The aim of this study is to investigate the regulation of Kaixin San-medicated serum(KXS-MS) on autophagy induced by Aβ_(25-35) in SH-SY5Y cells. The SH-SY5Y cell model of Aβ_(25-35)(25 μmol·L~(-1))-induced injury was established, and different concentrations of KXS-MS were added into the culture media of cells, which were then incubated for 24 h. Cell viability was measured by the methyl thiazolyl tetrazolium(MTT) assay. The protein levels of microtubule-associated protein 1 light chain 3(LC3)Ⅰ, LC3Ⅱ, protein kinase B(Akt), p-Akt, mammalian target of rapamycin(mTOR), and p-mTOR were assessed by Western blot. Furthermore, the combination of rapamycin(Rapa)/3-methyladenine(3-MA) and low concentration of KXS-MS was added to the culture medium of SH-SY5Y cells injured by Aβ_(25-35), and the cell viability and the expression levels of the above proteins were determined. The results showed that Aβ_(25-35) decreased the cell viability, up-regulated the expression levels of LC3Ⅱ and LC3Ⅱ/LC3Ⅰ, and down-regulated the expression levels of p-Akt, p-mTOR, p-Akt/Akt, and p-mTOR/mTOR. Compared with the Aβ_(25-35) model group, KXS-MS treatment attenuated Aβ_(25-35)-induced injury and enhanced the survival of SH-SY5Y cells. Meanwhile, KXS-MS down-regulated the LC3Ⅱ/LC3Ⅰ level and up-regulated the p-Akt/Akt and p-mTOR/mTOR levels. Compared with the low-concentration KXS-MS group, Rapa did not affect the cell survival and the levels of p-Akt and p-Akt/Akt, while it up-regulated the levels of LC3Ⅱ and LC3Ⅱ/LC3Ⅰ and down-regulated the levels of p-mTOR and p-mTOR/mTOR. 3-MA significantly reduced the cell survival rate and p-Akt, p-Akt/Akt level in the KXS-MS group, while it had no significant effect on the levels of LC3Ⅱ, LC3Ⅱ/LC3Ⅰ, p-mTOR, and p-mTOR/mTOR. The above results indicate that KXS-MS exhibits protective effects against Aβ_(25-35)-induced damage in SH-SY5Y cells by up-regulating Akt/mTOR activity to inhibit autophagy.
Humans ; Autophagy/drug effects* ; TOR Serine-Threonine Kinases/genetics* ; Amyloid beta-Peptides/toxicity* ; Proto-Oncogene Proteins c-akt/genetics* ; Drugs, Chinese Herbal/pharmacology* ; Cell Line, Tumor ; Cell Survival/drug effects* ; Peptide Fragments/toxicity* ; Microtubule-Associated Proteins/genetics*

OBJECTIVE: To investigate the mechanism of circular RNA RAD18 (CircRAD18 ) in regulating daunorubicin (DNR) resistance in acute myeloid leukemia (AML) cells through the miR-185-5p/hepatoma-derived growth factor ( HDGF) axis. METHODS: Real-time fluorescence quantitative PCR and immunoblotting were applied to detect the expression of CircRAD18 , miR-185-5p, and HDGF in human AML cell lines HL-60, U937, and human AML drug-resistant cell line KG1a. KG1a cells were cultured in vitro and randomly divided into control group, DNR group, DNR+negative control group, DNR+CircRAD18 knockdown group, and DNR+CircRAD18 knockdown+miR-185-5p inhibitor group. After transfection, real-time fluorescence quantitative PCR and immunoblotting were applied to detect the expression of CircRAD18 , miR-185-5p, and HDGF of cells, CCK-8 method and Ki-67 immunofluorescence staining were applied to detect cell proliferation, flow cytometry was applied to detect cell apoptosis, and immunoblotting was applied to detect the expression of cell proliferation, apoptosis and drug resistance related proteins in each group. The double luciferase reporter gene experiment was applied to detect the targeting regulation of CircRAD18 on miR-185-5p, and miR-185-5p on HDGF in KG1a cells. RESULTS: Compared with HL-60 and U937 cells, the expression of CircRAD18 , and HDGF mRNA and protein in KG1a cells increased (all P <0.05), while miR-185-5p decreased ( P <0.05). Compared with the control group, the CircRAD18 expression, HDGF mRNA and protein expression, cell viability, proliferation rate, and PCNA, Bcl-2, BCRP, and P-gp protein expression in the DNR+CircRAD18 knockdown group decreased (all P <0.05), while miR-185-5p expression, apoptosis rate, and Bax protein expression increased (all P <0.05). There were no obvious changes in all indicators of cells in the DNR group compared with control group ( P >0.05). Compared with the DNR group, the CircRAD18 expression, HDGF mRNA and protein expression, cell viability, proliferation rate, PCNA, Bcl-2, BCRP, and P-gp protein expression in the DNR+CircRAD18 knockdown group decreased (all P < 0.05), while miR-185-5p expression, apoptosis rate, and Bax protein expression increased (all P < 0.05). There were no obvious changes in all indicators of cells in the DNR+negative control group compared with DNR group (P >0.05). Compared with the DNR+CircRAD18 knockdown group, the HDGF mRNA and protein expression, cell viability, proliferation rate, PCNA, Bcl-2, BCRP, and P-gp protein expression in the DNR+CircRAD18 knockdown+miR-185-5p inhibitor group increased (all P < 0.05), while miR-185-5p expression, apoptosis rate, and Bax protein expression decreased (all P < 0.05). CircRAD18 was able to target and down-regulate the expression of miR-185-5p in KG1a cells, and miR-185-5p was able to target and down-regulate the HDGF expression. CONCLUSION Knocking down CircRAD18 can reduce HDGF expression by up-regulating miR-185-5p, thereby weakening DNR resistance in AML cells, inhibiting KG1a cell proliferation under DNR treatment, and promoting apoptosis.
Humans ; MicroRNAs/metabolism* ; Leukemia, Myeloid, Acute ; Daunorubicin/pharmacology* ; Drug Resistance, Neoplasm ; Apoptosis ; RNA, Circular ; Intercellular Signaling Peptides and Proteins/metabolism* ; Cell Proliferation ; HL-60 Cells ; Cell Line, Tumor

OBJECTIVE: To investigate the Wnt signaling pathway and miRNAs mechanism of extracts of Plastrum Testudinis (PT) in the treatment of osteoporosis (OP). METHODS: Thirty female Sprague Dawley rats were randomly divided into 5 groups by random number table method, including sham group, ovariectomized group (OVX), ovariectomized groups treated with high-, medium-, and low-dose PT (160, 80, 40 mg/kg per day, respectively), with 6 rats in each group. Except for the sham group, the other rats underwent bilateral ovariectomy to simulate OP and received PT by oral gavage for 10 consecutive weeks. After treatment, bone mineral density was measured by dual-energy X-ray absorptiometry; bone microstructure was analyzed by micro-computed tomography and hematoxylin and eosin staining; and the expressions of osteogenic differentiation-related factors were detected by immunochemistry, Western blot, and quantitative polymerase chain reaction. In addition, Dickkopf-1 (Dkk-1) was used to inhibit the Wnt signaling pathway in bone marrow mesenchymal stem cells (BMSCs) and miRNA overexpression was used to evaluate the effect of miR-214 on the osteogenic differentiation of BMSCs. Subsequently, PT extract was used to rescue the effects of Dkk-1 and miR-214, and its impacts on the osteogenic differentiation-related factors of BMSCs were evaluated. RESULTS: PT-M and PT-L significantly reduced the weight gain in OVX rats (P<0.05). PT also regulated the bone mass and bone microarchitecture of the femur in OVX rats, and increased the expressions of bone formation-related factors including alkaline phosphatase, bone morphogenetic protein type 2, collagen type I alpha 1, and runt-related transcription factor 2 when compared with the OVX group (P<0.05 or P<0.01). Meanwhile, different doses of PT significantly rescued the inhibition of Wnt signaling pathway-related factors in OVX rats, and increased the mRNA or protein expressions of Wnt3a, β-catenin, glycogen synthase kinase-3β, and low-density lipoprotein receptor-related protein 5 (P<0.05 or P<0.01). PT stimulated the osteogenic differentiation of BMSCs inhibited by Dkk-1 and activated the Wnt signaling pathway. In addition, the expression of miR-214 was decreased in OVX rats (P<0.01), and it was negatively correlated with the osteogenic differentiation of BMSCs (P<0.01). MiR-214 mimic inhibited Wnt signaling pathway in BMSCs (P<0.05 or P<0.01). Conversely, PT effectively counteracted the effect of miR-214 mimic, thereby activating the Wnt signaling pathway and stimulating osteogenic differentiation in BMSCs (P<0.05 or P<0.01). CONCLUSION PT stimulates bone formation in OVX rats through β-catenin-mediated Wnt signaling pathway, which may be related to inhibiting miR-214 in BMSCs.
Animals ; MicroRNAs/genetics* ; Female ; Rats, Sprague-Dawley ; Wnt Signaling Pathway/genetics* ; Osteogenesis/genetics* ; Mesenchymal Stem Cells/cytology* ; Cell Differentiation/drug effects* ; Bone Density/drug effects* ; Ovariectomy ; Osteoporosis/drug therapy* ; beta Catenin/metabolism* ; Rats ; Intercellular Signaling Peptides and Proteins/metabolism* ; Drugs, Chinese Herbal/pharmacology*

OBJECTIVE: To explore the mechanism of crocin, a major active component of Crocus sativus (Zanghonghua), in regulating amyloid beta (Aβ) generation, endoplasmic reticulum (ER) stress, and autophagy in neuronal cells, with potential therapeutic applications in Alzheimer's disease (AD). METHODS: Mouse neuroblastoma Neuron2a (N2a) cells stably transfected with the human amyloid precursor protein (APP) Swedish mutant was used as a cellular model for AD (N2a/APP). Control cells were vector transfected (N2a/vector). The effects of 3 different doses of crocin on reactive oxygen species (ROS) generation, cytosolic calcium, and apoptosis were evaluated by flow cytometry. Aβ levels were determined by enzyme-linked immunosorbent assay. APP processing and ER stress proteins expressions were determined by Western blot. Autophagosome formation was evaluated by autophagy detection kit and confocal microscope. RESULTS: Crocin inhibited APP expression in N2a/APP cells and promoted α-cleavage of APP processing, while modestly reduced beta-secretase 1 (BACE1) and presenilin 1 (PS1, P<0.05 or P<0.01). ER stress markers, including the binding immunoglobulin protein/78-kD glucose-regulated protein (Bip/GRP78) and C/EBP homologous protein (CHOP), were elevated in N2a/APP cells compared to N2a/vector cells (P<0.05). Crocin could effectively reduce the levels of ER stress (P<0.05 or P<0.01). In addition, crocin enhanced autophagy by promoting formation of autophagosome (P<0.05 or P<0.01). CONCLUSION Crocin significantly inhibited Aβ generation by promoting α-cleavage of APP processing, inhibiting ER stress-associated unfolded protein response, and regulating autophagy.
Endoplasmic Reticulum Stress/drug effects* ; Autophagy/drug effects* ; Animals ; Endoplasmic Reticulum Chaperone BiP ; Mice ; Amyloid beta-Peptides/metabolism* ; Amyloid beta-Protein Precursor/metabolism* ; Carotenoids/pharmacology* ; Humans ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism* ; Apoptosis/drug effects* ; Calcium/metabolism*

The cosmetic sector is a multibillion-dollar industry that requires constant attention being paid to innovative product development and engagement. Notably, its market value is projected to exceed 750 billion U.S. dollars by 2025, and it is expanding as novel, climate-friendly, green, and sustainable components from natural sources are incorporated. This review is written based on the numerous reports on the potential applications of food-derived peptides while focusing on their possible uses in the formulation of cosmeceutical and skincare products. First, the production methods of bioactive peptides linked to cosmeceutical uses are described. Then, we discuss the obtainment and characterization of different anti-inflammatory, antimicrobial, antioxidant, anti-aging, and other pleiotropic peptides with their specific mechanisms, from various food sources. The review concludes with salient considerations of the cost of production and pilot scale operation, stability, compatibility, user safety, site-specificity, and delivery methods, when designing or developing biopeptide-based cosmeceutical products.
Cosmeceuticals/chemistry* ; Peptides/pharmacology* ; Humans ; Antioxidants/pharmacology* ; Anti-Inflammatory Agents/pharmacology* ; Anti-Infective Agents/pharmacology* ; Cosmetics ; Skin Aging/drug effects*

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 effect of orexin-A-mediated regulation of ionotropic glutamate receptors for promoting motor function recovery in rats with spinal cord injury (SCI). METHODS: Thirty-six newborn SD rats (aged 7-14 days) were randomized into 6 groups (n=6), including a normal control group, a sham-operated group, and 4 SCI groups with daily intrathecal injection of saline, DNQX, orexin-A, or orexin-A+DNQX for 3 consecutive days after PCI. Motor function of the rats were evaluated using blood-brain barrier (BBB) score and inclined plane test 1 day before and at 1, 3, and 7 days after SCI. For patch-clamp experiment, spinal cord slices from newborn rats in the control, sham-operated, SCI, and SCI+orexin groups were prepared, and ventral horn neurons were acutely isolated to determine the reversal potential and dynamic indicators of glutamate receptor-mediated currents under glutamate perfusion. RESULTS: At 3 and 7 days after SCI, the orexin-A-treated rats showed significantly higher BBB scores and grip tilt angles than those with other interventions. Compared with those treated with DNQX alone, the rats receiving the combined treatment with orexin and DNQX had significantly higher BBB scores and grip tilt angles on day 7 after PCI. In the patch-clamp experiment, the ventral horn neurons from SCI rat models exhibited obviously higher reversal potential and greater rise slope of glutamate current with shorter decay time than those from sham-operated and orexin-treated rats. CONCLUSIONS Orexin-A promotes motor function recovery in rats after SCI possibly by improving the function of the ionotropic glutamate receptors.
Animals ; Spinal Cord Injuries/drug therapy* ; Rats ; Rats, Sprague-Dawley ; Receptors, Ionotropic Glutamate/metabolism* ; Recovery of Function/drug effects* ; Orexins/pharmacology* ; Male ; Female ; Animals, Newborn ; Neuropeptides/pharmacology* ; Intracellular Signaling Peptides and Proteins/pharmacology*

OBJECTIVES: To evaluate the inhibitory effect of Macrothele raveni crude venom against proliferation of different cancer cells and identify the active components in the venom. METHODS: Different cancer cell lines were treated with different concentrations of Macrothele raveni venom for 48 h, and cell proliferation and the half-maximal inhibitory concentrations (IC₅₀) of the venom were assessed with CCK-8 assay. The apoptosis rate of breast cancer MCF7 cells following the treatment was analyzed with flow cytometry, and the changes in cellular caspase-8 and caspase-9 expressions were detected. The crude venom was separated into protein, peptide, and small-molecule compound fractions using gel filtration chromatography and high-performance liquid chromatography (HPLC). The protein and peptide components were identified using proteomics analysis, and small-molecule compounds were structurally characterized using nuclear magnetic resonance (NMR), mass spectrometry (MS), and HPLC. RESULTS The crude venom exhibited strong concentration-dependent inhibitory effects on proliferation of MCF7 cells and nasopharyngeal carcinoma SUNE1 and HONE1 cells (IC₅₀ of 2.14±0.29, 1.57±0.14, and 2.85±0.15 µg/mL, respectively), with less potent inhibitory effects in gastric cancer HGC27 cells and colorectal cancer SW620 cells (IC₅₀ of 3.02±0.27 and 3.02±0.28 µg/mL, respectively). The crude venom significantly promoted MCF7 cell apoptosis likely via the caspase 8 signaling pathway. The protein fraction from the crude venom showed a weak inhibitory effect in MCF7 cells, whereas the peptide fraction exhibited a much stronger inhibitory effect (IC₅₀ of 6.41±0.31 µg/mL). The peptides in the peptide fraction, with relative molecular mass around 10 000, were homologous to those found in Macrothele gigas venom. The small-molecule fraction consisted mainly of nucleotide metabolites without obvious inhibitory effects in MCF7 cells, but its combination with the peptide fraction showed significantly enhanced inhibitory activity. Conclusion The inhibitory effects of Macrothele raveni venom, which vary significantly across different cancer cell lines, are attributed primarily to its peptide components, which may act synergistically with the nucleotide metabolites.
Humans ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Animals ; Cell Line, Tumor ; MCF-7 Cells ; Caspase 8/metabolism* ; Peptides/pharmacology* ; Caspase 9/metabolism*

Alzheimer's disease (AD) poses one of the most urgent medical challenges in the 21st century as it affects millions of people. Unfortunately, the etiopathogenesis of AD is not yet fully understood and the current pharmacotherapy options are somewhat limited. Here, we report a novel inhibitor, Compound 44, for targeting cholinesterases, amyloid-β (Aβ) aggregation, and glycogen synthase kinase 3β (GSK-3β) simultaneously with the aim of achieving symptomatic relief and disease modification in AD therapy. We found that Compound 44 had good inhibitory effects on all intended targets with IC₅₀s of submicromolar or better, significant neuroprotective effects in cell models, and beneficial improvement of cognitive deficits in the triple transgenic AD (3 × Tg AD) mouse model. Moreover, we showed that Compound 44 acts as an autophagy regulator by inducing nuclear translocation of transcription factor EB through GSK-3β inhibition, enhancing the biogenesis of lysosomes and elevating autophagic flux, thus ameliorating the amyloid burden and tauopathy, as well as mitigating the disease phenotype. Our results suggest that triple-target inhibition via Compound 44 could be a promising strategy that may lead to the development of effective therapeutic approaches for AD.
Animals ; Alzheimer Disease/genetics* ; Mice, Transgenic ; Glycogen Synthase Kinase 3 beta/metabolism* ; Disease Models, Animal ; Mice ; Amyloid beta-Peptides/metabolism* ; Cholinesterase Inhibitors/therapeutic use* ; Humans ; Autophagy/drug effects* ; Cognitive Dysfunction/pathology* ; Neuroprotective Agents/pharmacology*

Peptide-based therapies have attracted considerable interest in the treatment of cancer, diabetes, bacterial infections, and neurodegenerative diseases due to their promising therapeutic properties and enhanced safety profiles. This review provides a comprehensive overview of the major trends in peptide drug discovery and development, emphasizing preclinical strategies aimed at improving peptide stability, specificity, and pharmacokinetic properties. It assesses the current applications and challenges of peptide-based drugs in these diseases, illustrating the pharmaceutical areas where peptide-based drugs demonstrate significant potential. Furthermore, this review analyzes the obstacles that must be overcome in the future, aiming to provide valuable insights and references for the continued advancement of peptide-based drugs.
Humans ; Peptides/pharmacology* ; Animals ; Neoplasms/drug therapy* ; Drug Discovery ; Neurodegenerative Diseases/drug therapy* ; Diabetes Mellitus/drug therapy*