ABSTRACT
Bladder cancer is a common tumor that impacts the urinary system and marked by a significant fatality rate and an unfavorable prognosis. Promising antineoplastic properties are exhibited by brusatol, which is obtained from the dried ripe fruit of Brucea javanica. The present study aimed to evaluate the influence of brusatol on the progression of bladder cancer and uncover the molecular mechanism involved. We used Cell Counting Kit-8, colony formation and EdU assays to detect cell numbers, viability and proliferation. We used transwell migration assay to detect cell migration ability. The mechanism of brusatol inhibition of bladder cancer proliferation was studied by flow cytometry and western blotting. It was revealed that brusatol could reduce the viability and proliferation of T24 and 5637 cells. The transwell migration assay revealed that brusatol was able to attenuate the migration of T24 and 5637 cells. We found that treatment with brusatol increased the levels of reactive oxygen species, malondialdehyde and Fe2+, thereby further promoting ferroptosis in T24 and 5637 cells. In addition, treatment with RSL3 (an agonistor of ferroptosis) ferrostatin-1 (a selective inhibitor of ferroptosis) enhanced or reversed the brusatol-induced inhibition. In vivo, treatment with brusatol significantly suppressed the tumor growth in nude mice. Mechanistically, brusatol induced ferroptosis by upregulating the expression of ChaC glutathione-specific gamma-glutamylcyclotransferase (Chac1) and decreasing the expression of SLC7A11 and Nrf2 in T24 and 5637 cells. To summarize, the findings of this research demonstrated that brusatol hindered the growth of bladder cancer and triggered ferroptosis via the Chac1/Nrf2/SLC7A11 pathway.
Subject(s)
Amino Acid Transport System y+ , Cell Movement , Cell Proliferation , NF-E2-Related Factor 2 , Quassins , Urinary Bladder Neoplasms , Urinary Bladder Neoplasms/pathology , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/genetics , Quassins/pharmacology , Humans , NF-E2-Related Factor 2/metabolism , NF-E2-Related Factor 2/genetics , Animals , Mice , Cell Proliferation/drug effects , Cell Movement/drug effects , Cell Line, Tumor , Amino Acid Transport System y+/metabolism , Amino Acid Transport System y+/genetics , Mice, Nude , Signal Transduction/drug effects , Ferroptosis/drug effects , Xenograft Model Antitumor Assays , Reactive Oxygen Species/metabolism , Disease Progression , Mice, Inbred BALB C , Gene Expression Regulation, Neoplastic/drug effectsABSTRACT
Kidney stone, one of the oldest known diseases, has plagued humans for centuries, consistently imposing a heavy burden on patients and healthcare systems worldwide due to their high incidence and recurrence rates. Advancements in endoscopy, imaging, genetics, molecular biology and bioinformatics have led to a deeper and more comprehensive understanding of the mechanism behind nephrolithiasis. Kidney stone formation is a complex, multi-step and long-term process involving the transformation of stone-forming salts from free ions into asymptomatic or symptomatic stones influenced by physical, chemical and biological factors. Among the various types of kidney stones observed in clinical practice, calcareous nephrolithiasis is currently the most common and exhibits the most intricate formation mechanism. Extensive research suggests that calcareous nephrolithiasis primarily originates from interstitial subepithelial calcified plaques and/or calcified blockages in the openings of collecting ducts. These calcified plaques and blockages eventually come into contact with urine in the renal pelvis, serving as a nidus for crystal formation and subsequent stone growth. Both pathways of stone formation share similar mechanisms, such as the drive of abnormal urine composition, involvement of oxidative stress and inflammation, and an imbalance of stone inhibitors and promoters. However, they also possess unique characteristics. Hence, this review aims to provide detailed description and present recent discoveries regarding the formation processes of calcareous nephrolithiasis from two distinct birthplaces: renal interstitium and tubule lumen.
Subject(s)
Calcinosis , Kidney Calculi , Humans , Kidney Medulla/metabolism , Kidney Calculi/complications , Kidney Calculi/metabolism , Calcinosis/metabolism , Endoscopy , Inflammation/metabolismABSTRACT
Bladder cancer (BLCA) is ranked among the top ten most prevalent cancers worldwide and is the second most common malignant tumor within the field of urology. The limited effectiveness of immune targeted therapy in treating BLCA, due to its high metastasis and recurrence rates, necessitates the identification of new therapeutic targets. Secretogranin II (SCG2), a member of the chromaffin granin/secreted granin family, plays a crucial role in the regulated release of peptides and hormones. The role of SCG2 in the tumor microenvironment (TME) of lung adenocarcinoma and colon cancer has been established, but its functional significance in BLCA remains uncertain. This study aimed to investigate SCG2 expression in 15 bladder cancer tissue samples and their corresponding adjacent control tissues. The potential involvement of SCG2 in BLCA progression was assessed using various techniques, including analysis of public databases, immunohistochemistry, Western Blotting, immunofluorescence, wound-healing assay, Transwell assay, and xenograft tumor formation experiments in nude mice. This study provided novel evidence indicating that SCG2 plays a pivotal role in facilitating the proliferation, migration, and invasion of BLCA by activating the MEK/Erk and MEK/IKK/NF-κB signaling pathways, as well as by promoting M2 macrophage polarization. These findings propose the potential of SCG2 as a molecular target for immunotherapy in human BLCA.
Subject(s)
NF-kappa B , Urinary Bladder Neoplasms , Animals , Humans , Mice , Apoptosis , Chromogranins/therapeutic use , Mice, Nude , Mitogen-Activated Protein Kinase Kinases , NF-kappa B/genetics , NF-kappa B/metabolism , Secretogranin II/genetics , Secretogranin II/metabolism , Secretogranin II/therapeutic use , Tumor Microenvironment , Urinary Bladder Neoplasms/genetics , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/metabolismABSTRACT
BACKGROUND: Multiple myeloma (MM) is a malignancy in which plasma cells proliferate abnormally, and it remains incurable. The cells are characterized by high levels of endoplasmic reticulum stress (ERS) and depend on the ERS response for survival. Thus, we aim to find an ERS-related signature of MM and assess its diagnostic value. METHODS: We downloaded three datasets of MM from the Gene Expression Omnibus database. After identifying ERS-related differentially expressed genes (ERDEGs), we analyzed them using Gene Ontology enrichment analysis. A protein-protein interaction network, a transcription factor-mRNA network, a miRNA-mRNA network and a drug-mRNA network were constructed to explore the ERDEGs. The clinical application of these genes was identified by calculating the infiltration of immune cells and using receiver operating characteistic analyses. Finally, qPCR was performed to further confirm the roles of ERDEGs. RESULTS: We obtained nine ERDEGs of MM. Gene Ontology enrichment indicated that the ERDEGs played a role in the endoplasmic reticulum membrane. Additionally, the protein-protein interaction network showed interaction among the ERDEGs, and there were 20 proteins, 107 transcription factors, 42 drugs or molecular compounds and 51 miRNAs which were likely to interact with the nine genes. In addition, immune cell infiltration analyses showed that there was a strong correlation between the nine genes and immune cells, and these potential biomarkers exhibited good diagnostic values. Finally, the expression of ERDEGs in MM cells was different from that in healthy donor samples. CONCLUSION: The nine ERS-related genes, CR2, DHCR7, DNAJC3, KDELR2, LPL, OSBPL3, PINK1, VCAM1 and XBP1 are potential biomarkers of MM, and this supports further clinical development of the diagnosis and treatment of MM.
Subject(s)
MicroRNAs , Multiple Myeloma , Humans , Multiple Myeloma/genetics , Endoplasmic Reticulum Stress/genetics , Gene Ontology , MicroRNAs/genetics , Biomarkers , RNA, Messenger/genetics , Vesicular Transport ProteinsABSTRACT
The engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis.
Subject(s)
CRISPR-Cas Systems , Calcium Oxalate , Kidney , Animals , Humans , Male , Mice , Calcium Oxalate/metabolism , CRISPR-Cas Systems/genetics , Eukaryotic Initiation Factor-4G/metabolism , Eukaryotic Initiation Factor-4G/genetics , Ferritins , Ferroptosis/genetics , Gene Editing/methods , HEK293 Cells , Kidney/metabolism , Kidney/pathology , Kidney Calculi/genetics , Kidney Calculi/metabolism , Oxidoreductases/metabolism , Oxidoreductases/genetics , Protein Biosynthesis/genetics , RNA, Guide, CRISPR-Cas Systems/genetics , RNA, Guide, CRISPR-Cas Systems/metabolismABSTRACT
Breast cancer (BC) has threatened women worldwide for a long time, and novel treatments are needed. Ferroptosis is a new form of regulated cell death that is a potential therapeutic target for BC. In this study, we identified Escin, a traditional Chinese medicine, as a possible supplement for existing chemotherapy strategies. Escin inhibited BC cell growth in vitro and in vivo, and ferroptosis is probable to be the main cause for Escin-induced cell death. Mechanistically, Escin significantly downregulated the protein level of GPX4, while overexpression of GPX4 could reverse the ferroptosis triggered by Escin. Further study revealed that Escin could promote G6PD ubiquitination and degradation, thus inhibiting the expression of GPX4 and contributing to the ferroptosis. Moreover, proteasome inhibitor MG132 or G6PD overexpression could partially reverse Escin-induced ferroptosis, when G6PD knockdown aggravated that. In vivo study also supported that downregulation of G6PD exacerbated tumor growth inhibition by Escin. Finally, our data showed that cell apoptosis was dramatically elevated by Escin combined with cisplatin in BC cells. Taken together, these results suggest that Escin inhibits tumor growth in vivo and in vitro via regulating the ferroptosis mediated by G6PD/GPX4 axis. Our findings provide a promising therapeutic strategy for BC.
Subject(s)
Breast Neoplasms , Ferroptosis , Female , Humans , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cisplatin/pharmacology , Cisplatin/therapeutic use , Escin , Ferroptosis/genetics , ApoptosisABSTRACT
Crop domestication for increasing growth rates and yields appears to have altered the features of adaxial and abaxial stomata, but its effect on leaf water use efficiency (WUE) have not been experimentally verified. In this study, we characterized stomatal anatomy and carbon isotope discrimination (δ13C) in 32 wild and 36 domesticated genotypes of cotton grown under agricultural field conditions. The results showed that domesticated genotypes possessed lower WUE, as indicated by low or more negative δ13C compared with wild genotypes. Higher theoretical maximum stomatal conductance (gsmax) after domestication was accounted for by more stomata rather than significantly enlarged stomata. Specifically, abaxial stomatal density was higher whilst there was no change in the adaxial density. The size of both adaxial and abaxial stomata was greater due to larger guard cells but without there being any increase in pore size. However, there was a negative relationship between δ13C and stomatal size across wild and domesticated genotypes, especially on the abaxial leaf surface, because bigger stomata resulted in a lower maximum stomatal response rate to fluctuating canopy light, resulting in increased water loss. Overall, our results indicate that cotton domestication has resulted in substantial variation in stomatal anatomy, and that WUE and drought tolerance can potentially be improved in future breeding by decreasing the size of abaxial stomata to produce a faster stomatal response and hence a reduction in unnecessary water loss.
Subject(s)
Plant Stomata , Water , Plant Stomata/physiology , Domestication , Plant Breeding , Plant Leaves/physiologyABSTRACT
BACKGROUND: The ossification of renal tubular epithelial cells (RTECs) plays an important initial role in the formation of kidney stones, but its specific mechanism is still unclear. The JAK2/STAT3 signaling pathway is important for bone cell differentiation. Accordingly, we explored the role and mechanism of the JAK2/STAT3 signaling pathway in the ossification of RTECs. METHODS: We used oxalate or ethylene glycol to construct kidney stone models in vitro and in vivo, and investigated the expression of osteogenic-specific genes, osteogenesis ability, and JAK2/STAT3 signaling in the kidney stone models by western blotting, qRT-PCR, immunofluorescence, and immunohistochemistry. Then, genetic engineering or drugs were used to inhibit the expression or activation of JAK2, and the expression of osteogenic-specific genes and the osteogenic ability of the RTECs were determined again. RESULTS: In the in vitro and in vivo kidney stone models, the expression of osteogenic specific genes in the RTECs was significantly upregulated, the osteogenic capacity was significantly increased, and the expression of p-JAK2 (phospho-JAK2) and p-STAT3 (phospho-STAT3) was significantly increased. When the expression or activation of JAK2 was inhibited, the ossification of RTECs and the formation of kidney stones was reversed. CONCLUSIONS: During the formation of kidney stones, RTECs undergo obvious ossification, and the JAK2/STAT3 signaling pathway plays a key positive regulatory role in this process.
Subject(s)
Kidney Calculi , Osteogenesis , Cell Differentiation , Humans , Janus Kinase 2/metabolism , Oxalates , STAT3 Transcription Factor/metabolism , Signal TransductionABSTRACT
A machine learning approach has been applied to virtual screening for lysine specific demethylase 1 (LSD1) inhibitors. LSD1 is an important anti-cancer target. Machine learning models to predict activity were constructed using Morgan molecular fingerprints. The dataset, consisting of 931 molecules with LSD1 inhibition activity, was obtained from the ChEMBL database. An evaluation of several candidate algorithms on the main dataset revealed that the support vector regressor gave the best model, with a coefficient of determination (R2) of 0.703. Virtual screening, using this model, identified five predicted potent inhibitors from the ZINC database comprising more than 300,000 molecules. The virtual screening recovered a known inhibitor, RN1, as well as four compounds where activity against LSD1 had not previously been suggested. Thus, we performed a machine-learning-enabled virtual screening of LSD1 inhibitors using only the structural information of the molecules.
Subject(s)
Enzyme Inhibitors/pharmacology , Histone Demethylases/antagonists & inhibitors , Lysine/pharmacology , Machine Learning , Databases, Factual , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Histone Demethylases/metabolism , Humans , Lysine/chemistry , Molecular StructureABSTRACT
BACKGROUND: M2 macrophages have important roles in diseases such as tumours, cardiovascular diseases and renal diseases. This study aimed to determine the effects and protective mechanism of M2 macrophages against oxidative stress injury and apoptosis induced by calcium oxalate crystals (CaOx) in renal tubular epithelial cells (HK-2) under coculture conditions. METHODS: THP-1 cells were induced to differentiate into M2 macrophages by using phorbol-12-myristate-13-acetate, IL-4 and IL-13. Morphological features were observed by microscopy. Phenotypic markers were identified by reverse transcription-polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay (ELISA). HK-2 cells were treated with 0.5 mg/mL CaOx crystals and co-cultured with M2 macrophages or apocynin. The viability of HK-2 cells was detected by CCK-8 assay. The lactate dehydrogenase (LDH) activity of HK-2 cells was analysed using a microplate reader. The apoptosis of HK-2 cells was examined by flow cytometry and Hoechst 33258 staining. Reactive oxygen species (ROS) expression and mitochondrial membrane potential in HK-2 cells were detected by a fluorescence microplate reader. Western blot analysis was conducted to detect the expression of p47phox, Bcl-2, cleaved caspase-3, cytochrome c, p38 MAPK, phospho-p38 MAPK, Akt and phospho-Akt. RESULTS: The results of morphology, reverse transcription-polymerase chain reaction, Western blot and ELISA showed that THP-1 cells were successfully polarised to M2 macrophages. The results of co-culture suggested that M2 macrophages or apocynin significantly increased the cell viability and decreased the LDH activity and apoptosis rate after HK-2 cells were challenged with CaOx crystals. The expression of the p47phox protein and the concentration of ROS were reduced, the release of mitochondrial membrane potential and the expression of the Bcl-2 protein were upregulated and the protein expression of cleaved caspase-3 and cytochrome c was downregulated. The expression of the phosphorylated form of p38 MAPK increased. Under coculture conditions with M2 macrophages, the Akt protein of HK-2 cells treated with CaOx crystals was dephosphorylated, but the phosphorylated form of Akt was not reduced by apocynin. CONCLUSIONS: M2 macrophages reduced the oxidative stress injury and apoptosis of HK-2 cells by downregulating the activation of NADPH oxidase, reducing the production of ROS, inhibiting the phosphorylation of p38 MAPK and enhancing the phosphorylation of Akt. We have revealed one of the possible mechanisms by which M2 macrophages reduce the formation of kidney stones.
Subject(s)
Apoptosis/drug effects , Calcium Oxalate/pharmacology , Kidney Tubules/drug effects , Macrophages/physiology , Oxidative Stress , Acetophenones/pharmacology , Antioxidants/pharmacology , Cell Line , Coculture Techniques , Epithelial Cells/drug effects , Epithelial Cells/pathology , Humans , Kidney Calculi , Kidney Tubules/injuries , Kidney Tubules/pathology , Phosphorylation , Reactive Oxygen Species/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND/AIMS: Nephrolithiasis is a common and frequently occurring disease, its exact pathogenesis is remains unclear. Emerging data suggest that autophagy plays a vital role in the pathophysiological processes of kidney diseases. Therefore, this study was designed to investigate the potential role of autophagy in the formation of calcium oxalate (CaOx) kidney stones in rat model. METHODS: Thirty-two rats were randomly divided into four groups (eight rats/group): untreated control group, stone model group, rapamycin-treated group, chloroquine-treated group. Rat models of CaOx nephrolithiasis was administration of 0.75% ethylene glycol (EG) in their drinking water for 4 weeks. Western blot and transmission electron microscope (TEM) were used to detect the expression of autophagy related protein LC3-II, BECN1 and p62 and autophagic vacuoles respectively. Renal function was evaluated by measuring the levels of serum CRE and BUN. Renal tubular injury markers NGAL and Kim-1 was determined by ELISA kits. Von Kossa staining was used to assess crystal deposits and histological tissue injury. TUNEL staining was employed to assess apoptosis of the renal tubular cell. RESULTS: Compare with the controls, the expression of autophagy related protein LC3-II, BECN1 and number of autophagic vacuoles were increased significantly, whereas the p62 protein level was decreased in the stone model group. The levels of apoptosis, serum CRE and BUN, NGAL and Kim-1 in the stone model group were increased compared with the control group and crystals deposition and renal injury were increased significantly. However, the levels of autophagy, kidney injury and crystal deposition were decreased by chloroquine but increased by rapamycin. CONCLUSION: These findings suggested that rats were administration of ethylene glycol could lead to the formation of CaOx nephrolithiasis and autophagy activation. Inhibiting autophagy could be an effective therapeutic approach for decreasing the formation of nephrolithiasis.
Subject(s)
Autophagy/drug effects , Ethylene Glycol/pharmacology , Kidney/injuries , Nephrolithiasis/pathology , Animals , Calcium Oxalate , Chloroquine/pharmacology , Crystallization , Kidney Calculi/etiology , Nephrolithiasis/etiology , Rats , Sirolimus/pharmacologyABSTRACT
Calcifying nanoparticles have been linked to various types of human disease, but how they contribute to disease processes is unclear. Here, we examined whether and how calcifying nanoparticles isolated from patients with kidney stones are cytotoxic to human bladder cancer cells. Calcifying nanoparticles were isolated from midstream urine of patients with renal calcium oxalate stones and examined by electron microscopy. Human bladder cancer cells (EJ cells) were cultured in the presence of calcifying nanoparticles or nanohydroxyapatites for 12 and 72 h and examined for toxicity using the Cell Counting Kit-8, for autophagy using transmission electron microscopy and confocal microscopy, and for apoptosis using fluorescence microscopy, transmission electron microscopy, and flow cytometry. Changes in protein expression were analyzed by Western blotting. The results showed that the size and shape of the isolated calcifying nanoparticles were as expected. Calcifying nanoparticles were cytotoxic to EJ cells, more so than nanohydroxyapatites, and this was due, at least in part, to the production of intracellular reactive oxygen species. Transmission electron microscopy showed that calcifying nanoparticles were packaged into vesicles and autolysosomes. Calcifying nanoparticles induced greater autophagy and apoptosis than nanohydroxyapatites. Our findings demonstrate that calcifying nanoparticles can trigger bladder cancer cell injury by boosting reactive oxygen species production and stimulating autophagy and apoptosis.
Subject(s)
Apoptosis/drug effects , Autophagy/drug effects , Calcifying Nanoparticles/administration & dosage , Urinary Bladder Neoplasms/drug therapy , Calcifying Nanoparticles/chemistry , Cell Line, Tumor , Cell Survival/drug effects , Flow Cytometry , Humans , Kidney Calculi/chemistry , Kidney Calculi/metabolism , Microscopy, Confocal , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Reactive Oxygen Species/metabolism , Urinary Bladder Neoplasms/pathologyABSTRACT
There has recently been significant interest within the vibrational spectroscopy community to apply quantitative spectroscopic imaging techniques to histology and clinical diagnosis. However, many of the proposed methods require collecting spectroscopic images that have a similar region size and resolution to the corresponding histological images. Since spectroscopic images contain significantly more spectral samples than traditional histology, the resulting data sets can approach hundreds of gigabytes to terabytes in size. This makes them difficult to store and process, and the tools available to researchers for handling large spectroscopic data sets are limited. Fundamental mathematical tools, such as MATLAB, Octave, and SciPy, are extremely powerful but require that the data be stored in fast memory. This memory limitation becomes impractical for even modestly sized histological images, which can be hundreds of gigabytes in size. In this paper, we propose an open-source toolkit designed to perform out-of-core processing of hyperspectral images. By taking advantage of graphical processing unit (GPU) computing combined with adaptive data streaming, our software alleviates common workstation memory limitations while achieving better performance than existing applications.
Subject(s)
Image Processing, Computer-Assisted , Software , Spectrum Analysis , AlgorithmsABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Urolithiasis represents a predominant concern within urology due to its high recurrence rate and consequential surgical complications. Traditional Chinese Medicine (TCM), with a history spanning over 2000 years in treating kidney diseases, not only offers a less invasive and cost-effective option for treating and preventing urolithiasis, but also serves as a pharmacological treasure trove for the development of anti-urolithic drugs. AIM OF THE STUDY: With the continuous deepening of research on the anti-urolithic effects of Chinese medicines, the pharmacological mechanisms of TCMs against urolithiasis are continuously evolving. Therefore, it is essential to summarize the current research status, clinical effectiveness, and mechanisms of TCM in treating and preventing urolithiasis, to ascertain its potential in anti-urolithic treatments, and to provide a reference for future anti-urolithiasis drug research. METHODS: The electronic databases such as PubMed, Web of Science, and China National Knowledge Infrastructure (CNKI) have been utilized to retrieve relevant literature spanning from 2000 to September 2023, using keywords "Traditional Chinese Medicine" and "Urolithiasis". Then we conducted a visual analysis of the current status of related research, as well as a systematic organization of the therapeutic effects and underlying mechanisms of anti-urolithic TCMs. RESULTS: Through the organization of research models, therapeutic effects, and active ingredients of 31 potential anti-urolithic TCMs, we have systematically summarized the underlying mechanisms of TCMs in management of urolithiasis. Mechanistically, Chinese herbs facilitate stone expulsion by enhancing diuresis, instigating anti-spasmodic effects, and promoting ureteral peristalsis when addressing calculi. They also harbor the potential to dissolve pre-existing stones. In terms of stone recurrence prevention, TCM compounds obstruct stone formation through targeting the sequence of crystal adhesion, nucleation, growth, and aggregation to inhibit stone formation. Additionally, TCM's significant roles include stifling oxidative stress, augmenting urinary stone inhibitors, and harmonizing oxalate metabolism, all of which are critical actions in stone prevention. CONCLUSION: The anti-urolithic mechanism of TCM is multifaceted. Investigating the anti-urolithiasis mechanisms of TCM not only illuminates the potential of Chinese medicine in treating and preventing urolithiasis, but also uncovers active molecules and targets for drug treatment against calculus formation.
Subject(s)
Drugs, Chinese Herbal , Medicine, Chinese Traditional , Urolithiasis , Urolithiasis/drug therapy , Urolithiasis/prevention & control , Medicine, Chinese Traditional/methods , Humans , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Drugs, Chinese Herbal/chemistry , AnimalsABSTRACT
Background: Nephrolithiasis, a common and chronic urological condition, exerts significant pressure on both the general public and society as a whole. The precise mechanisms of nephrolith formation remain inadequately comprehended. Nevertheless, the utilization of proteomics methods has not been employed to examine the development of renal calculi in order to efficiently hinder and manage the creation and reappearance of nephrolith. Nowadays, with the rapid development of proteomics techniques, more efficient and more accurate proteomics technique is utilized to uncover the mechanisms underlying diseases. The objective of this study was to investigate the possible alterations of HK-2 cells when exposed to varying amounts of calcium oxalate (CaOx). The aim was to understand the precise development of stone formation and recurrence, in order to find effective preventive and treatment methods. Methods: To provide a complete view of the proteins involved in the development of nephrolithiasis, we utilized an innovative proteomics method called 4D-LFQ proteomic quantitative techniques. HK-2 cells were selected as our experimental subjects. Three groups (n = 3) of HK-2 cells were treated with intervention solutions containing 0 (negative control, NC), 1 mM, and 2 mM CaOx, respectively. For the proteins that showed differential expression, various analyses were conducted including examination of Gene Ontology (GO), Clusters of Orthologous Groups of proteins (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, enrichment analysis of protein domains, and hierarchical clustering analysis. The STRING database was used to identify the interaction network of the chosen proteins. Candidate proteins were validated using parallel reaction monitoring (PRM) in the end. Results: All three groups verified the repeatability of samples. According to the results of 4D-LFQ proteomic quantitative analysis, there were 120, 262, and 81 differentially expressed proteins (DEPs) in the 1 mM-VS-NC, 2 mM-VS-NC, and 2 mM-VS-1mM conditions, respectively. According to GO annotation, the functional enrichment analysis indicates that the differentially expressed proteins (DEPs) were notably enriched in promoting cell migration and the extracellular matrix, among other functions. Analysis of enrichment, based on the KEGG pathway, revealed significant enrichment of DEPs in complement and coagulation cascades, as well as in ECM-receptor (extracellular matrix-receptor) interaction and other related pathways. 14 DEPs of great interest were selected as candidate proteins, including FN1, TFRC, ITGA3, FBN1, HYOU1, SPP1, HSPA5, COL6A1, MANF, HIP1R, JUP, AXL, CTNNB1 and DSG2.The data from PRM demonstrated the variation trend of 14 DEPs was identical as 4D-LFQ proteomic quantitative analysis. Conclusion: Proteomics studies of CaOx-induced HK-2 cells using 4D-LFQ proteomic quantitative analysis and PRM may help to provide crucial potential target proteins and signaling pathways for elucidating the mechanism of nephrolithiasis and better treating nephrolithiasis.
ABSTRACT
The CRISPR-Cas system, initially for DNA-level gene editing and transcription regulation, has expanded to RNA targeting with the Cas13d family, notably the RfxCas13d. This advancement allows for mRNA targeting with high specificity, particularly after catalytic inactivation, broadening the exploration of translation regulation. This study introduces a CRISPR-dCas13d-eIF4G fusion module, combining dCas13d with the eIF4G translation regulatory element, enhancing target mRNA translation levels. This module, using specially designed sgRNAs, selectively boosts protein translation in targeted tissue cells without altering transcription, leading to notable protein expression upregulation. This system is applied to a kidney stone disease model, focusing on ferroptosis-linked GPX4 gene regulation. By targeting GPX4 with sgRNAs, its protein expression is upregulated in human renal cells and mouse kidney tissue, countering ferroptosis and resisting calcium oxalate-induced cell damage, hence mitigating stone formation. This study evidences the CRISPR-dCas13d-eIF4G system's efficacy in eukaryotic cells, presenting a novel protein translation research approach and potential kidney stone disease treatment advancements.
Subject(s)
CRISPR-Cas Systems , Calcium Oxalate , Disease Models, Animal , Eukaryotic Initiation Factor-4G , Ferroptosis , Ferroptosis/genetics , Mice , Animals , Calcium Oxalate/metabolism , CRISPR-Cas Systems/genetics , Humans , Eukaryotic Initiation Factor-4G/genetics , Eukaryotic Initiation Factor-4G/metabolism , Kidney Calculi/genetics , Kidney Calculi/metabolism , Protein Biosynthesis/genetics , Phospholipid Hydroperoxide Glutathione Peroxidase/genetics , Phospholipid Hydroperoxide Glutathione Peroxidase/metabolismABSTRACT
Multiple myeloma (MM) is the second most prevalent hematologic malignancy which remains uncurable. Numerous drugs have been discovered to inhibit MM cells. Indisulam, an aryl sulfonamide, has a potent anti-myeloma activity in vitro and in vivo. This study aims to explore the new mechanism of indisulam and investigate its potential use in combination with melphalan. We examined DNA damage in MM cells through various methods such as western blotting (WB), immunofluorescence, and comet assay. We also identified the role of topoisomerase IIα (TOP2A) using bioinformatic analyses. The impact of indisulam on the RNA and protein levels of TOP2A was investigated through qPCR and WB. Cell proliferation and apoptosis were assessed using CCK-8 assays, Annexin V/PI assays and WB. We predicted the synergistic effect of the combination treatment based on calculations performed on a website, and further explored the effect of indisulam in combination with melphalan on MM cell lines and xenografts. RNA sequencing data and basic experiments indicated that indisulam caused DNA damage and inhibited TOP2A expression by decreasing transcription and promoting degradation via the proteasome pathway. Functional experiments revealed that silencing TOP2A inhibited cell proliferation and induced apoptosis and DNA damage. Finally, Indisulam/melphalan combination treatment demonstrated a strong synergistic anti-tumor effect compared to single-agent treatments in vitro and in vivo. These findings suggest that combination therapies incorporating indisulam and melphalan have the potential to enhance treatment outcomes for MM.
Subject(s)
Melphalan , Multiple Myeloma , Humans , Melphalan/pharmacology , Melphalan/therapeutic use , Multiple Myeloma/drug therapy , Multiple Myeloma/genetics , Multiple Myeloma/pathology , Cell Line, Tumor , Sulfonamides/pharmacology , Sulfonamides/therapeutic useABSTRACT
Oxalate is an organic dicarboxylic acid that is a common component of plant foods. The kidneys are essential organs for oxalate excretion, but excessive oxalates may induce kidney stones. Jupiter microtubule associated homolog 2 (JPT2) is a critical molecule in Ca2+ mobilization, and its intrinsic mechanism in oxalate exposure and kidney stones remains unclear. This study aimed to reveal the mechanism of JPT2 in oxalate exposure and kidney stones. Genetic approaches were used to control JPT2 expression in cells and mice, and the JPT2 mechanism of action was analyzed using transcriptomics and untargeted metabolomics. The results showed that oxalate exposure triggered the upregulation of JPT2, which is involved in nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca2+ mobilization. Transcriptomic analysis revealed that cell adhesion and macrophage inflammatory polarization were inhibited by JPT2 knockdown, and these were dominated by phosphatidylinositol 3-kinase (PI3K)/AKT signaling, respectively. Untargeted metabolomics indicated that JPT2 knockdown inhibited the production of succinic acid semialdehyde (SSA) in macrophages. Furthermore, JPT2 deficiency in mice inhibited kidney stones mineralization. In conclusion, this study demonstrates that oxalate exposure facilitates kidney stones by promoting crystal-cell adhesion, and modulating macrophage metabolism and inflammatory polarization via JPT2/PI3K/AKT signaling.
ABSTRACT
Oxalate-induced damage to renal tubular epithelial cells (RTECs) is an essential factor in the incident kidney stone, but the specific mechanism is unclear. Recent research has pinpointed interacting areas within the endoplasmic reticulum and mitochondria, called mitochondria-associated membranes (MAMs). These studies have linked endoplasmic reticulum stress (ERS) and oxidative imbalance to kidney disease development. The sigma-1 receptor (S1R), a specific protein found in MAMs, is involved in various physiological processes, but its role in oxalate-induced kidney stone formation remains unclear. In this study, we established cellular and rat models of oxalate-induced kidney stone formation to elucidate the S1R's effects against ERS and apoptosis and its mechanism in oxalate-induced RTEC injury. We found that oxalate downregulated S1R expression in RTECs and escalated oxidative stress and ERS, culminating in increased apoptosis. The S1R agonist dimemorfan up-regulated S1R expression and mitigated ERS and oxidative stress, thereby reducing apoptosis. This protective effect was mediated through S1R inhibition of the CHOP pathway. Animal experiments demonstrated that S1R's activation attenuated oxalate-induced kidney injury and alleviated kidney stone formation. This is the first study to establish the connection between S1R and kidney stones, suggesting S1R's protective role in inhibiting ERS-mediated apoptosis to ameliorate kidney stone formation.
Subject(s)
Apoptosis , Endoplasmic Reticulum Stress , Endoplasmic Reticulum , Epithelial Cells , Kidney Tubules , Mitochondria , Nephrolithiasis , Receptors, sigma , Sigma-1 Receptor , Receptors, sigma/metabolism , Animals , Endoplasmic Reticulum Stress/drug effects , Apoptosis/drug effects , Rats , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Kidney Tubules/metabolism , Kidney Tubules/pathology , Nephrolithiasis/metabolism , Mitochondria/metabolism , Mitochondria/drug effects , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/drug effects , Male , Oxidative Stress/drug effects , Rats, Sprague-DawleyABSTRACT
Mycotoxins are toxic compounds that pose a serious threat to animal health and food safety. Therefore, there is an urgent need for safe and efficient methods of detoxifying mycotoxins. As biotechnology has continued to develop, methods involving biological enzymes have shown great promise. Biological enzymatic methods, which can fundamentally destroy the structures of mycotoxins and produce degradation products whose toxicity is greatly reduced, are generally more specific, efficient, and environmentally friendly. Mycotoxin-degrading enzymes can thus facilitate the safe and effective detoxification of mycotoxins which gives them a huge advantage over other methods. This article summarizes the newly discovered degrading enzymes that can degrade four common mycotoxins (aflatoxins, zearalenone, deoxynivalenol, and ochratoxin A) in the past five years, and reveals the degradation mechanism of degrading enzymes on four mycotoxins, as well as their positive effects on animal production. This review will provide a theoretical basis for the safe treatment of mycotoxins by using biological enzyme technology.