p-PI3K)/PI3K, phosphorylated Akt(p-Akt)/Akt, phosphorylated mTOR(p-mTOR)/mTOR, and P62 were significantly increased. Beclin1 protein expression was decreased. Comparied with the model group, the number of chondrocytes in the knee joint of rats in each group of Syngnathus extract increased, and the arrangement of chondrocytes was relatively neat. The cartilage layer was restored, and the serum levels of IL-1β, IL-6, and TNF-α, as well as the mRNA expression levels of PI3K, Akt, and mTOR in cartilage tissue were significantly reduced. The protein expression levels of p-PI3K/PI3K, p-Akt/Akt, p-mTOR/mTOR, and P62 were significantly reduced in the rats in the middle-dose and high-dose groups of Syngnathus extract, and the Beclin1 protein expression was significantly increased. The protein expression levels of p-PI3K/PI3K, p-Akt/Akt, and P62 in rats in the low-dose group of Syngnathus extract were significantly reduced. In summary, Syngnathus extract may be used to treat knee osteoarthritis by inhibiting the expression of PI3K/Akt/mTOR signaling pathway, so as to alleviate the inflammatory response in the organism, enhance the autophagy activity of chondrocytes, and reduce the apoptosis of chondrocytes.
Animals ; TOR Serine-Threonine Kinases/genetics* ; Male ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/genetics* ; Rats ; Osteoarthritis, Knee/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Phosphatidylinositol 3-Kinases/genetics* ; Humans

This study aims to evaluate the therapeutic effect of Alpiniae Oxyphyllae Fructus-Saposhnikoviae Radix(AOF-SR) in a diabetic kidney disease(DKD) mouse model, explore its potential mechanism in regulating the NOD-like receptor protein 3(NLRP3) inflammasome via phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway, and provide new theoretical support for traditional Chinese medicine(TCM) intervention in DKD. Using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), the active ingredients and potential targets of AOF-SR were screened and its molecular mechanisms were investigated through molecular docking, molecular dynamics simulations, and experimental validation. The db/db mice were randomly divided into four groups: model group, low-dose AOF-SR group, high-dose AOF-SR group, and canagliflozin group. The db/m mice served as normal group. After one week of acclimatization, the mice underwent drug intervention. Starting from one week after treatment, body weight, blood glucose levels, and 24-hour urinary protein(24hUP) were measured every two weeks. After 13 weeks of administration, tissue collection and indicator detection were performed. Blood glucose, 24hUP, urinary microalbumin(mAlb), serum creatinine(Scr), and blood urea nitrogen(BUN) levels were determined. Pathological changes in kidney tissue were observed using hematoxylin-eosin(HE) staining. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of serum IL-1β, IL-18, and caspase-1, while RT-qPCR was employed to measure the mRNA expression levels of IL-1β, IL-18, caspase-1, and NLRP3. Western blot was used to assess the protein expression levels of NLRP3, PI3K, p-Akt, Akt, p-mTOR, and mTOR. Network pharmacology analysis indicated that wogonin, pinocembrin, hancinol, and kaempferol were the core compounds for drug treatment of the disease. Molecular docking and molecular dynamics simulations showed that core compounds, particularly wogonin, could specifically bind to PIK3R1, thereby regulating the PI3K/Akt/mTOR pathway. The experimental results indicated that both low and high doses of AOF-SR and canagliflozin significantly reduced blood glucose, 24hUP, mAlb, Scr, and BUN levels in db/db mice, while improving kidney pathological damage and inflammatory cell infiltration. Moreover, the treatments reduced the mRNA expression levels of caspase-1, IL-1β, and IL-18 in the kidneys of db/db mice, as well as the secretion of these factors in the serum. The drugs also inhibited the mRNA and protein expression levels of NLRP3 in the kidneys of db/db mice and decreased the protein levels of PI3K, p-Akt/Akt, and p-mTOR/mTOR. In conclusion, AOF-SR may improve kidney inflammation in DKD mice by regulating the PI3K/Akt/mTOR signaling pathway and inhibiting NLRP3 inflammasome activation.
Animals ; Mice ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Signal Transduction/drug effects* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Diabetic Nephropathies/metabolism* ; Inflammasomes/drug effects* ; Male ; Drugs, Chinese Herbal/chemistry* ; Humans ; Mice, Inbred C57BL