As the main bioactive component of musk, muscone has been reported to have marked protective effects in treating acute ischemic stroke (AIS). However, the specific anti-stroke mechanism of muscone still needs further research. In the current investigation, the PC12 cells OGD/R and the rat transient MCAO/R models were utilized as the AIS models. Serum hepatic and renal functional indexes (ALT, AST, BUN, and Cr) and cell viability were determined to select the appropriate muscone concentrations for in vitro and in vivo experiments. TTC, Hematoxylin and eosin (H&E), and Live/Dead staining were utilized to evaluate the protective effects of muscone in injured tissues and cells. Western blotting analysis, TUNEL staining, propidium iodide, and annexin V staining were applied to detect the anti-apoptotic effect of muscone. Double-label immunofluorescence staining of T-cell intracellular antigen-1 (TIA1) and Ras-GAP SH3 domain-binding protein 1 (G3BP1) was performed to observe whether muscone regulated the SG formation level. Molecular docking, TIA1 silencing and TIA1 overexpression experiments were employed to investigate the molecular mechanism underlying the regulation of SG formation by muscone. The 2, 3, 5-Triphenyl-tetrazolium chloride (TTC) staining and live/dead staining showed the AIS injury level of MCAO/R rat and the OGD/R PC12 cells were attenuated by muscone administration. The muscone significantly minimized the apoptosis rate in MCAO/R rats and OGD/R PC12 cells following flow cytometry analysis, western blotting analysis, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. The double-label immunofluorescence staining data revealed that muscone promoted the SG formation level in OGD/R PC12 cells and the cortex MCAO/R rats. The results of molecular docking, TIA1 silencing and TIA1 overexpression experiments revealed that muscone could bind to TIA1 protein and regulate its expression level, thereby promoting the formation of stress granules and exerting a protective effect against AIS injury. This study indicated that the significant protective effect of muscone in reducing apoptosis levels might be via promoting SG formation under AIS conditions. This study further explores the therapeutic effect and anti-apoptosis mechanism of muscone in AIS, which may provide a potential candidate drug for the clinical treatment of AIS injury.

Ethnopharmacological relevance: Traditional Chinese medicine views kidney shortage as a significant contributor to the aetiology of Parkinson's disease (PD), a neurodegenerative condition that is closely linked to aging. In clinical, patients with Parkinson's disease are often treated with Testudinis Carapax et Plastrum (Plastrum Testudinis, PT), a traditional Chinese medication that tonifies the kidney. Previous research has demonstrated that ethyl stearate (PubChem CID: 8122), an active component of Plastrum Testudinis Extracted with ethyl acetate (PTE), may encourage neural stem cells (NSCs) development into dopaminergic (DAergic) neurons. However, the effectiveness and mechanism of cotransplantation of ethyl stearate and NSCs in treating PD model rats still require further investigation.Aim of the study: PD is a neurodegenerative condition marked by the loss and degradation of dopaminergic neurons in the substantia nigra of the midbrain. Synaptic damage is also a critical pathology in PD. Because of their self-renewal, minimal immunogenicity, and capacity to differentiate into dopaminergic (DAergic) neurons, NSCs are a prospective treatment option for Parkinson's disease cell transplantation therapy. However, encouraging transplanted NSCs to differentiate into dopaminergic neurons and enhancing synaptic plasticity in vivo remains a significant challenge in improving the efficacy of NSCs transplantation for PD. This investigation seeks to examine the efficacy of cotransplantation of NSCs and ethyl stearate in PD model rats and its mechanism related to synaptic plasticity. Materials and methods: On 6-hydroxydopamine-induced PD model rats, we performed NSCs transplantation therapy and cotransplantation therapy involving ethyl stearate and NSCs. Rotating behavior induced by apomorphine (APO) and pole climbing tests were used to evaluate behavioral changes. Using a variety of methods, including Western blotting (WB), immunofluorescence analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction (qRT-PCR), we examined the function and potential molecular mechanisms of ethyl stearate in combined NSCs transplantation therapy. Results: In the rat PD model, cotransplantation of ethyl stearate with NSCs dramatically reduced motor dysfunction, restored TH protein levels, and boosted dopamine levels in the striatum, according to our findings. Furthermore, the expression levels of SYN1 and PSD95, markers of synaptic plasticity, and BDNF, closely related to synaptic plasticity, were significantly increased. Cotransplantation with ethyl stearate and NSCs also increased the expression levels of Dopamine Receptor D1 (Drd1), an important receptor in the dopamine neural circuit, accompanied by an increase in MMP9 levels, ERK1/2 phosphorylation levels, and c-fos protein levels.Conclusions: According to the results of our investigation, cotransplantation of ethyl stearate and NSCs significantly improves the condition of PD model rats. We found that cotransplantation of ethyl stearate and NSCs may promote the expression of MMP9 by regulating the Drd1-ERK-AP-1 pathway, thus improving synaptic plasticity

Geraniin, a chemical component of the traditional Chinese medicine geranii herba, possesses anti-inflammatory and anti-oxidative activities. However, its anti-inflammatory role in managing NLRP3 inflammasome and pyroptosis remains to be elucidated. To investigate the anti-inflammation mechanism of geraniin, LPS-primed macrophages were incubated with classical activators of NLRP3 inflammasome (such as ATP, Nigericin, or MSU crystals), and MSU crystals were injected into the ankle joints of mice to establish an acute gouty arthritis model. The propidium iodide (PI) staining results showed that geraniin could restrain cell death in the ATP- or nigericin-stimulated bone marrow-derived macrophages (BMDMs). Geraniin decreased the release of lactate dehydrogenase (LDH) and interleukin (IL)-1β from cytoplasm to cell supernatant. Geraniin also inhibited the expression of caspase-1 p20, IL-1β in cell supernatant and N-terminal of gasdermin D (GSDMD-NT) while blocking the oligomerization of ASC to form speck. The inhibitory effects of geraniin on caspase-1 p20, IL-1β, GSDMD-NT, and ASC speck were not observed in NLRP3 knockout (NLRP3-/-) BMDMs. Hence, the resistance of geraniin to inflammasome and pyroptosis was contingent upon NLRP3 presence. Geraniin reduced reactive oxygen species (ROS) production and maintained mitochondrial membrane potential while preventing interaction between ASC and NLRP3 protein. Additionally, geraniin diminished MSU crystal-induced mouse ankle joint swelling and IL-1β expression. Geraniin blocked the recruitment of neutrophils and macrophages to the synovium of joints. Our results demonstrate that geraniin prevents the assembly of ASC and NLRP3 through its antioxidant effect, thereby inhibiting inflammasome activation, pyroptosis, and IL-1β release to provide potential insights for gouty arthritis targeted therapy.

Our recent study demonstrated that knockout of microRNA-301a attenuates migration and phagocytosis in macrophages. Considering that macrophages and Schwann cells synergistically clear the debris of degraded axons and myelin during Wallerian degeneration, which is a prerequisite for nerve regeneration, we hypothesized that microRNA-301a regulates Wallerian degeneration and nerve regeneration via impacts on Schwann cell migration and phagocytosis. Herein, we found low expression of microRNA-301a in intact sciatic nerves, with no impact of the microRNA-301a knockout on nerve structure and function. By contrast, we found significant upregulation of microRNA-301a in injured sciatic nerves. We established a sciatic nerve crush model in microRNA-301a knockout mice, which exhibited attenua9ted morphological and functional regeneration following sciatic nerve crush injury. The microRNA-301a knockout also led to significantly inhibited Wallerian degeneration in an in vivo sciatic nerve-transection model and in an in vitro nerve explant block model. Schwann cells with the microRNA-301a knockout showed inhibition of phagocytosis and migration, which was reversible under transfection with microRNA-301a mimics. Rescue experiments involving transfection of microRNA-301a-knockout Schwann cells with microRNA-301a mimics or treatment with the C-X-C motif receptor 4 inhibitor WZ811 indicated the mechanistic involvement of the Yin Yang 1/C-X-C motif receptor 4 pathway in the role of microRNA-301a. Combined with our previous findings in macrophages, we conclude that microRNA-301a plays a key role in peripheral nerve injury and repair by regulating the migratory and phagocytic capabilities of Schwann cells and macrophages via the Yin Yang 1/C-X-C motif receptor 4 pathway.

Background Depression is a psychiatric disorder which affects many aspects of social life of patients; however, the molecular biological mechanisms underlying its development are not fully understood. Atypical phase separation is a mechanism contributing to the occurrence of neurological disorders. The interaction between autophagy and iron metabolism have been implicated in the pathogenesis of neurological diseases.Methods To explore the pathogenesis of depression, we analyzed super-enhancers (SEs) in the prefrontal cortex of depression model rats using chromatin immunoprecipitation sequencing (ChIP-seq). The role of the upstream protein Bromodomain Containing 2 (BRD2) in super-enhancers (SEs) was investigated by synthesizing liquid-liquid phase separation proteins in vitro which were examined through Fluorescence recovery after photobleaching (FRAP) analysis. Moreover, the markers of ferritinophagy in cortisol-stimulated primary cortical neuron cells and PC12 cell models were explored to the occurrence of ferritinophagy in depression models.Results Rats exposed to chronic mild stress (CMS) exhibited decreased activation of autophagy-related 7 (ATG7) mediated by SEs. In rats subjected to CMS and in cellular models of depression, ferritinophagy was detected. Arid5a stimulated ATG7 activation via SEs and was influenced by the BET family and liquid-liquid phase separation. Ferritinophagy dysfunction in cellular models of depression was dependent on ATG7. BRD2 exhibited aberrant phase separation in CMS rats. The BET inhibitor JQ1 alleviated depressive behavior in rats subjected to CMS.Conclusions The ATG7 genes are activated by SEs in cellular and animal models of depression. The increased transcriptional activity of ATG7 can increase the risk of ferritinophagy. Moreover, the phase separation of BRD2 promotes ATG7 expression via SEs.

In this study, homoisoflavone methylophiopogonanone A (MOA) was investigated for its inhibitory effect on ferroptosis of H9c2 cells using a set of cellular assays, such as BODIPY-probed and H2DCFDA-probed flow cytometry analyses, cell counting kit-8 analysis (CCK-8), and lactate dehydrogenase (LDH) release analysis. All these cellular assays adopted Fer-1 as the positive control. Subsequently, MOA and Fer-1 were subjected to two antioxidant assays, i.e., 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•)-scavenging and 2,2′-azinobis(3-ethylbenzo-thiazoline-6-sulfonic acid radical (ABTS•+)-scavenging. Finally, MOA, along with Fer-1, were systematically analyzed for molecular docking and dynamics simulations using a set of software tools. The experimental results revealed that MOA could inhibit ferroptosis of H9c2 cells but did not effectively scavenge PTIO• and ABTS•+ free radicals. Two molecular simulation methods or algorithms suggested that MOA possessed similar binding affinity and binding free energy (∆Gbind) to Fer-1. Visual analyses indicated various hydrophobic interactions between MOA and one of the seven enzymes, including superoxide dismutase (SOD), dihydroorotate dehydrogenase (DHODH), ferroportin1 (FPN), ferroptosis suppressor protein 1 (FSP1), glutathione peroxidase 4 (GPX4), nicotinamide adenine dinucleotide phosphate (NADPH), and solute carrier family 7 member 11 (SLC7A11). Based on these experimental and molecular simulation results, it is concluded that MOA, a homoisoflavonoid with meta-di-OHs, can inhibit ferroptosis in H9c2 cells. Its inhibitory effect is mainly attributed to the regulation of enzymes rather than direct free radical scavenging. The regulation of enzymes primarily depends on hydrophobic interactions rather than H-bond formation. During the process, flexibility around position 9 allows MOA to adjust to the enzyme binding site. All these findings provide foundational information for developing MOA and its derivatives as potential drugs for myocardial diseases.

Osteoarthritis (OA) represents a significant burden on global healthcare systems that causes pain and functional impairment by affecting joint tissue. Ubiquitin-specific peptidase 53 (USP53), a member of the ubiquitin-specific protease (USP) family, is involved in the progression of various disease states, but its role in OA has not been investigated. This study employed bioinformatics analysis to identify 92 common genes were identified in the positivity related modules of OA. Further studies demonstrated that the DEGs enriched in 20 pathways, mainly including the Focal adhesion, PI3K-Akt signaling pathway, ECM-receptor interaction, Human papillomavirus infection and Pyrimidine metabolism in KEGG enrichment analysis. Furthermore, machine learning results show that USP53, CA12 and PON could be effective biomarkers for OA diagnosis. Preliminary laboratory investigations showed that compared with the control group, the expression of USP53 in the OA group showed significant changes in vivo. The clinical specimen test results showed the protein expression of USP53 in cartilage tissue of OA patients. All in all, this study indicated the hub genes USP53 could be a potential biomarker for OA, thus providing a novel insight into the modulation of ubiquitin in OA clinical diagnosis and treatment.

BACKGROUND:Acute-on-chronic liver failure (ACLF) constitutes a prevalent manifestation of liver failure within clinical settings. This condition manifests swiftly and is characterized by an exceedingly elevated fatality rate.OBJECTIVE:While numerous investigations have delved into the role of RNA methylation in ferroptosis, the impact of such methylation on ACLF-associated ferroptosis remains notably underexplored. This study aimed to elucidate the molecular mechanism underlying the efficacy of Niujiao Dihuang Jiedu decoction (NDD) in mitigating ferroptosis in ACLF, with a specific focus on RNA 5-methylcytosine (m5C) methylation.MATERIALS AND METHODS:An ACLF rat model was established alongside an erastin-induced ferroptosis model in LO2 cells. Both in vitro and in vivo experiments were conducted to substantiate NDD's influence on ferroptosis. The modifying influence of methylase NOL1/NOP2/sun domain (NSUN5) upon SLC7A11, a key ferroptosis-associated gene, was probed through dot blot, immunofluorescence co-localization, and RNA binding protein immunoprecipitation (RIP) experiments.RESULTS:Serological and hepatic histopathological findings indicated NDD's discernible therapeutic impact on ACLF. Furthermore, ferroptosis phenotype experiments revealed NDD's proficiency in effectively impeding the occurrence and development of ferroptosis. Dot blot assays demonstrated a reduction in the overall RNA m5C levels during cellular ferroptosis. Furthermore, through immunofluorescence co-localization and RIP techniques, we found that the propensity of methylase NSUN5 to associate with SLC7A11 mRNA, thereby enhancing its protein translation and conferring resistance against ferroptosis.CONCLUSION:RNA methylation is involved in the process of ACLF-associated ferroptosis, and NDD can inhibit ACLF-associated ferroptosis by fostering SLC7A11 m5C methylation.

Atractylenolide-III (AT-III) is well known as its role in antioxidant and anti-inflammatory. Present study was aimed to figure out its effects on osteoarthritis and potential mechanisms. Rat model, human osteoarthritis cartilage explants as well as rat/human chondrocyte cultures were prepared to test AT-III's effects on osteoarthritis progression and chondrocyte senescence. Potential targeted molecules of AT-III were predicted using network pharmacology and molecular docking, assessed by Western blotting and then verified with rescue experiments. AT-III treatment alleviated osteoarthritis severity (shown by OARSI grading score and micro-CT) and chondrocyte senescence (indexed by levels of SA-β-gal, P16, P53, MMP13, ROS and ratio of healthy/collapsed mitochondrial membrane potentials). Network pharmacology and molecular docking suggested that AT-III might play role through NF-κB pathway. Further experiments revealed that AT-III reduced phosphorylation of IKKα/β, IκBα and P65 in NF-κB pathway. As well as nuclear translocation of p65. Both in vivo and in vitro experiments indicated that AT-III's effects on osteoarthritis and anti-senescence were reversed by an NF-κB agonist. AT-III could alleviate osteoarthritis by inhibiting chondrocyte senescence through NF-κB pathway, which indicated that AT-III is a prospective drug for osteoarthritis treatment.

Diabetic cardiomyopathy (DCM) is a cardiovascular disease which has been reported as a major cause of mortality worldwide for several years. Berberine (BBR) is a natural compound extracted from a Chinese herb, with a clinically reported anti‑DCM effect; however, its molecular mechanisms have not yet been fully elucidated. The present study indicated that BBR markedly alleviated DCM by inhibiting IL‑1β secretion and the expression of gasdermin D (Gsdmd) at the post‑transcriptional level. Considering the importance of microRNAs (miRNAs/miRs) in the regulation of the post‑transcriptional process of specific genes, the ability of BBR to upregulate the expression levels of miR‑18a‑3p by activating its promoter (‑1,000/‑500) was examined. Notably, miR‑18a‑3p targeted Gsdmd and abated pyroptosis in high glucose‑treated H9C2 cells. Moreover, miR‑18a‑3p overexpression inhibited Gsdmd expression and improved biomarkers of cardiac function in a rat model of DCM. On the whole, the findings of the present study indicate that BBR alleviates DCM by inhibiting miR‑18a‑3p‑mediated Gsdmd activation; thus, BBR may be considered a potential therapeutic agent for the treatment of DCM.

Background and Objectives: Osteoarthritis (OA) is a degenerative disease that leads to the progressive destruction of articular cartilage. Current clinical therapeutic strategies are moderately effective at relieving OA-associated pain but cannot induce chondrocyte differentiation or achieve cartilage regeneration. We investigated the ability of wedelolactone, a biologically active natural product that occurs in Eclipta alba (false daisy), to promote chondrogenic differentiation. Methods and Results: Real-time reverse transcription-polymerase chain reaction, immunohistochemical staining, and immunofluorescence staining assays were used to evaluate the effects of wedelolactone on the chondrogenic differentiation of mesenchymal stem cells (MSCs). RNA sequencing, microRNA (miRNA) sequencing, and isobaric tags for relative and absolute quantitation analyses were performed to explore the mechanism by which wedelolactone promotes the chondrogenic differentiation of MSCs. We found that wedelolactone facilitates the chondrogenic differentiation of human induced pluripotent stem cell-derived MSCs and rat bone-marrow MSCs. Moreover, the forkhead box O (FOXO) signaling pathway was upregulated by wedelolactone during chondrogenic differentiation, and a FOXO1 inhibitor attenuated the effect of wedelolactone on chondrocyte differentiation. We determined that wedelolactone reduces enhancer of zeste homolog 2 (EZH2)-mediated histone H3 lysine 27 trimethylation of the promoter region of FOXO1 to upregulate its transcription. Additionally, we found that wedelolactone represses miR-1271-5p expression, and that miR-1271-5p post-transcriptionally suppresses the expression of FOXO1 that is dependent on the binding of miR1271-5p to the FOXO1 3'-untranscribed region.Conclusions: These results indicate that wedelolactone suppresses the activity of EZH2 to facilitate the chondrogenic differentiation of MSCs by activating the FOXO1 signaling pathway. Wedelolactone may therefore improve cartilage regeneration in diseases characterized by inflammatory tissue destruction, such as OA.

ETHNOPHARMACOLOGICAL RELEVANCE:Angelica Sinensis Radix (ASR) is a commonly used Chinese medicine known for its effects on tonifying blood, promoting blood circulation, and alleviating pain associated with menstrual regulation. Additionally, it has been used in the treatment of vascular cognitive impairment (VCI). The primary pharmacodynamic agent within ASR is volatile oil of Angelica Sinensis Radix (VOASR), which has demonstrated efficacy in combating cognitive impairment, although its mechanism remains unclear.OBJECTIVE:This study aimed to elucidate the potential molecular mechanisms underlying VOASR's improvement of cognitive function in cerebral ischemic mice.METHODS:A model of cerebral ischemic mice was established through unilateral common carotid artery occlusion (UCCAO) surgery, followed by intervention with VOASR. Cognitive function was assessed using the Morris water maze (MWM) test, while RT-qPCR was utilized to measure the differential expression of miR-301a-3p in the hippocampus. To evaluate cognitive function and hippocampal protein differences, wild-type mice and miR-301a-3p knockout mice were subjected to the MWM test and iTRAQ protein profiling. The relationship between miR-301a-3p and potential target genes was validated through a Dual-Luciferase Reporter experiment. RT-qPCR and Western blot were employed to determine the differential expression of Ppp2ca and synaptic plasticity-related proteins in the mouse hippocampus.RESULTS:Intervention with VOASR significantly improved cognitive impairment in cerebral ischemic mice and reduced the expression of miR-301a-3p in the hippocampus. Our findings suggest that miR-301a-3p may regulate cognitive function by targeting Ppp2ca. Furthermore, VOASR intervention led to an increase in the expression of Ppp2ca and synaptic plasticity-related proteins.CONCLUSION:Our study indicates that VOASR may be involved in regulating cognitive function by inhibiting miR-301a-3p, consequently increasing the expression of Ppp2ca and synaptic plasticity proteins. These results provide a new target and direction for the treatment of cognitive dysfunction.

The development of natural peptides as direct Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid2-related factor 2 (Nrf2) protein-protein interaction (PPI) inhibitors for antioxidant and anti-ferroptotic purposes has attracted increasing interest from chemists. Radix Angelicae sinensis (RAS) is a widely used traditional Chinese medicine with antioxidant capability. However, few studies have screened Keap1-Nrf2 PPI inhibitory RAS peptides (RASPs). This study optimized the extraction and hydrolysis protocols of RAS protein using response surface methodology coupled with Box-Behnken design. The molecular weight distribution of the prepared hydrolysates was analysed to obtain active fractions. Subsequently, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry was employed to identify RASPs. Various in vitro and in silico assays were conducted to evaluate the antioxidant and anti-ferroptotic effects of RASPs. The results revealed that at least 50 RASPs could be obtained through the optimized protocols. RASPs containing active residues effectively scavenged 2,2-diphenyl-1-picrylhydrazyl radical and 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid radical cation. They also showed cytoprotective effect against erastin-induced ferroptosis in HT22 cells, which was characterized by the activation of Nrf2 and weakened under the incubation of an Nrf2 inhibitor. Moreover, RASPs could bind to Keap1 and then dissociate Nrf2 in molecular dynamics simulations. In conclusion, RASPs exhibit antioxidant activity through hydrogen atom transfer and electron transfer mechanisms. Importantly, they also inhibit ferroptosis by directly inhibiting Keap1-Nrf2 PPI.

Lacasse can transform phytophenol into furanocyclic dimers, including coumaran linkage dimer and 3,7-dioxabicyclo[3.3.0]octane linkage dimer. However, the substrate specificity and diastereoselective process remain unknown until now. The study therefore selected a series of monomeric phytophenol isomers or analogues as substrates to interact with laccase from Trametes versicolor (Lac TV). The products were rapidly identified by ultra-performance liquid chromatography coupled with high-resolution mass-spectrometry. On this basis, 9 dimerization reactions were established for a systematical structure–function relationship analysis. Through analysis, it can be concluded that, phytophenol furanocyclic dimerization requires a specific substrate structure, i.e., a C=C bond sitting at the -OH para -position and also conjugating with the phenolic core. The coumaran linkage dimerization presents “7,8 diastereoselectivity” character; while the 3,7-dioxabicyclo[3.3.0]octane linkage dimerization shows not only “7,8 diastereoselectivity” character but also “ cis- 8H,8’H” character. The former character is attributed to the intramolecular nucleophilic addition which always prevents the Re -attack. The latter character however is caused by a substantial energy gap between normal envelop conformation and twist envelop conformation. All these findings can help to efficiently utilize Lac TV tool for phytophenol biosynthesis in green chemistry field.

ObjectiveMicroRNAs (miRNAs) play a key role in the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes. Our previous study found that novel-miR-81 can relieve osteoarthritis, but its role in chondrogenic differentiation of BMSCs remains unclear. The purpose of this study was to explore the role of novel-miR-81 in chondrogenic differentiation of BMSCs.MethodsWe used a model in which transforming growth factor (TGF)-β3-induced BMSCs differentiation into chondrocytes. We detected the expression Sox9, Collagen Ⅱ, Aggrecan, novel-miR-81, and Rac2 by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blot was performed to detect the expression of Sox9, Collagen Ⅱ, and Rac2. Dual-luciferase reporter gene assay confirmed that the association between novel-miR-81 and Rac2. In addition, the ectopic chondrocyte differentiation of BMSCs was performed subcutaneously in nude mice. The effect of novel-miR-81 and Rac2 on ectopic chondrogenic differentiation of BMSCs was determined by immunohistochemical staining.ResultsNovel-miR-81 upregulated in chondrogenic differentiation of BMSCs. Rac2 was a key target of novel-miR-81. Mimic novel-miR-81 and siRac2 upregulated the expression of Sox9, Collagen Ⅱ, and Aggrecan.ConclusionNovel-miR-81 promotes the chondrocytes differentiation of BMSCs by inhibiting the expression of target gene Rac2, which provides potential targets for BMSCs transplantation to repair cartilage defects.

Bone marrow mesenchymal stem cells (BMSCs) have the ability to differentiate into chondrocytes. In the differentiation of BMSCs into chondrocytes, micro-RNAs (miRNAs) play an important role. rno-miR-90 is a new miRNA discovered by our research team, and its role in chondrogenic differentiation of BMSCs is unknown. This study aimed to investigate whether rno-miR-90 could promote chondrogenic differentiation of BMSCs by regulating secreted protein acidic and rich in cysteine-related modular calcium binding 2 (Smoc2). First, BMSCs chondroblast differentiation was successfully induced in vitro by classical induction method of transforming growth factor (TGF)-β3. On this basis, we transfected rno-miR-90 mimic and inhibitor, and confirmed that rno-miR-90 mimic could promote the differentiation of BMSCs into chondrocytes by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. In addition, we demonstrated that Smoc2 was a target gene of rno-miR-90 by dual-luciferase reporter assay, and confirmed that rno-miR-90 mimic could inhibit the expression of Smoc2 by RT-qPCR and western blotting. In order to further prove the targeting relationship between rno-miR-90 and Smoc2, we constructed three interfering fragments of Smoc2, and proved that silencing Smoc2 could promote the differentiation of BMSCs into chondrocytes at the transcriptional and protein levels. Finally, we constructed a carrier scaffold for ectopic chondrogenic differentiation in vivo, and confirmed that rno-miR-90 mimic and siSmoc2 could promote chondrogenic differentiation of BMSCs by Alcian blue staining and immunohistochemistry. In summary, our results suggested that rno-miR-90 could promote chondrogenic differentiation of BMSCs by down-regulating the expression of Smoc2. rno-miR-90 mimic and Smoc2 may be therapeutic targets of osteoarthritis.

Abstract Parkinson's disease (PD) is a highly prevalent neurodegenerative disorder associated with abnormal brain iron metabolism. Ferroptosis is a newly discovered form of iron-dependent cell death. In recent years, many studies have identified ferroptosis as an important pathogenic mechanism of PD. Nevertheless, the underlying mechanisms remain unclear. A 6-hydroxydopamine (6-OHDA) stimulated the PD rat model and the PD cell model were used in this research. The experimental results showed that the level of kinesin 1 heavy chain (KIF5A) decreases, and the level of ferroptosis increases after 6-OHDA stimulation. A PD cell model is consistent with these results. The overexpression of KIF5A in SH-SY5Y cells significantly reduces intracellular lipid peroxidation, Fe2+ accumulation, and ferroptosis. In contrast, knockdown of KIF5A exacerbated lipid peroxidation and Fe2+ accumulation, and cellular ferroptosis was more severe. Therefore, this study provides new views and potential for studying treatment targets of PD by demonstrating that KIF5A protects cells from ferroptosis in a PD model.

BACKGROUND:In recent years, the ability of neural stem cells (NSCs) transplantation to treat Parkinson's disease (PD) has attracted attention. However, it is still a challenge to promote the migration of NSCs to the lesion site and their directional differentiation into dopaminergic neurons in PD. C-C motif chemokine ligand 5 (CCL5) and C-C motif chemokine receptor 5 (CCR5) are expressed in the brain and are important regulators of cell migration. It has been reported that ethyl stearate (PubChem CID: 8122) has a protective effect in 6-OHDA-induced PD rats.METHODS:Parkinson's disease rats were injected with 6-hydroxydopamine (6-OHDA) into the right substantia nigra, and striatum followed by 8 μL of an NSC cell suspension containing 100 μM ethyl stearate and 8 × 105 cells in the right striatum. The effect of transplantation NSCs combined with ethyl stearate was assessed by evaluating apomorphine (APO)-induced turning behavior and performance in the pole test. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting (WB), and immunofluorescence staining were also performed.RESULTS:NSCs transplantation combined with ethyl stearate ameliorated the behavioral deficits of PD rats. PD rats that received transplantation NSCs combined with ethyl stearate exhibited increased expression of tyrosine hydroxylase (TH) and an increased number of green fluorescent protein (GFP)-positive cells. Furthermore, GFP-positive cells migrated into the substantia nigra and differentiated into dopaminergic neurons. The expression of CCL5 and CCR5 was significantly increased after transplantation NSCs combined with ethyl stearate.CONCLUSIONS:These findings suggest that NSCs transplantation combined with ethyl stearate can improve the motor behavioral performance of PD rats by promoting NSCs migration from the striatum to the substantia nigra via CCL5/CCR5 and promoting the differentiation of NSCs into dopaminergic neurons.

Ferroptosis is a type of cell death induced by the iron-dependent accumulation of lipid hydroperoxides and reactive oxygen species (ROS) in cells. Inhibiting ferroptosis is important for improving the survival of transplanted bone marrow-derived mesenchymal stem cells (BMSCs). Although it is known that NOP2/Sun RNA methyltransferase 5 (NSUN5) post-transcriptionally regulates ferroptosis in BMSCs through RNA methylation, the precise mechanisms underlying these effects have not been reported. In this study, we demonstrate that NSUN5 is downregulated in erastin-induced ferroptosis in BMSCs. Ferroptosis was inhibited by the overexpression of NSUN5 or ferritin heavy chain/light-chain (FTH1/FTL) and was enhanced by NSUN5 knockdown. RNA immunoprecipitation experiments revealed that NSUN5 binds to FTH1/FTL, while NSUN5 depletion reduced the levels of 5-methylcytosine in FTH1/FTL RNA and increased intracellular iron concentrations, resulting in the downregulation of glutathione peroxidase 4 (GPX4) and the accumulation of ROS and lipid peroxidation products. Co-immunoprecipitation experiments demonstrated that the recognition of FTH1 and FTL by NSUN5 is dependent on the recruitment of tumor necrosis factor receptor-associated protein 1 (TRAP1). These results suggested that the NSUN5-FTH1/FTL pathway mediates ferroptosis in BMSCs and that the therapeutic targeting of components of this pathway may promote resistance to ferroptosis and improve the survival of transplanted BMSCs.

Background/ObjectivesNon-ergot dopamine agonist (NEDA) are recommended as the first-line treatment for patients with early Parkinson's disease (PD) because of their efficacy in treating PD motor symptoms. However, systematic evaluations of the risk of motor complications induced by NEDA and risk factors potentially associated with motor complications are still lacking.MethodsMedline, Embase, the Cochrane Central Register of Controlled Trials, and Web of Science were searched for potentially eligible randomized controlled trials. The incidence of motor complications (dyskinesia, motor fluctuations), impulsive-compulsive behaviors and adverse events and clinical disability rating scale (UPDRS) scores were evaluated using standard meta-analytic methods. Metaregression was conducted on the incidence of motor complications (dyskinesia) with treatment duration and NEDA dose as covariates.ResultsPatients treated with NEDA had significantly lower UPDRS total scores, motor scores and activity of daily living (ADL) scores than those receiving a placebo (weighted mean difference (WMD) −4.81, 95% CI −6.57 to −3.05; WMD −4.901, 95% CI −7.03 to −2.77; WMD −1.52, 95% CI −2.19 to −0.84, respectively). Patients in the NEDA and NEDA+open Levodopa (LD) groups had lower odds for dyskinesia than patients in the LD group (OR = 0.21, 95% CI: 0.15–0.29; OR = 0.31, 95% CI 0.24–0.42, respectively). Metaregressions indicated that the mean LD dose of the NEDA group increased, and the odds of developing dyskinesia increased (p = 0.012). However, the odds of developing dyskinesia in the NEDA group were not related to treatment duration (p = 0.308). PD patients treated with NEDA or NEDA+open LD had a lower risk of wearing-off implications than those treated with LD (all p < 0.05). No significant difference was found between the NEDA and placebo groups in impulsive-compulsive behavior development (p > 0.05). Patients in the NEDA group were more likely to suffer somnolence, edema, constipation, dizziness, hallucinations, nausea and vomiting than those in the placebo or LD group.ConclusionNEDA therapy reduces motor symptoms and improves ADLs in early PD. The odds of developing motor complications were lower with NEDA than with LD, and dyskinesia increased with increasing LD equivalent dose and was not influenced by NEDA treatment duration. Therefore, long-term treatment with an appropriate dosage of NEDA might be more suitable than LD for early PD patients.RegistrationPROSPERO CRD42021287172.