The Role and Mechanism of Paeoniae Radix Alba in Tumor Therapy.

Tumors have a huge impact on human life and are now the main cause of disease-related deaths. The main means of treatment are surgery and radiotherapy, but they are more damaging to the organism and have a poor postoperative prognosis. Therefore, we urgently need safe and effective drugs to treat tumors. In recent years, Chinese herbal medicines have been widely used in tumor therapy as complementary and alternative therapies. Medicinal and edible herbs are popular and have become a hot topic of research, which not only have excellent pharmacological effects and activities, but also have almost no side effects. Therefore, as a typical medicine and food homology, some components of Paeoniae Radix Alba (PRA, called Baishao in China) have been shown to have good efficacy and safety against cancer. Numerous studies have also shown that Paeoniae Radix Alba and its active ingredients treat cancer through various pathways and are also one of the important components of many antitumor herbal compound formulas. In this paper, we reviewed the literature on the intervention of Paeoniae Radix Alba in tumors and its mechanism of action in recent years and found that there is a large amount of literature on its effect on total glucosides of paeony (TGP) and paeoniflorin (PF), as well as an in-depth discussion of the mechanism of action of Paeoniae Radix Alba and its main constituents, with a view to promote the clinical development and application of Paeoniae Radix Alba in the field of antitumor management.

m6A methyltransferase METTL14­mediated RP1­228H13.5 promotes the occurrence of liver cancer by targeting hsa­miR­205/ZIK1.

The aim of the present study was to explore the association between N6­methyladenosine (m6A) modification regulatory gene­related long noncoding (lnc)RNA RP1­228H13.5 and cancer prognosis through bioinformatics analysis, as well as the impact of RP1­228H13.5 on cell biology­related behaviors and specific molecular mechanisms. Bioinformatics analysis was used to construct a risk model consisting of nine genes. This model can reflect the survival time and differentiation degree of cancer. Subsequently, a competing endogenous RNA network consisting of 3 m6A­related lncRNAs, six microRNAs (miRs) and 201 mRNAs was constructed. A cell assay confirmed that RP1­228H13.5 is significantly upregulated in liver cancer cells, which can promote liver cancer cell proliferation, migration and invasion, and inhibit liver cancer cell apoptosis. The specific molecular mechanism may be the regulation of the expression of zinc finger protein interacting with K protein 1 (ZIK1) by targeting the downstream hsa­miR­205. Further experiments found that the m6A methyltransferase 14, N6­adenosine­methyltransferase subunit mediates the regulation of miR­205­5p expression by RP1­228H13.5. m6A methylation regulatory factor­related lncRNA has an important role in cancer. The targeting of hsa­miR­205 by RP1­228H13.5 to regulate ZIK1 may serve as a potential mechanism in the occurrence and development of liver cancer.

Banxia-Shengjiang drug pair inhibits gastric cancer development and progression by improving body immunity.

To investigate the mechanism of action of Banxia-Shengjiang drug pair on the inhibition of gastric cancer (GC) using network pharmacology and bioinformatics techniques. The action targets of the Banxia (Pinellia ternata (Thunb.) Makino) -Shengjiang (Zingiber officinale Roscoe) drug pair obtained from the TCMSP database were intersected with differentially expressed genes (DEGs) and GC-related genes, and the intersected genes were analyzed for pathway enrichment to identify the signaling pathways and core target genes. Subsequently, the core target genes were analyzed for clinical relevance gene mutation analysis, methylation analysis, immune infiltration analysis and immune cell analysis. Finally, by constructing the PPI network of hub genes and corresponding active ingredients, the key active ingredients of the Banxia-Shengjiang drug pair were screened for molecular docking with the hub genes. In this study, a total of 557 target genes of Banxia-Shengjiang pairs, 7754 GC-related genes and 1799 DEGs in GC were screened. Five hub genes were screened, which were PTGS2, MMP9, PPARG, MMP2, and CXCR4. The pathway enrichment analyses showed that the intersecting genes were associated with RAS/MAPK signaling pathway. In addition, the clinical correlation analysis showed that hub genes were differentially expressed in GC and was closely associated with immune infiltration and immunotherapy. The results of single nucleotide variation (SNV) and copy number variation (CNV) indicated that mutations in the hub genes were associated with the survival of gastric cancer patients. Finally, the PPI network and molecular docking results showed that PTGS2 and MMP9 were potentially important targets for the inhibition of GC by Banxia-Shengjiang drug pair, while cavidine was an important active ingredient for the inhibition of GC by Banxia-Shengjiang drug pair. Banxia-Shengjiang drug pair may regulate the immune function and inhibit GC by modulating the expression of core target genes such as RAS/MAPK signaling pathway, PTGS2 and MMP9.

Pan-cancer analysis of NUDT21 and its effect on the proliferation of human head and neck squamous cell carcinoma.

BACKGROUND: Based on bioinformatics research of NUDT21 in pan-cancer, we aimed to clarify the mechanism of NUDT21 in HHNC by experiment. METHODS: The correlation between differential expression of NUDT21 in pan-cancer and survival prognosis, genomic instability, tumor stemness, DNA repair, RNA methylation and with immune microenvironment were analyzed by the application of different pan-cancer analysis web databases. In addition, immunohistochemistry staining and genetic detection of NUDT21 in HHNCC tumor tissues by immunohistochemistry and qRT-PCR. Then, through in vitro cell experiments, NUDT21 was knocked down by lentivirus to detect the proliferation, cycle, apoptosis of FaDu and CNE-2Z cells, and finally by PathScan intracellular signaling array reagent to detect the apoptotic protein content. RESULTS: Based on the pan-cancer analysis, we found that elevated expression of NUDT21 in most cancers was significantly correlated with TMB, MSI, neoantigens and chromosomal ploidy, and in epigenetics, elevated NUDT21 expression was strongly associated with genomic stability, mismatch repair genes, tumor stemness, and RNA methylation. Based on immunosuppressive score, we found that NUDT21 plays an essential role in the immunosuppressive environment by suppressing immune checkpointing effect in most cancers. In addition, using HHNSCC as a study target, PCR and pathological detection of NUDT21 in tumor tissues was significantly increased than that in paracancerous normal tissues. In vitro cellular assays, silencing NUDT21 inhibited proliferation and promoted apoptosis in FaDu and CNE-2Z cells, and blocked the cell cycle in the G2/M phase. Therefore, the experiments confirmed that NUDT21 promotes the proliferation of FaDu by suppressing the expression of apoptotic.

Molecular mechanism of Gan-song Yin inhibiting the proliferation of renal tubular epithelial cells by regulating miR-21-5p in adipocyte exosomes.

ETHNOPHARMACOLOGICAL RELEVANCE: Gan-song Yin is derived from the classic ancient prescription " Gan-song pill " for the treatment of wasting-thirst in Ningxia combined with the characteristic "fragrant medicine". It is clinically used for the treatment of early renal fibrosis caused by diabetic nephropathy. Previous studies have shown that it has a good effect and great potential in the prevention and treatment of diabetic nephropathy, but its mechanism research is still limited. AIM OF THE STUDY: To investigate the mechanism of GSY to improve DN by interfering with miR-21-5p and glycolipid metabolism in adipocyte exosomes using 3T3-L1 and TCMK-1 co-culture system. MATERIALS AND METHODS: The co-culture system of 3T3-L3 and TCMK-1 was established, the IR model was established, and the stability, lipid drop change, glucose consumption, triglyceride content, cell viability, cell cycle and apoptosis level, protein content and mRNA expression of the IR model were detected. RESULTS: GSY inhibited 3T3-L1 activity, increased glucose consumption and decreased TG content. Decreased TCMK-1 cell viability, inhibited apoptosis, cell cycle arrest occurred in G0/G1 phase and S phase. Adipocyte IR model and co-culture system were stable within 48 h. After GSY intervention, lipid droplet decomposition and glucose consumption increased. The TG content of adipocytes increased, while the TG content of co-culture system decreased. GSY can regulate the expression of TGF-ß1/SMAD signaling pathway protein in IR state. After GSY intervention, the expression of miR-21-5p was increased in 3T3-L1 and Exo cells, and decreased in TCMK-1 cells. CONCLUSIONS: GSY can regulate TGF-ß1/SMAD signaling pathway through the secretion of miR-21-5p from adipocytes, protect IR TCMK-1, regulate the protein and mRNA expression levels of PPARγ, GLUT4, FABP4, and improve glucose and lipid metabolism.

Research progress of ginger in the treatment of gastrointestinal tumors.

Cancer seriously endangers human health. Gastrointestinal cancer is the most common and major malignant tumor, and its morbidity and mortality are gradually increasing. Although there are effective treatments such as radiotherapy and chemotherapy for gastrointestinal tumors, they are often accompanied by serious side effects. According to the traditional Chinese medicine and food homology theory, many materials are both food and medicine. Moreover, food is just as capable of preventing and treating diseases as medicine. Medicine and food homologous herbs not only have excellent pharmacological effects and activities but also have few side effects. As a typical medicinal herb with both medicinal and edible uses, some components of ginger have been shown to have good efficacy and safety against cancer. A mass of evidence has also shown that ginger has anti-tumor effects on digestive tract cancers (such as gastric cancer, colorectal cancer, liver cancer, laryngeal cancer, and pancreatic cancer) through a variety of pathways. The aim of this study is to investigate the mechanisms of action of the main components of ginger and their potential clinical applications in treating gastrointestinal tumors.

Exploring the targets and molecular mechanism of glycyrrhetinic acid against diabetic nephropathy based on network pharmacology and molecular docking.

BACKGROUND: Diabetic nephropathy (DN) stands as the most prevalent chronic microvascular complication of diabetes mellitus. Approximately 50% of DN patients progress to end-stage renal disease, posing a substantial health burden. AIM: To employ network pharmacology and molecular docking methods to predict the mechanism by which glycyrrhetinic acid (GA) treats DN, subsequently validating these predictions through experimental means. METHODS: The study initially identified GA targets using Pharm Mapper and the TCMSP database. Targets relevant to DN were obtained from the Genecards, OMIM, and TTD databases. The Venny database facilitated the acquisition of intersecting targets between GA and DN. The String database was used to construct a protein interaction network, while DAVID database was used to conducted Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and Gene Ontology (GO) analysis. Molecular docking experiments were performed using Autodock software with selected proteins. Experimental validation was conducted using renal proximal tubular cells (HK-2) as the study subjects. A hyperglycemic environment was simulated using glucose solution, and the effect of GA on cell viability was assessed through the cell counting kit-8 method. Flow cytometry was employed to detect cell cycle and apoptosis, and protein immunoblot (western blot) was used to measure the expression of proteins of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and insulin resistance pathway, including insulin receptor (INSR), PI3K, p-PI3K, AKT, p-AKT, and glycogen synthase kinase-3 (GSK3). RESULTS: A total of 186 intersecting targets between GA and DN were identified, which were associated with 144 KEGG-related enrichment pathways, 375 GO biological process entries, 45 GO cellular component entries, and 112 GO cellular function entries. Molecular docking demonstrated strong binding of GA to mitogen-activated protein kinase (MAPK)-1, SRC, PIK3R1, HSP90AA1, CASPASE9, HARS, KRAS, and MAPK14. In vitro experiments revealed that GA inhibited HK-2 cell viability, induced cell cycle arrest at the G2/M phase, and reduced apoptosis with increasing drug concentration. Western blot analysis showed that GA differentially up-regulated GSK3 protein expression, up-regulated AKT/p-AKT expression, down-regulated INSR, AKT, p-AKT, PI3K, and p-PI3K protein expression, and reduced p-PI3K/PI3K levels under high glucose conditions. CONCLUSION: GA may protect renal intrinsic cells by modulating the PI3K/AKT signaling pathway, thereby inhibiting HK-2 cell viability, reducing HK-2 cell apoptosis, and inducing cell cycle arrest at the G0/G1 phase.

Exploring the mechanism of Dahuang-Tusizi drug pair in the treatment of diabetes nephropathy based on network pharmacology and immune infiltration analysis.

The study aimed to explore the key targets and molecular mechanisms of Dahuang-Tusizi drug pair (DTDP) in the treatment of diabetes nephropathy (DN) based on the GEO database by using network pharmacology combined with molecular docking and immune infiltration. The active components of the DTDP were screened using the Traditional Chinese Medicine Systems Pharmacology database and the Swiss Target Prediction database. The differential genes of DN were retrieved from GEO databases. Next, the intersecting targets of drug and disease were imported into the String database for protein-protein interactions network analysis, and the core targets were identified through topological analysis. Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes enrichment analyses were performed with the help of the Metascape database and gene set enrichment analysis database. Subsequently, molecular docking was performed to verify the binding activity of the key component and the key target. The Nephroseq V5 database was used to verify the clinical relevance of DN and core genes. Finally, the Using CIBERSORT Algorithm to analyze the immune Infiltration of DN Gene Chip. The network analysis showed that 25 active ingredients of DTDP were associated with 22 targets in DN. The key active ingredients (Sesamin, quercetin, EUPATIN, matrine, beta-sitosterol, isorhamnetin, etc.) and the core targets (JUN, EGF, CD44, FOS, KDR, CCL2, PTGS2, and MMP2) were further identified. Enrichment analysis revealed signaling pathways including TNF, MAPK, and IL-17 signaling pathway. Molecular docking results showed that there was a strong affinity between the key components and core targets. The results of immune infiltration found that the proportion of macrophages in DN tissues was significantly increased. Our findings demonstrated that the characteristics of DTDP in treating DN are "multiple components, multiple targets and multiple pathways." We predicted that DTDP may inhibit inflammation related pathways by regulating key genes, reducing macrophage infiltration. Thus, inhibiting inflammatory response to reduce glomerular damage and delay the development of DN.

Klotho/FGF23 Axis Regulates Cardiomyocyte Apoptosis and Cytokine Release through ERK/MAPK Pathway.

Coronary artery disease (CAD) as a major cardiovascular disease is the leading global cause of mortality, Klotho/FGF23 axis involved in development of cardiovascular disease, while the function and underlying mechanism of Klotho/FGF23 axis in CAD is unclear. Blood samples from 67 CAD patients with coronary artery bypass graft (CABG) surgery were collected, and the level of Klotho and FGF23 of those patients was measured by using an ELISA kit. Cardiomyocyte was isolated from 0 to 3 days Sprague Dawley (SD) rats. Expression of Klotho, FGF23 and the cardiomyocyte marker α-sarcomeric actin (α-SA), myosin heavy chain (MHC) and cardiac troponin I (cTnI) was assessed by immunofluorescence staining. Expression of Klotho and FGF23 mRNA was detected by qRT-PCR. Apoptosis and cell cycle were measured by flow cytometry. Cell viability was detected by using CCK-8. The protein expression of ERK/MAPK pathway related protein and cytokines production was measured by western blotting. The levels of Klotho in CAD patients increased after CABG surgery, while FGF23 decreased. Isolated cardiomyocyte morphology and structure were completed, and with stabilized beating within culture for 15 days, besides, α-SA, MHC, and cTnI proved positive. After transfected Lenti-Klotho and Lenti-FGF23 into isolated cardiomyocyte, fluorescence staining showed that the transfection was successful, and qRT-PCR results showed that the expression levels of Klotho and FGF23 mRNA significant increased compared with NEG (empty vector) group. Immunofluorescence staining results showed that compared with NEG group, there was a higher Klotho positive rate and lower FGF23 positive rate in Klotho overexpression (Klotho) group, while, there was a higher FGF23 positive rate and lower Klotho positive rate in FGF23 overexpression (FGF23) group. In addition, the expression of p-ERK1/2 and p-P38 increased in Klotho group but decreased in FGF23 group. Furthermore, overexpression of Klotho inhibited cardiomyocyte apoptosis, increased S phase fraction, promoted proliferation and elevated expression of transforming growth factor ß1 (TGF-ß1), nuclear factor-kappa B (NF-κB), angiotensin-II (AT-II), and activator protein-1 (AP-1), overexpression of FGF23 showed the opposite effect, however, ERK agonist (TPA) and inhibitor (U0126) reversed the effect caused by overexpression of Klotho and FGF23 separately. Klotho/FGF23 axis play a critical role in CAD progression through regulating ERK/MAPK pathway in Cardiomyocyte.

MiR-204-3p overexpression inhibits gastric carcinoma cell proliferation by inhibiting the MAPK pathway and RIP1/MLK1 necroptosis pathway to promote apoptosis.

BACKGROUND: Gastric carcinoma (GC) is the third most frequent cause of cancer-related death, highlighting the pressing need for novel clinical treatment options. In this regard, microRNAs (miRNAs) have emerged as a promising therapeutic strategy. Studies have shown that miRNAs can regulate related signaling pathways, acting as tumor suppressors or tumor promoters. AIM: To explore the effect of miR-204-3p on GC cells. METHODS: We measured the expression levels of miR-204-3p in GC cells using quantitative real-time polymerase chain reaction, followed by the delivery of miR-204-3p overexpression and miR-204-3p knockdown vectors into GC cells. CCK-8 was used to detect the effect of miR-204-3p on the proliferation of GC cells, and the colony formation ability of GC cells was detected by the clonal formation assay. The effects of miR-204-3p on GC cell cycle and apoptosis were detected by flow cytometry. The BABL/c nude mouse subcutaneous tumor model using MKN-45 cells was constructed to verify the effect of miR-204-3p on the tumorigenicity of GC cells. Furthermore, the study investigated the effects of miR-204-3p on various proteins related to the MAPK signaling pathway, necroptosis signaling pathway and apoptosis signaling pathway on GC cells using Western blot techniques. RESULTS: Firstly, we found that the expression of miR-204-3p in GC was low. When treated with the lentivirus overexpression vector, miR-204-3p expression significantly increased, but the lentivirus knockout vector had no significant effect on miR-204-3p. In vitro experiments confirmed that miR-204-3p overexpression inhibited GC cell viability, promoted cell apoptosis, blocked the cell cycle, and inhibited colony formation ability. In vivo animal experiments confirmed that miR-204-3p overexpression inhibited subcutaneous tumorigenesis ability in BABL/c nude mice. Simultaneously, our results verified that miR-204-3p overexpression can inhibit GC cell proliferation by inhibiting protein expression levels of KRAS and p-ERK1/2 in the MAPK pathway, as well as inhibiting protein expression levels of p-RIP1 and p-MLK1 in the necroptosis pathway to promote the BCL-2/BAX/Caspase-3 apoptosis pathway. CONCLUSION: MiR-204-3p overexpression inhibited GC cell proliferation by inhibiting the MAPK pathway and necroptosis pathway to promote apoptosis of GC cells. Thus, miR-204-3p may represent a new potential therapeutic target for GC.

The Role and Mechanism of Perilla frutescens in Cancer Treatment.

Perilla frutescens is an annual herb of the Labiatae family and is widely grown in several countries in Asia. Perilla frutescens is a plant that is used medicinally in its entirety, as seen in its subdivision into perilla seeds, perilla stalks, and perilla leaves, which vary more markedly in their chemical composition. Several studies have shown that Perilla frutescens has a variety of pharmacological effects, including anti-inflammatory, antibacterial, detoxifying, antioxidant, and hepatoprotective. In the absence of a review of Perilla frutescens for the treatment of cancer. This review provides an overview of the chemical composition and molecular mechanisms of Perilla frutescens for cancer treatment. It was found that the main active components of Perilla frutescens producing cancer therapeutic effects were perilla aldehyde (PAH), rosmarinic acid (Ros A), lignan, and isoestrogen (IK). In addition to these, extracts of the leaves and fruits of Perilla frutescens are also included. Among these, perilla seed oil (PSO) has a preventive effect against colorectal cancer due to the presence of omega-3 polyunsaturated fatty acids. This review also provides new ideas and thoughts for scientific innovation and clinical applications related to Perilla frutescens.

Induced Pluripotent Stem Cells and Their Applications in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that results in the loss of motor function in the central nervous system (CNS) and ultimately death. The mechanisms underlying ALS pathogenesis have not yet been fully elucidated, and ALS cannot be treated effectively. Most studies have applied animal or single-gene intervention cell lines as ALS disease models, but they cannot accurately reflect the pathological characteristics of ALS. Induced pluripotent stem cells (iPSCs) can be reprogrammed from somatic cells, possessing the ability to self-renew and differentiate into a variety of cells. iPSCs can be obtained from ALS patients with different genotypes and phenotypes, and the genetic background of the donor cells remains unchanged during reprogramming. iPSCs can differentiate into neurons and glial cells related to ALS. Therefore, iPSCs provide an excellent method to evaluate the impact of diseases on ALS patients. Moreover, patient-derived iPSCs are obtained from their own somatic cells, avoiding ethical concerns and posing only a low risk of immune rejection. The iPSC technology creates new hope for ALS treatment. Here, we review recent studies on iPSCs and their applications in disease modeling, drug screening and cell therapy in ALS, with a particular focus on the potential for ALS treatment.

miR-193a-3p Enhanced the Chemosensitivity to Trametinib in Gallbladder Carcinoma by Targeting KRAS and Downregulating ERK Signaling.

Objective: In this study, the authors identified miR-193a-3p as a tumor-suppressing microRNA, and its effects on the chemosensitivity to trametinib in gallbladder carcinoma (GBC) were evaluated. Materials and Methods: The levels of miR-193a-3p in clinical GBC tissues and GBC cells were determined by quantitative real-time polymerase chain reaction. The protein levels of KRAS, ERK, and phosphorylated ERK (p-ERK) were examined by Western blot. Dual-luciferase reporter assays were performed to confirm the interaction between miR-193a-3p and KRAS. The effect of miR-193a-3p knockdown or overexpression on the malignant behaviors and chemosensitivity of GBC was determined by 3-(4,5-dimethlthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide and flow cytometry assays in vitro and further examined in a xenograft model. Results: The levels of miR-193a-3p were significantly decreased in GBC cell lines, especially with KRAS mutations. In addition, miR-193a-3p overexpression retarded cell proliferation of GBC, but induced cell apoptosis. Moreover, miR-193a-3p overexpression significantly improved the chemosensitivity of GBC to trametinib both in in vitro assays and in vivo xenograft mouse model. Further mechanisms disclosed that KRAS was a target of miR-193a-3p and levels of p-ERK were increased by treatment with miR-193a-3p inhibitor in GBC. Conclusions: These data suggested that miR-193a-3p enhanced the chemosensitivity to trametinib in GBC with wild-type KRAS or KRAS mutations by directly targeting KRAS and finally downregulated ERK signaling.

Anti-tumor effect of coix seed based on the theory of medicinal and food homology.

Coix seed is a dry and mature seed of Coix lacryma-jobi L.var.ma-yuen (Roman.) Stapf in the Gramineae family. Coix seed has a sweet, light taste, and a cool nature. Coix seed enters the spleen, stomach, and lung meridians. It has the effects of promoting diuresis and dampness, strengthening the spleen to prevent diarrhea, removing arthralgia, expelling pus, and detoxifying and dispersing nodules. It is used for the treatment of edema, athlete's foot, poor urination, spleen deficiency and diarrhea, dampness and obstruction, lung carbuncle, intestinal carbuncle, verruca, and cancer. The medicinal and health value is high, and it has been included in the list of medicinal and food sources in China, which has a large development and application space. This article reviews the current research achievements in the processing methods and anti-tumor activities of Coix seed and provides examples of its clinical application in ancient and modern times, aiming to provide reference for further research on Coix seed and contribute to its clinical application and development. Through the analysis of the traditional Chinese patent medicines, and simple preparations and related health food of Coix seed queried by Yaozhi.com, the source, function, and dosage form of Coix seed were comprehensively analyzed, with a view of providing a reference for the development of Coix seed medicine and food.

Wnt5a protects motor neurons in amyotrophic lateral sclerosis by regulating the Wnt/Ca2+ signaling pathway.

OBJECTIVES: We aimed to detect the expression profile of downstream signaling molecules of non-canonical Wnt pathway in SOD1G93A transgenic mice (ALS mice) and SOD1G93A mutant motor neuron-like hybrid (NSC-34) cells. Characterizing the molecular mechanism of the Wnt5a-mediated non-canonical Wnt/Ca2+ signaling pathway in motor neuron (MN) degeneration may provide a feasible approach to effective treatment of amyotrophic lateral sclerosis (ALS). METHODS: The expressions of CaMKII-α, CaMKII-ß and TAK1 in the spinal cord of SOD1G93A ALS transgenic mice at different ages were determined using western blotting and immunofluorescence. The level of Ca2+ and cell apoptosis were assessed with flow cytometry and cell viability was evaluated using MTS assay. Cell proliferation was analyzed by the EdU cell proliferation assay. Neurite length was measured after treatment with retinoic acid. RESULTS: CaMKII-α, CaMKII-ß, and TAK1 were down-regulated in the spinal cord of ALS mice. Ca2+ level and CaMKII-α, CaMKII-ß, and TAK1 were down-regulated in SOD1G93A mutant NSC-34 cells. Expression of Ca2+, CaMKII-α, CaMKII-ß, and TAK1 were up-regulated in SOD1G93A mutant NSC-34 cells after Wnt5a overexpression and down-regulated after Wnt5a knockdown. Overexpression of Wnt5a promoted cell viability and proliferation but inhibited cell apoptosis. Contrastingly, Wnt5a knockdown inhibited cell viability and proliferation but promoted cell apoptosis. CaMKII inhibitor KN-93 and CaMKII activator oleic acid reversed changes in cell viability, proliferation, apoptosis, and neurite outgrowth induced by Wnt5a overexpression and knockdown. CONCLUSIONS: This study demonstrates that Wnt5a protects MNs in ALS by regulating cell viability, proliferation, apoptosis, and neurite growth through the Wnt/Ca2+ signaling pathway. Our data indicate that the non-canonical Wnt/Ca2+ signaling pathway regulated by Wnt5a is involved in MN degeneration in ALS.

Spatiotemporal evolution of pyroptosis and canonical inflammasome pathway in hSOD1G93A ALS mouse model.

BACKGROUND: Evidences indicate that inflammasome compounds participate in amyotrophic lateral sclerosis (ALS), a fatal progressive motoneuron degenerative disease. Researchers have observed the expressions of nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) related inflammasome components in specific regions of the central nervous system in different ALS models, but the cellular spatiotemporal evolution of this canonical inflammasome pathway and pyroptosis during ALS progression are unclear. METHODS: The spinal cords of hSOD1G93A mice (ALS mice) and age-matched littermates (CON mice) were dissected at pre-symptomatic stage (60 d), early- symptomatic stage (95 d), symptomatic stage (108 d) and late-symptomatic stage (122 d) of the disease. By using Nissl staining, double immunofluorescence labelling, qRT-PCR or western blot, we detected morphology change and the expression, cellular location of GSDMD, NLRP3, caspase-1 and IL-1ß in the ventral horn of lumbar spinal cords over the course of disease. RESULTS: Neural morphology changes and GSDMD+/NeuN+ double positive cells were observed in ventral horn from ALS mice even at 60 d of age, even though there were no changes of GSDMD mRNA and protein expressions at this stage compared with CON mice. With disease progression, compared with age-matched CON mice, increased expressions of GSDMD, NLRP3, activated caspase-1 and IL-1ß were detected. Double immunofluorescence labeling revealed that NLRP3, caspase-1, IL-1ß positive signals mainly localized in ventral horn neurons at pre- and early-symptomatic stages. From symptomatic stage to late-symptomatic stage, robust positive signals were co-expressed in reactive astrocytes and microglia. CONCLUSIONS: Early activation of the canonical NLRP3 inflammasome induced pyroptosis in ventral horn neurons, which may participate in motor neuron degeneration and initiate neuroinflammatory processes during ALS progression.

The Impact of Mitochondrial Dysfunction in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and highly fatal neurodegenerative disease. Although the pathogenesis of ALS remains unclear, increasing evidence suggests that a key contributing factor is mitochondrial dysfunction. Mitochondria are organelles in eukaryotic cells responsible for bioenergy production, cellular metabolism, signal transduction, calcium homeostasis, and immune responses and the stability of their function plays a crucial role in neurons. A single disorder or defect in mitochondrial function can lead to pathological changes in cells, such as an impaired calcium buffer period, excessive generation of free radicals, increased mitochondrial membrane permeability, and oxidative stress (OS). Recent research has also shown that these mitochondrial dysfunctions are also associated with pathological changes in ALS and are believed to be commonly involved in the pathogenesis of the disease. This article reviews the latest research on mitochondrial dysfunction and its impact on the progression of ALS, with specific attention to the potential of novel therapeutic strategies targeting mitochondrial dysfunction.

Circulating miR-141 as a potential biomarker for diagnosis, prognosis and therapeutic targets in gallbladder cancer.

MicroRNA-141(miR-141) has been reported to play vital roles in the regulation of carcinogenesis and cancer progression. However, the biological function of miR-141 in GBC has received less attention. The aim of this study was to estimate the potential value of the expression level of miR-141 as a diagnostic and prognostic blood-based biomarker in gallbladder cancer (GBC) patients. Meanwhile, to explore its biological role in GBC cells. RT-PCR was employed to confirm the expression of miR-141 in ten paired tissue samples (10 GBC tissues and 10 adjacent normal gallbladder tissues), GBC cell lines and peripheral blood specimens from 98 GBC patients and 60 healthy controls. MTT assay was used to evaluate the GBC cells proliferation and flow cytometry was used to detect the cell apoptosis. Receiver operating characteristic curve analysis and the area under the curve (AUC) were used to evaluate the value of miR-141 plasma levels for GBC diagnosis. Finally, clinicopathological and survival data of all GBC patients were collected and analyzed. Here, we confirmed that the expression of miR-141 were upregulated in primary gallbladder cancer cells and tissues compared with human gallbladder epithelial cells and adjacent normal tissues (P < 0.0001). Meanwhile, we found that downregulated expression of miR-141 by miR-141 inhibitor could induce apoptosis and inhibit proliferation of GBC cells. Additionally, elevated plasma miR-141 expression was also detected in the peripheral blood of GBC patients compared with healthy controls (P < 0.0001). The AUC value of miR-141 for GBC diagnosis was 0.894 (95% CI 0.843-0.945), which was more valuable than those including carcinoembryonic antigen (CEA) (0.713, 95% CI 0.633-0.793), carbohydrate antigen 125 (CA125) (0.837, 95% CI 0.776-0.899) and carbohydrate antigen 19-9 (CA19-9) (0.869, 95% CI 0.813-0.924). The high expression level of miR-141 in plasma was significantly associated with tumor invasion (P = 0.008), lymph node metastasis (P < 0.0001) and advanced pathologic tumor/node/metastasis (pTNM) stage (P = 0.009). More importantly, high plasma miR-141 expression was an independent prognostic factor for predicting poorer long-term survival in GBC patients. Elevated expression of circulating miR-141 in peripheral blood might be a potential novel biomarker for diagnosis and prognosis of GBC patients. Downregulated expression of miR-141 could inhibit proliferation and induce apoptosis of GBC cells, that provide a potential therapeutic target for GBC.

The mechanism of the WNT5A and FZD4 receptor mediated WNT/ß-catenin pathway in the degeneration of ALS spinal cord motor neurons.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with unknown etiology, characterized by motor neuron degeneration, and there is no highly effective treatment. The canonical WNT/ß-catenin signaling pathway has a critical role in the physiological and pathophysiological processes of the central nervous system. In this study, we investigated the regulatory mechanism of the WNT/ß-catenin signaling pathway from the perspective of ligand-receptor binding and its relationship with the degeneration of ALS motor neurons. We used hSOD1-G93A mutant ALS transgenic mice and hSOD1-G93A mutant NSC34 cells combined with morphological and molecular biology techniques to determine the role of the WNT/ß-catenin pathway in ALS. Our findings demonstrated that WNT5A regulates the WNT/ß-catenin signaling pathway by binding to the FZD4 receptor in the pathogenesis of ALS and affects the proliferation and apoptosis of ALS motor neurons. Therefore, these findings may lead to the development of novel therapies to support the survival of ALS motor neurons.

The Biogenesis of miRNAs and Their Role in the Development of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects upper and lower motor neurons. As there is no effective treatment for ALS, it is particularly important to screen key gene therapy targets. The identifications of microRNAs (miRNAs) have completely changed the traditional view of gene regulation. miRNAs are small noncoding single-stranded RNA molecules involved in the regulation of post-transcriptional gene expression. Recent advances also indicate that miRNAs are biomarkers in many diseases, including neurodegenerative diseases. In this review, we summarize recent advances regarding the mechanisms underlying the role of miRNAs in ALS pathogenesis and its application to gene therapy for ALS. The potential of miRNAs to target diverse pathways opens a new avenue for ALS therapy.