OBJECTIVE: To observe the effects of acupuncture on podocyte autophagy and long non-coding RNA SOX2 overlapping transcript (LncRNA SOX2OT)/mammalian target of rapamycin C1 (mTORC1)/Unc-51-like kinase 1 (ULK1) pathway in rats with diabetic kidney disease (DKD), and to explore the mechanism by which acupuncture reduces urinary protein. METHODS: A total of 40 SPF-grade male Sprague-Dawley rats were randomly divided into a control group (n=10) and a modeling group (n=30). The DKD model was established by feeding a high-fat, high-sugar diet combined with intraperitoneal injection of streptozotocin (STZ) in the modeling group. Twenty rats with successful DKD model were randomly divided into a model group (n=10) and an acupuncture group (n=10). The acupuncture group received "spleen and stomach-regulating" acupuncture at bilateral "Zusanli" (ST36), "Fenglong" (ST40), "Yinlingquan" (SP9), and "Zhongwan" (CV12), 30 min per session, once daily, five times per week, for four weeks. The general condition, fasting blood glucose (FBG), 2-hour postprandial glucose (2hPG), serum creatinine (SCr), blood urea nitrogen (BUN), 24-hour urinary protein quantification, and urine albumin-to-creatinine ratio (UACR) were compared before and after the intervention. After intervention, urinary podocyte injury marker SPON2 was measured by ELISA. Podocyte autophagosomes and glomerular basement membrane ultrastructure in renal tissue were observed via transmission electron microscopy. Podocyte apoptosis was assessed by TUNEL staining. The protein expression of microtubule-associated protein 1 light chain 3Ⅱ (LC3-Ⅱ), mTORC1, ULK1, Beclin-1, and p62 in renal tissue was detected by Western blot. LncRNA SOX2OT expression in renal tissue was measured by real-time PCR. RESULTS: After the intervention, compared with the control group, the model group exhibited increased food and water intake, increased urine output, weight loss, and loose stools; compared with the model group, the food and water intake, urine volume, and loose stools were improved in the acupuncture group. Compared with the control group, FBG, 2hPG, SCr, BUN, 24-hour urinary protein quantification, UACR, and urinary SPON2 were all higher in the model group (P<0.01); compared with the model group, the FBG, 2hPG, SCr, BUN, 24-hour urinary protein quantification, UACR, and urinary SPON2 were all lower in the acupuncture group (P<0.01). Compared with the control group, the model group showed reduced podocyte autophagosomes and thickened glomerular basement membrane; compared with the model group, the acupuncture group had increased podocyte autophagosomes and less thickened basement membrane. Compared with the control group, the podocyte apoptosis index (AI) was higher in the model group (P<0.01); compared with the model group, the AI was lower in the acupuncture group (P<0.01). Compared with the control group, the expression of ULK1, Beclin-1, and LC3-Ⅱ proteins was lower, and the expression of mTORC1 and p62 proteins was higher in the model group (P<0.01). Compared with the model group, the expression of ULK1, Beclin-1, and LC3-Ⅱ proteins was higher, and the expression of mTORC1 and p62 proteins was lower in the acupuncture group (P<0.01). Compared with the control group, the LncRNA SOX2OT expression was lower in the model group (P<0.01). Compared with the model group, LncRNA SOX2OT expression was higher in the acupuncture group (P<0.01). CONCLUSION The "spleen and stomach-regulating" acupuncture method could improve renal function in DKD rats, reduce blood glucose and urinary protein excretion, alleviate podocyte injury, and enhance podocyte autophagy. The mechanism may be related to modulation of the renal LncRNA SOX2OT/mTORC1/ULK1 pathway.
Animals ; Podocytes/cytology* ; Diabetic Nephropathies/physiopathology* ; Rats, Sprague-Dawley ; Male ; Rats ; Mechanistic Target of Rapamycin Complex 1/genetics* ; Autophagy ; Acupuncture Therapy ; Autophagy-Related Protein-1 Homolog/genetics* ; RNA, Long Noncoding/metabolism* ; Humans ; Signal Transduction

Anastasis is a phenomenon described as a cellular escape from ethanol-induced cell death. Although the relevant mechanism has not yet been fully elucidated, anastasis is thought to play a role in drug resistance in cancer cells. To date, the regulation of anastasis in normal and cancerous cells has not been clarified. The current cancer treatment strategies are expected to selectively attack cancer cells without negatively affecting normal cell proliferation. Inspired by the anti-cancer potential of bee venom, this study is the first to evaluate whether bee venom has similar selectivity in producing an anastatic effect. The results indicated that bee venom induces anastasis in normal cells (Michigan Cancer Foundation-10A (MCF10A), Adult Retinal Pigment Epithelium cell line-19 (ARPE-19), and National Institutes of Health 3T3 cell line (NIH3T3)) but causes irreversible cell death in breast cancer cells (M.D. Anderson-Metastatic Breast-231 (MDA-MB-231) and Michigan Cancer Foundation-7 (MCF7)). Liver cancer (HepG2) cells were moderately more resistant to permanent cell death after bee venom treatment compared to breast cancer cells. However, cisplatin caused permanent non-selective cell death in both normal and cancerous cells. The selectivity indices after bee venom treatment were higher compared to cisplatin. Taken together, bee venom was shown to induce selective anastasis only in normal cells, not in cancer cells, which suggests that bee venom has significant potential in selective cancer therapy, especially for breast cancer, via promoting the recovery and maintenance of viability of normal cells.
Bee Venoms/pharmacology* ; Humans ; Animals ; Mice ; Cell Survival/drug effects* ; Breast Neoplasms/pathology* ; Female ; Cell Line, Tumor ; NIH 3T3 Cells ; Antineoplastic Agents/pharmacology* ; Cisplatin/pharmacology* ; Cell Death/drug effects* ; Hep G2 Cells ; MCF-7 Cells

OBJECTIVES: To investigate the protective effect of Yiqi Yangyin Huazhuo Tongluo Formula (YYHT) against high glucose-induced injury in mouse renal podocytes (MPC5 cells) and the possible mechanism. METHODS: Adult Wistar rats were treated with 19, 38, and 76 g/kg YYHT or saline via gavage for 7 days to prepare YYHT-medicated or blank sera for treatment of MPC5 cells cultured in high glucose (30 mmol/L) prior to transfection with a miR-21a-5p inhibitor or a miR-21a-5p mimic. The changes in miR-21a-5p expressions and the mRNA levels of FoxO1, PINK1, and Parkin in the treated cells were detected with qRT-PCR, and the protein levels of nephrin, podocin, FoxO1, PINK1, and Parkin were detected with Western blotting. Autophagic activity in the cells were evaluated with MDC staining. The effect of miR-21a-5p mimic on FoxO1 transcription and the binding of miR-21a-5p to FoxO1 were examined with luciferase reporter gene assay and radioimmunoprecipitation assay. RESULTS: MPC5 cells exposed to high glucose showed significantly increased miR-21a-5p expression, lowered expressions of FoxO1, PINK1, and Parkin1 mRNAs, and reduced levels of FoxO1, PINK1, parkin, nephrin, and podocin proteins and autophagic activity. Treatment of the exposed cells with YYHT-medicated sera and miR-21a-5p inhibitor both significantly enhanced the protein expressions of nephrin and podocin, inhibited the expression of miR-21a-5p, increased the mRNA and protein expressions of FoxO1, PINK1 and Parkin, and upregulated autophagic activity of the cells. Transfection with miR-21a-5p mimic effectively inhibited the transcription of FoxO1 and promoted the binding of miR-21a-5p to FoxO1 in MPC5 cells, and these effects were obviously attenuated by treatment with YYHT-medicated sera. CONCLUSIONS YYHT-medicated sera alleviate high glucose-induced injury in MPC5 cells by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.
Animals ; MicroRNAs/genetics* ; Podocytes/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Autophagy/drug effects* ; Rats, Wistar ; Protein Kinases/metabolism* ; Rats ; Forkhead Box Protein O1 ; Mice ; Mitochondria/drug effects* ; Ubiquitin-Protein Ligases/metabolism* ; Glucose ; Diabetic Nephropathies ; Male ; Membrane Proteins/metabolism* ; Intracellular Signaling Peptides and 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 active ingredients in Hedyotis diffusa-Scutellaria barbata D. Don and the main biological processes and signaling pathways mediating their inhibitory effect on primary hepatocellular carcinoma (HCC). METHODS: The core intersecting genes of HCC and the two drugs were screened from TCMSP, Uniport, Genecards, and String databases using Cytoscape software, and GO and KEGG enrichment analyses of the intersecting genes were conducted. Molecular docking between the active ingredients of the drugs and the core genes was carried out using Pubcham, RCSB and Autoduckto to identify the active ingredients with the highest binding energy, whose inhibitory effect on HepG2 cells was verifies using CCK-8 assay, flow cytometry and Western blotting. RESULTS: TP53 and ESR1 were identified as the core genes of HCC and the two drugs. GO and KEGG analyses showed that the two genes were mainly involved in regulation of apoptotic signaling pathway, cell population proliferation, methane raft, and protein kinase activity, and participated in the signaling pathways of apoptosis, proteoglycans in cancer, PI3K Akt signaling pathway, and hepatitis B. Molecular docking studies showed that the active ingredients of the drugs could be docked with TP53 and ESR1 genes under natural conditions, and ursolic acid had the highest binding energy to ESR1 (-4.98 kcal/mol). The results of CCK-8 assay, flow cytometry and Western blotting all demonstrated significant inhibitory effect of ursolic acid on HepG2 cells. CONCLUSIONS The inhibitory effect of Hedyotis diffusa-scutellariae barbatae on HCC is mediated by multiple active ingredients in the two drugs.
Humans ; Molecular Docking Simulation ; Liver Neoplasms/drug therapy* ; Hep G2 Cells ; Network Pharmacology ; Carcinoma, Hepatocellular/drug therapy* ; Hedyotis/chemistry* ; Signal Transduction/drug effects* ; Cell Proliferation/drug effects* ; Tumor Suppressor Protein p53/metabolism* ; Apoptosis/drug effects* ; Estrogen Receptor alpha/metabolism* ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVES: To investigate the therapeutic mechanism of nodakenin for Crohn's disease (CD)-like colitis in mice. METHODS: Using a colonic organoid model with lipopolysaccharide (LPS)- and ATP-induced pyroptosis, we investigated the effects of nodakenin on pyroptosis, intestinal barrier function and inflammatory response by detecting key pyroptosis-regulating factors and assessing changes in permeability and pro-inflammatory factors. In a mouse model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis, the therapeutic effect of nodakenin was evaluated by measuring changes in body weight, DAI score, colonic histopathologies, inflammation score, intestinal barrier function and intestinal epithelial cell pyroptosis. The mechanism of nodakenin protection against pyroptosis of intestinal epithelial cells was explored using network pharmacology analysis and in vivo and in vitro experiments. RESULTS: In LPS- and ATP-induced colonic organoids, treatment with nodakenin significantly inhibited the expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11, improved intestinal FITC-dextran (FD4, 4000) permeability, and decreased the levels of IL-1β and IL-18. In the mouse model of TNBS-induced colitis, nodakenin treatment significantly alleviated weight loss, reduced DAI score, inflammatory cell infiltration and inflammation score, and decreased serum FD4 and I-FABP levels and bacteria translocation to the mesenteric lymph nodes, spleen and liver. The mice with nodakenin treatment had also lowered expressions of NLRP3, GSDMD-N, cleaved caspase-1 and caspase-11 in the intestinal mucosa. Network pharmacology analysis suggested that the inhibitory effect of nodakenin on colitis was associated with the PI3K/Akt pathway. In both the colonic organoid model and mouse models of colitis, nodakenin effectively inhibited the activation of the PI3K/Akt pathway, and the application of IGF-1, a PI3K/Akt pathway activator, strongly attenuated the protective effect of nodakenin against intestinal epithelial cell pyroptosis and intestinal barrier dysfunction. CONCLUSIONS Nodakenin protects intestinal barrier function and alleviates CD-like colitis in mice at least partly by inhibiting PI3K/Akt signaling to reduce intestinal epithelial cell pyroptosis.
Animals ; Pyroptosis/drug effects* ; Mice ; Trinitrobenzenesulfonic Acid ; Colitis/drug therapy* ; Epithelial Cells/drug effects* ; Intestinal Mucosa/cytology* ; Disease Models, Animal ; Coumarins/pharmacology* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Crohn Disease/drug therapy*

OBJECTIVES: To reprogram human placental fibroblasts (HPFs) into chemically induced epithelioid-like cells (ciEP-Ls) using a combination of small-molecule compounds. METHODS: HPFs cultured under normoxic conditions were identified using immunofluorescence assay, PCR and chromosomal karyotyping. Under hypoxic conditions (37 ℃, 5% O₂), HPFs were cultured in a medium containing small-molecule compounds C6TSEDRVAJZ (CHIR99021, 616452, TTNPB, SAG, EPZ5676, DZNep, Ruxolitinib, VTP50469, Afuresertib, JNK-IN-8, and EZM0414), and the cell morphology was observed daily. The expression levels of epithelial cell markers in the induced cells were detected by immunofluorescence, Western blotting and PCR. Chromosomal karyotyping of the induced cells was performed and the induction efficiency was calculated. RESULTS: Before induction, HPFs showed positive expressions of fibroblast surface markers CD34 and vimentin and were negative for epithelial surface markers. PCR results showed high expressions of fibroblast-specific genes S100A4 and COL1A1 in HPFs with a normal human diploid karyotype. After one day of induction, the HPFs underwent morphological changes from a multinodular spindle shape to a round or polygonal shape, which was morphologically characteristic of ciEP-Ls. On day 4 of induction, the cells exhibited high expressions of the epithelial cell markers E-cadherin and Lin28A. RT-qPCR results also showed that the cells expressed the epithelial markers Smad3, GLi3, PAX8, WT1, KRT19, and KRT18 with significantly down-regulated expressions of all the fibroblast surface markers and a normal human diploid karyotype. The reprogramming efficiency of HPFs into ciEP-Ls ranged from (64.53±2.8)% to (68.10±3.6)%. CONCLUSIONS The small-molecule compound combination C6TSEDRVAJZ is capable of inducing HPFs into ciEP-Ls under hypoxic conditions with a high induction efficiency.
Humans ; Fibroblasts/drug effects* ; Pregnancy ; Female ; Cell Differentiation/drug effects* ; Pyrimidines/pharmacology* ; Placenta/cytology* ; Cells, Cultured ; Pyridines/pharmacology* ; Pyrazoles/pharmacology* ; Epithelial Cells/cytology*

OBJECTIVES: To explore the expression of hexokinase 2 (HK2) in colorectal cancer (CRC) and its possible mechanisms for regulating tumor cell behaviors and tumor immune microenvironment. METHODS: We analyzed HK2 expression in CRC and its impact on patient prognosis and tumor immune microenvironment using public databases. HK2 expression was also examined in 8 CRC and paired adjacent tissues using immunohistochemistry, Western blotting and RT-qPCR. In cultured CRC cell lines CT26 and HCT116 with low HK2 expression, the effects of lentivirus-mediated HK2 overexpression and JAK/STAT3 inhibitors on cell proliferation, migration, and invasion were assessed using CCK-8 assay, colony formation assay and Transwell assay and in a subcutaneous tumor-bearing mouse model; the changes were also observed in MC38 and CACO2 cells with high HK2 expressions following treatment with HK2 inhibitor 3-BP. Western blotting was performed to verify the relationship between HK2 and JAK/STAT signaling pathway protein expressions. RESULTS: Informatics analyses suggested that HK2 expression was significantly higher in CRC tissues than in adjacent tissues (P<0.001), and patients with high HK2 expressions had worse prognosis (P=0.09). In the 8 clinical CRC tissues, HK2 expressions were significantly higher in the tumor tissues than in the adjacent tissues (P<0.01). In CT26 and HCT116 cells, HK2 overexpression significantly enhanced cell proliferation, migration and invasion, while in HK2-overexpressing MC38 and CACO2 cells, inhibiting HK2 with 3-BP strongly suppressed these changes. HK2 overexpression promoted STAT3 phosphorylation, and JAK/STAT3 inhibitors effectively suppressed tumor cell proliferation, migration and invasion. TIMER and MCPcounter analyses indicated correlations between HK2 and immune cells, and TCGA and GEO analyses suggested significant positive correlations between HK2 and the immune checkpoints including PDCD1. CONCLUSIONS HK2 is upregulated in CRC to promote tumor cell proliferation, migration and invasion possibly by activating the JAK-STAT signaling pathway and modulating tumor immune microenvironment.
Humans ; Colorectal Neoplasms/metabolism* ; Cell Proliferation ; Hexokinase/genetics* ; Tumor Microenvironment ; Cell Movement ; Signal Transduction ; Animals ; STAT3 Transcription Factor/metabolism* ; Mice ; Neoplasm Invasiveness ; Cell Line, Tumor ; Janus Kinases/metabolism* ; HCT116 Cells ; Caco-2 Cells

OBJECTIVES: To investigate the regulatory effects of Aurora-A in regulating proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) of cervical cancer cells and the role of actin-related protein 2/3 complex subunit 4 (ARPC4) in mediating its effects. METHODS: The plasmids pCDH-NC, pCDH-Aurora-A, and shRNA-ARPC4 were used for inducing Aurora-A overexpression or ARPC4 knockdown in HeLa cells. The cells were divided into vector group, Aurora-A overexpression group, Aurora-A overexpression+ARPC4 knockdown group, and Aurora-A overexpression+NF‑κBp65 inhibitor group and transfected with the corresponding plasmids. The proliferation, colony-forming ability, migration and invasion of the treated Hela cells was evaluated using EdU immunofluorescence assay, crystal violet staining, scratch assay, Transwell assay, and Matrigel assay. Western blotting was performed to detect the changes in cellular expressions of EMT-related proteins and expression levels of NF-κBp65 and ARPC4. RESULTS: The expression of ARPC4 was significantly decreased in HeLa cells with Aurora-A knockdown and increased in Aurora-A-overexpressing cells. Aurora-A overexpression obviously promoted proliferation, migration, and invasion abilities of HeLa cells, and these effects was significantly antagonized by ARPC4 knockdown. In Aurora-A-overexpressing cells, the phosphorylation level of NF-κBp65 and the expression level of ARPC4 were increased significantly, and application of the NF‑κBp65 inhibitor obviously lowered the expression level of ARPC4. CONCLUSIONS Aurora-A overexpression upregulates the expression of ARPC4 by activating the NF-κBp65 signaling pathway, thereby promoting migration, invasion and EMT of HeLa cells.
Humans ; Uterine Cervical Neoplasms/metabolism* ; Female ; HeLa Cells ; Epithelial-Mesenchymal Transition ; Signal Transduction ; Cell Movement ; Neoplasm Invasiveness ; Cell Proliferation ; Aurora Kinase A/metabolism* ; Transcription Factor RelA/metabolism* ; Neoplasm Metastasis

OBJECTIVES: To investigate the subcellular localization and biological functions of transmembrane protein 240 (TMEM240). METHODS: NCBI BLAST and TMHMM bioinformatics software were used for protein sequence analysis and prediction of transmembrane domain of TMEM240. Brain tissues from male C57BL/6 mice (18-20 days old) were examined for distribution of TMEM240 using in situ hybridization, and qPCR and Western blotting were used to detect TMEM240 expression in different mouse tissues and in cortical neurons at different time points (n=3). In the in vitro experiment, HepG2 and Neuro-2a cells were transfected with plasmids for overexpression of TMEM240, and subcellular localization of TMEM240 was analyzed using cell imaging. In primary cultures of cortical neurons isolated from C57BL/6 mice, TMEM240 expression and its biological functions were investigated using qPCR, Western blotting, and immunofluorescence staining. RESULTS: Human and mouse TMEM240 proteins share a 97.69% similarity in the protein sequences, and both are transmembrane proteins with two transmembrane domains. TMEM240 mRNA and protein were highly expressed in mouse brain tissues and cortical neurons. In isolated mouse cortical neurons, TMEM240 expression reached the peak level after primary culture for 9 days and distributed in scattered spots within the cells. In HepG2 cells, TMEM240 was characterized as intracellular membrane structures and showed 80% colocalization with peroxisomes. In Neuro-2a cells, TMEM240 overexpression caused significant enhancement of the expressions of Rho GDP dissociation inhibitor β (ARHGDIB) at both the mRNA and protein levels. CONCLUSIONS TMEM240 is a novel intracellular subcellular structure specifically expressed in neurons with significant potential for targeted cellular function regulation.
Animals ; Humans ; Mice ; Peroxisomes/metabolism* ; Membrane Proteins/genetics* ; Mice, Inbred C57BL ; Neurons/metabolism* ; Male ; rho-Specific Guanine Nucleotide Dissociation Inhibitors ; Hep G2 Cells ; Brain/metabolism*