OBJECTIVE: To observe the effects of the "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture on key regulatory factors during mitochondrial apoptosis of testicular tissue in asthenozoospermia mice, and explore the potential mechanism of the protective effect of acupuncture on reproductive function. METHODS: Thirty C57BL/6 male mice were randomly divided into a blank group, a model group and an acupuncture group, 10 mice in each group. In the model and the acupuncture groups, the intraperitoneal injection of cyclophosphamide (30 mg•kg^-1•d^-1) was delivered for 7 days to prepare the asthenozoospermia model. After the success of modeling, the modeled mice in the acupuncture group were intervened with "Zhibian" (BL54)-toward-"Shuidao" (ST28) acupuncture, once daily and the needles were retained for 20 min. The duration of the intervention was 2 weeks. The general condition of each mouse was observed, and the body mass was recorded before modeling, after modeling and after intervention completion. After intervention, the testicular mass was recorded and the weight coefficient was calculated, and the mouse sperm quality was examined; the serum contents of testosterone (T), follicle stimulating hormone (FSH) and luteinizing hormone (LH) were detected using ELISA, the morphology of testicular tissue was observed using HE, the mitochondrial ultra-microstructure of testicular tissue was observed under transmission electrone microscopy, the mitochondrial membrane potential level of testicular tissue was detected using JC-1 staining, the positive rate of apoptosis cell of testicular tissue was observed using TUNEL; and the mRNA and protein expression of b-cell lymphocytoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), cytochrome c (Cyt C), apoptotic protease-activating factor1 (Apaf-1), Caspase-9 and Caspase-3 of testicular tissue was detected using real-time quantitative fluorescence PCR and Western blot methods separately; and the positive expression of Cleaved Caspase-3 of the testicular tissue was detected using immunohistochemistry. RESULTS: Compared with the blank group, the mice were in listless spirits, had shaggy hairs, the reduced appetite and movement, and weight loss in the model group (P<0.01); the testicular mass and the weight coefficient decreased (P<0.01); the total number of sperms, sperm motility, and sperm viability were declined (P<0.01); while the levels of serum T, FSH, and LH were dropped (P<0.01). The morphology of seminiferous tubules in testicular tissue was abnormal, the number of spermatogenic cells and the number of mitochondria decreased, the inner mitochondrial crest was fractured and lost, and vacuoles appeared. The level of mitochondrial membrane potential was reduced (P<0.01); and the positive rate of apoptosis cell in testicular tissue increased (P<0.01). The mRNA and protein expression of Bax, Cyt C, Apaf-1, Caspase-9 and Caspase-3 was elevated (P<0.01, P<0.05), the mRNA and protein expression of Bcl-2 was dropped (P<0.01), and the average absorbance value of Cleaved Caspase-3 increased (P<0.01). When compared with the model group, in the acupuncture group, the general condition of mice was improved, the testicular mass and the weight coefficient elevated (P<0.01); the total number of sperms, sperm motility, and sperm viability increased (P<0.01); while the levels of serum T, FSH, and LH rose (P<0.01). The pathological morphology of testicular tissue and the inner mitochondrial ultra-microstructure were ameliorated, the level of mitochondrial membrane potential was elevated (P<0.01); the positive rate of apoptosis cell was reduced (P<0.01). The mRNA and protein expression of Bax, Cyt C, Apaf-1, Caspase-9 and Caspase-3 was dropped (P<0.01, P<0.05), the mRNA and protein expression of Bcl-2 elevated (P<0.05), and the average absorbance value of Cleaved Caspase-3 declined (P<0.01). CONCLUSION "Zhibian" (BL54)-toward- "Shuidao" (ST28) acupuncture may ameliorate mouse reproductive function by inhibiting mitochondrial apoptosis pathway, alleviating testicular tissue damage in the asthenospermia mice induced by cyclophosphamide.
Animals ; Male ; Mice ; Apoptosis ; Acupuncture Therapy ; Mitochondria/metabolism* ; Asthenozoospermia/genetics* ; Humans ; Testis/metabolism* ; Mice, Inbred C57BL ; Spermatozoa/metabolism* ; Acupuncture Points ; Sperm Motility ; Testosterone/blood* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; Caspase 3/genetics* ; Follicle Stimulating Hormone/blood* ; Reproduction ; Cytochromes c/genetics* ; bcl-2-Associated X Protein/genetics* ; Apoptotic Protease-Activating Factor 1/genetics*

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: 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*

The Kirsten rat sarcoma viral oncogene homolog ( KRAS ) mutation is one of the most prevalent activating alterations in cancer. It indicates a poor overall prognosis due to its highly invasive nature. Although several KRAS inhibitors have been developed in recent years, a significant clinical challenge has emerged as a substantial proportion of patients eventually develop resistance to these therapies. Therefore, identifying determinants of drug resistance is critical for guiding treatment strategies. This review provides a comprehensive overview of the mutation landscape and molecular mechanisms of KRAS activity in various cancers. Meanwhile, it summaries the progress and prospects of small molecule KRAS inhibitors undergoing clinical trials. Furthemore, this review explores potential strategies to overcome drug resistance, with the ultimate goal of steering toward patient-centric precision oncology in the foreseeable future.
Humans ; Drug Resistance, Neoplasm/drug effects* ; Proto-Oncogene Proteins p21(ras)/metabolism* ; Mutation/genetics* ; Neoplasms/genetics* ; Antineoplastic Agents/therapeutic use*

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*

This study aimed to explore the therapeutic mechanisms of Colquhounia Root Tablets(CRT) in treating diabetic kidney disease(DKD) by integrating biomolecular network mining with animal model verification. By analyzing clinical transcriptomics data, an interaction network was constructed between candidate targets of CRT and DKD-related genes. Based on the topological eigenvalues of network nodes, 101 core network targets of CRT against DKD were identified. These targets were found to be closely related to multiple pathways associated with type 2 diabetes, immune response, and metabolic reprogramming. Given that immune-inflammatory imbalance driven by metabolic reprogramming is one of the key pathogenic mechanisms of DKD, and that many core network targets of CRT are involved in this pathological process, receptor for advanced glycation end products(RAGE)-reactive oxygen species(ROS)-phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT)-nuclear factor-κB(NF-κB)-NOD-like receptor family pyrin domain containing 3(NLRP3) signaling axis was selected as a candidate target for in-depth research. Further, a rat model of DKD induced by a high-sugar, high-fat diet and streptozotocin was established to evaluate the pharmacological effects of CRT and verify the expression of related targets. The experimental results showed that CRT could effectively correct metabolic disturbances in DKD, restore immune-inflammatory balance, and improve renal function and its pathological changes by inhibiting the activation of the RAGE-ROS-PI3K-AKT-NF-κB-NLRP3 signaling axis. In conclusion, this study reveals that CRT alleviates the progression of DKD through dual regulation of metabolic reprogramming and immune-inflammatory responses, providing strong experimental evidence for its clinical application in DKD.
Animals ; Diabetic Nephropathies/metabolism* ; Receptor for Advanced Glycation End Products/genetics* ; NF-kappa B/genetics* ; Signal Transduction/drug effects* ; Rats ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics* ; Proto-Oncogene Proteins c-akt/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Phosphatidylinositol 3-Kinases/genetics* ; Reactive Oxygen Species/metabolism* ; Humans ; Plant Roots/chemistry* ; Rats, Sprague-Dawley ; Tablets/administration & dosage*

This study aimed to investigate the effect of Dahuang Zhechong Pills(DHZCP) in delaying heart aging(HA) and explore the potential mechanism. Network pharmacology and molecular docking were employed to explore the targets and potential mechanisms of DHZCP in delaying HA. Furthermore, in vitro experiments were conducted with the DHZCP-containing serum to verify key targets and pathways in D-galactose(D-gal)-induced aging of cardiomyocytes. Active components of DHZCP were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCSMP), and relevant targets were predicted. HA-related targets were screened from the GeneCards, Online Mendelian Inheritance in Man(OMIM), and DisGeNET. The common targets shared by the active components of DHZCP and HA were used to construct a protein-protein interaction network in STRING 12.0, and core targets were screened based on degree in Cytoscape 3.9.1. Metaspace was used for Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses of the core targets to predict the mechanisms. Molecular docking was performed in AutoDock Vina. The results indicated that a total of 774 targets of the active components of DHZCP and 4 520 targets related to HA were screened out, including 510 common targets. Core targets included B-cell lymphoma 2(BCL-2), serine/threonine kinase 1(AKT1), and hypoxia-inducible factor 1 subunit A(HIF1A). The GO and KEGG enrichment analyses suggested that DHZCP mainly exerted its effects via the phosphatidylinositol 3-kinase(PI3K)/AKT signaling pathway, HIF-1α signaling pathway, longevity signaling pathway, and apoptosis signaling pathway. Among the pathways predicted by GO and KEGG enrichment analyses, the PI3K/AKT/HIF-1α signaling pathway was selected for verification. The cell-counting kit 8(CCK-8) assay showed that D-gal significantly inhibited the proliferation of H9c2 cells, while DHZCP-containing serum increased the viability of H9c2 cells. SA-β-gal staining revealed a significant increase in the number of blue-green positive cells in the D-gal group, which was reduced by DHZCP-containing serum. TUNEL staining showed that DHZCP-containing serum decreased the number of apoptotic cells. After treatment with DHZCP-containing serum, the protein levels of Klotho, BCL-2, p-PI3K/PI3K, p-AKT1/AKT1, and HIF-1α were up-regulated, while those of P21, P16, BCL-2 associated X protein(Bax), and cleaved caspase-3 were down-regulated. The results indicated that DHZCP delayed HA via multiple components, targets, and pathways. Specifically, DHZCP may delay HA by reducing apoptosis via activating the PI3K/AKT/HIF-1α signaling pathway.
Proto-Oncogene Proteins c-akt/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Signal Transduction/drug effects* ; Apoptosis/drug effects* ; Myocytes, Cardiac/cytology* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Animals ; Rats ; Humans ; Molecular Docking Simulation ; Aging/metabolism* ; Protein Interaction Maps/drug effects* ; Heart/drug effects* ; Network Pharmacology

This study explores the therapeutic differences and mechanisms of salt-processed Phellodendri Chinensis Cortex in improving insulin resistance(IR) based on network pharmacology, molecular docking, and cellular experiments. The components and intersection targets of Phellodendri Chinensis Cortex in improving IR were collected from databases, and a "drug-component-target-disease" network and protein-protein interaction(PPI) network were constructed to screen core components and targets. A total of 29 active components and 240 intersection targets were identified, of which 13 were core targets. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were used to identify key signaling pathways, and molecular docking was performed to validate the binding activity between core components and targets. An IR model in HepG2 cells was induced using insulin combined with high glucose, and the effects of Phellodendri Chinensis Cortex before and after salt-processing on cell glucose consumption were evaluated. The expression of proteins related to the mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT) signaling pathways was detected by Western blot. The cellular experimental results showed that, compared with the model group, glucose consumption in the drug-treated groups was significantly increased(P<0.01), the phosphorylation level of extracellular regulated protein kinase(ERK) was decreased(P<0.05), the phosphorylation levels of PI3K and AKT were increased, and the expression of glucose transporter 4(GLUT4) was also upregulated(P<0.05). Furthermore, the effect of salt-processed Phellodendri Chinensis Cortex was better than that of raw Phellodendri Chinensis Cortex. The study demonstrates that Phellodendri Chinensis Cortex, both before and after salt-processing, improves IR by regulating the expression of related proteins in the MAPK and PI3K-AKT signaling pathways, with enhanced effects after salt-processing.
Humans ; Network Pharmacology ; Phellodendron/chemistry* ; Insulin Resistance ; Drugs, Chinese Herbal/chemistry* ; Hep G2 Cells ; Signal Transduction/drug effects* ; Molecular Docking Simulation ; Protein Interaction Maps/drug effects* ; Proto-Oncogene Proteins c-akt/genetics* ; Phosphatidylinositol 3-Kinases/genetics* ; Glucose/metabolism*