Chondrocyte-targeted exosome-mediated delivery of Nrf2 alleviates cartilaginous endplate degeneration by modulating mitochondrial fission.

BACKGROUND: Cartilaginous endplate (CEP) degeneration, which is an important contributor to intervertebral disc degeneration (IVDD), is characterized by chondrocyte death. Accumulating evidence has revealed that dynamin-related protein 1 (Drp1)-mediated mitochondrial fission and dysfunction lead to apoptosis during CEP degeneration and IVDD. Exosomes are promising agents for the treatment of many diseases, including osteoporosis, osteosarcoma, osteoarthritis and IVDD. Despite their major success in drug delivery, the full potential of exosomes remains untapped. MATERIALS AND METHODS: In vitro and in vivo models of CEP degeneration were established by using lipopolysaccharide (LPS). We designed genetically engineered exosomes (CAP-Nrf2-Exos) expressing chondrocyte-affinity peptide (CAP) on the surface and carrying the antioxidant transcription factor nuclear factor E2-related factor 2 (Nrf2). The affinity between CAP-Nrf2-Exos and CEP was evaluated by in vitro internalization assays and in vivo imaging assays. qRT‒PCR, Western blotting and immunofluorescence assays were performed to examine the expression level of Nrf2 and the subcellular localization of Nrf2 and Drp1. Mitochondrial function was measured by the JC-1 probe and MitoSOX Red. Mitochondrial morphology was visualized by MitoTracker staining and transmission electron microscopy (TEM). After subendplate injection of the engineered exosomes, the degree of CEP degeneration and IVDD was validated radiologically and histologically. RESULTS: We found that the cargo delivery efficiency of exosomes after cargo packaging was increased by surface modification. CAP-Nrf2-Exos facilitated chondrocyte-targeted delivery of Nrf2 and activated the endogenous antioxidant defence system in CEP cells. The engineered exosomes inhibited Drp1 S616 phosphorylation and mitochondrial translocation, thereby preventing mitochondrial fragmentation and dysfunction. LPS-induced CEP cell apoptosis was alleviated by CAP-Nrf2-Exo treatment. In a rat model of CEP degeneration, the engineered exosomes successfully attenuated CEP degeneration and IVDD and exhibited better repair capacity than natural exosomes. CONCLUSION: Collectively, our findings showed that exosome-mediated chondrocyte-targeted delivery of Nrf2 was an effective strategy for treating CEP degeneration.

HIF-1α-induced expression of the m6A reader YTHDF1 inhibits the ferroptosis of nucleus pulposus cells by promoting SLC7A11 translation.

The nucleus pulposus is in a hypoxic environment in the human body, and when intervertebral disc degeneration (IVDD) occurs, the hypoxic environment is disrupted. Nucleus pulposus cell (NPC) ferroptosis is one of the causes of IVDD. N6-methyladenosine (m6A) and its reader protein YTHDF1 regulate cellular activities by affecting RNA metabolism. However, the regulation of ferroptosis in NPCs by m6A-modified RNAs under hypoxic conditions has not been as well studied. In this study, through in vitro and in vivo experiments, we explored the underlying mechanism of HIF-1α and YTHDF1 in regulating ferroptosis in NPCs. The results indicated that the overexpression of HIF-1α or YTHDF1 suppressed NPC ferroptosis; conversely, the knockdown of HIF-1α or YTHDF1 increased ferroptosis levels in NPCs. Luciferase reporter assays and chromatin immunoprecipitation demonstrated that HIF-1α regulated YTHDF1 transcription by directly binding to its promoter region. Polysome profiling results showed that YTHDF1 promoted the translation of SLC7A11 and consequently the expression of the anti-ferroptosis protein GPX4 by binding to m6A-modified SLC7A11 mRNA. In conclusion, HIF-1α-induced YTHDF1 expression reduces NPC ferroptosis and delays IVDD by promoting SLC7A11 translation in a m6A-dependent manner.

Engineered exosomes enriched in netrin-1 modRNA promote axonal growth in spinal cord injury by attenuating inflammation and pyroptosis.

BACKGROUND: Spinal cord injury (SCI) brings a heavy burden to individuals and society, and there is no effective treatment at present. Exosomes (EX) are cell secreted vesicles containing molecules such as nucleic acids and proteins, which hold promise for the treatment of SCI. Netrin-1 is an axon guidance factor that regulates neuronal growth. We investigated the effects of engineered EX enriched in netrin-1 chemically synthetic modified message RNA (modRNA) in treating SCI in an attempt to find a novel therapeutic approach for SCI. METHODS: Netrin-1 modRNA was transfected into bone marrow mesenchymal stem cells to obtain EX enriched with netrin-1 (EX-netrin1). We built an inflammatory model in vitro with lipopolysaccharide (LPS) in vitro to study the therapeutic effect of EX-netrin1 on SCI. For experiments in vitro, ELISA, CCK-8 assay, immunofluorescence staining, lactate dehydrogenase release experiments test, real-time quantitative polymerase chain reaction, and western blot were conducted. At the same time, we constructed a rat model of SCI. MRI, hematoxylin-eosin and Nissl staining were used to assess the extent of SCI in rats. RESULTS: In vitro experiments showed that EX had no effect on the viability of oligodendrocytes and PC12 cells. EX-netrin1 could attenuate LPS-induced inflammation and pyroptosis and accelerate axonal/dentritic growth in PC12 cells/oligodendrocytes. In addition, netrin-1 could activate the PI3K/AKT/mTOR signalling pathway upon binding to its receptor unc5b. When Unc5b and PI3K were inhibited, the effect of EX-netrin1 was weakened, which could be reversed by PI3K or mTOR activator. Our in vivo experiments indicated that EX-netrin1 could promote recovery in rats with SCI. CONCLUSION: We found that EX-netrin1 regulated inflammation, pyroptosis and axon growth in SCI via the Unc5b/PI3K/AKT/mTOR pathway, which provides a new strategy for the treatment of SCI.

Sulforaphane Delays Intervertebral Disc Degeneration by Alleviating Endoplasmic Reticulum Stress in Nucleus Pulposus Cells via Activating Nrf-2/HO-1.

Intervertebral disc degeneration (IVDD) is one of the main causes of low back pain, which brings heavy burdens to individuals and society. The mechanism of IVDD is complex and diverse. One of the important reasons is that the abnormal accumulation of reactive oxygen species (ROS) in nucleus pulposus cells (NPCs) leads to endoplasmic reticulum stress (ERS), which causes increased apoptosis of NPCs. Nuclear factor E2-related factor 2 (Nrf-2) and its downstream antioxidant proteins are key molecular switches for sensing oxidative stress and regulating antioxidant responses in cells. Sulforaphane (SFN), a natural compound derived from Brassicaceae plants, is a Nrf-2 agonist that displays potent antioxidant potential in vitro and in vivo. Here, we used advanced glycation end products (AGEs) to construct an in vitro degeneration model of NPCs, and we found that AGEs elevated ROS level in NPCs and caused severe ERS and apoptosis. While SFN can promote the entry of Nrf-2 into the nucleus and increase the expression level of heme oxygenase 1 (HO-1) in vitro, thus clearing the accumulated ROS in cells and alleviating ERS and cell apoptosis. Moreover, the protection of SFN on NPCs was greatly attenuated after HO-1 was inhibited. We also used AGEs to construct a rat IVDD model. Consistent with the in vitro experiments, SFN could attenuate ERS in NPCs in vivo and delay disc degeneration in rats. This study found that SFN can be used as a new and promising agent for the treatment of IVDD.

Roles of circular RNAs in the pathogenesis of intervertebral disc degeneration (Review).

Lower back pain (LBP) is an extremely common symptom and is recognized as a leading contributor to disability and disease burden globally. Intervertebral disc degeneration (IDD) represents a major cause of LBP. However, the molecular mechanisms involved in the pathogenesis of IDD remain unclear, and currently available treatments, including conservative and surgical options, fail to effectively delay, stop or reverse the progression of IDD. Circular RNAs (circRNAs) are a newly discovered group of covalently closed, single-stranded and endogenous non-coding RNAs. A growing body of research has revealed that a number of circRNAs are widely and aberrantly expressed in IDD tissues. Furthermore, they play important roles in the pathogenesis of IDD, including proliferation, apoptosis, senescence, mitophagy, inflammation and extracellular matrix metabolism, mainly by acting as sponges for microRNAs. The present review aims to summarize the current understanding on the mechanisms of circRNA-mediated regulation in IDD.

Diagnostic potential of trace metals concentration in expressed prostatic secretion and serum of patients with category IV prostatitis.

BACKGROUND: The National Institutes of Health (NIH) category IV prostatitis is a painless prostate gland inflammation, just as its name implies, this type of prostatitis is related with inflammation of the prostate, but most men are not conscious of it. However, category IV prostatitis is fairly common in general populations and reported having indirect relationships with prostate cancer. METHOD: We analyzed the concentration of zinc (Zn), copper (Cu), calcium (Ca) and magnesium (Mg) in expressed prostatic secretion (EPS) and serum of patients with category IV prostatitis and healthy controls, investigating the diagnostic potential of different metals in category IV prostatitis using a flame atomic absorption spectrometer (FAAS). RESULTS: Metal concentration combined clinical characteristics analysis suggested that average level of Zn, Ca, Mg were significantly lower in the EPS of patients with category IV prostatitis (P-value< 0.000), while Cu level raised obviously (P-value< 0.000). And in the serum, mean concentrations of Ca was also found to increase significantly in the patients with category IV prostatitis compared to healthy controls. Moreover, the correlation analysis indicated that age showed a positive correlation with EPS Zn, Ca, Mg concentration (P-value< 0.05), while albumin correlates with EPS Zn, Ca, Mg concentration reversely (P-value< 0.05) in patients with category IV prostatitis. CONCLUSION: Our report revealed that determination of the metal elements zinc, copper, calcium and magnesium in the serum and EPS could be a new and promising strategy for the rapid diagnosis of category IV prostatitis.

A narrative review of malignant eosinophilic pleural effusion: incidence, etiology and prognostic significance.

Eosinophilic pleural effusion (EPE) is defined as a pleural fluid with an eosinophilic count exceeding 10% and currently considered to be mainly caused by malignancy. However, the incidence, etiology and prognostic significance of malignant eosinophilic pleural effusion (MEPE) have not been studied extensively yet. Thus, the objective of this review was to summarize medical studies regarding MEPE to 2020 throughout an extensive search of PubMed. Overall, MEPE was a disease associated with multiplecytokines-mediated immunity and varied from 4% to 92% of patients who had EPE. The discrepancy of the MEPE prevalence among studies could be explained by the development of diagnostic technology, disparity of study population, or various disease spectrum over time. Data summarized in this review demonstrated that the incidence of malignancy was lower in EPEs than in non-EPEs (29.7% vs. 32.9%). Additionally, MEPE could be a manifestation of a great variety of tumor subtypes, among which lung cancer was the most common cause and accounted for more than 34% of cases. The second common causes were non-Hodgkin lymphoma and metastatic cancers with unknown primary site which were observed in around 5% of cases, respectively. The presence of eosinophils in the pleural effusion may be associated with a positive prognosis of MEPE. Besides, the prognosis of MEPE may be related to the percentage of eosinophils in the pleural fluid. More extensive studies, however, are warranted to validate these findings.

The association between herpes simplex virus type 1 infection and Alzheimer's disease.

There is growing evidence demonstrating the relationship between herpes simplex virus type 1 (HSV-1) infection and Alzheimer's disease (AD). We searched PubMed, Embase, and Cochrane databases for relevant articles. The Newcastle-Ottawa Scale (NOS) was used to evaluate the qualities of these studies. Pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated using random-effects models. We also performed subgroup analyses stratified by apolipoprotein ε4 (APOE ε4), NOS score, and the method of confirming AD. A total of 21 studies between 1990 and 2020 were identified. The pooled OR suggested that HSV-1 infection is a risk factor of AD: pooled OR 1.40 (95% CI: 1.13-1.75; I2 = 3%, P = 0.42). In the subgroup analyses, the pooled ORs of HSV-1 infection associated with AD were 0.75 (95% CI: 0.24-2.37) among the APOE ε4-positive individuals; 0.85 (95% CI: 0.61-1.17) among the APOE ε4-negative individuals; 1.51 (95% CI: 1.10-2.06) in the high NOS score studies; 1.23 (95% CI: 0.85-1.76) in the moderate NOS score studies; 1.47 (95% CI: 1.16-1.87) in the clinical diagnosis group, and 1.20 (95% CI: 0.77-1.87) in the autopsy group. Our up-to-date systematic review and meta-analysis suggest that HSV-1 infection is a risk factor of AD.

Association of napping and all-cause mortality and incident cardiovascular diseases: a dose-response meta analysis of cohort studies.

BACKGROUND: Napping is a habit prevalent worldwide and occurs from an early age. However, the association between napping and the risk of incident cardiovascular disease (CVD) and all-cause mortality remains unclear. METHODS: We conducted a systematic search of Medline, Embase, and Cochrane databases from inception to December 2019 for cohort studies investigating the association between napping and the risk of incident CVD and/or all-cause mortality. Overall estimates were calculated using random-effect models with inverse variance weighting. Dose-response meta-analysis was performed using restricted cubic spline models. RESULTS: A total of 313,651 participants (57.8% female, 38.9% took naps) from 20 cohort studies were included in the analysis. All-cause mortality was associated with napping overall (HR 1.19, 95% CI 1.12-1.26). Pooled analysis detected no association between daytime nap and incident CVD. However, in subgroup analysis including only participants who were female (HR 1.31, 95% CI 1.09-1.58), older (HR 1.36, 95% CI 1.07-1.72), or took a long nap (HR 1.34, 95% CI 1.05-1.63), napping was significantly associated with a higher risk of CVD. Dose-response analysis showed a J-curve relation between nap time and incident CVD. The HR decreased from 0 to 25 min/day, followed by a sharp increase in the risk at longer times. A positive linear relationship between nap time and all-cause mortality was also observed. CONCLUSIONS: Long napping was associated with increased risks of incident CVD and all-cause mortality. Further, large-scale studies and genetic studies need to confirm our conclusion and investigate the underlying mechanisms driving these associations.

Diagnostic value of ultrasound-guided needle biopsy in undiagnosed pleural effusions: A systematic review and meta-analysis.

BACKGROUND AND OBJECTIVE: Undiagnosed pleural effusions (UPEs) are a common problem of respiratory medicine, leading to an increased diagnostic burden globally. However, the most efficient and cost-effective approaches to UPEs remain controversial. This study aimed to assess the diagnostic value of ultrasound-guided needle biopsy (UGNB) in UPEs. METHODS: We conducted a search of PubMed, Embase, the Cochrane Library and reference lists of retrieved studies with no publication data limitation. Articles that investigated the diagnostic accuracy of UGNB in UPEs were included. The quality of eligible studies was assessed using Quality Assessment of Diagnostic Accuracy Studies-2. The diagnostic value of UGNB was evaluated by calculating the pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds rate, and the area under the curve for the summary receiver operating characteristic curve using a random effects model. RESULTS: Seven studies comprising 165 patients with UPEs met the inclusion criteria. UGNB had a pooled sensitivity of 83% (95% confidence intervals [CI], 75% - 89%), a specificity of 100% (95% CI, 90% - 100%), a positive likelihood ratio of 8.89 (95% CI, 3.29 - 24.02), a negative likelihood ratio of 0.23 (95% CI, 0.16 - 0.33), a diagnostic odds rate of 51.47 (95% CI, 14.70 - 180.16), and an area under the curve of 0.94. Six pneumothorax cases (3.6%), 5 local wound infections (3.0%), and 1 empyema case (less than 1%) were observed. There was no significant heterogeneity or publication bias in this study. CONCLUSIONS: Based on current evidence, UGNB is a safe and convenient procedure with a high accuracy for diagnosing UPEs. However, physicians should still be cautious in interpreting negative UGNB results.

Methylation of S100A8 is a promising diagnosis and prognostic marker in hepatocellular carcinoma.

The abnormality of DNA methylation is one of the major epigenetic alterations in the human hepatocellular carcinoma (HCC). We have assessed the global genomic DNA methylation profiles in human HCC patients by using the Infinium Human Methylation27 BeadChip. A CpG loci of S100A8 was found to be significantly hypomethylated in HCC.Pooled meta-analysis of five validation public datasets demonstrated its methylation level was significantly lower for HCC compared to paired adjacent normal tissues. Quantitative pyrosequencing analysis also showed that the S100A8 methylation level was decreased in cancer tissues (31.90%±13.31%) than that in the paired adjacent normal tissues (65.33%±3.64%, p<0.01). The area under the ROC curve (AUC) value was 0.950 (p<0.01). Kaplan-Meier survival curves revealed that hypomethylation of S100A8 was associated with shortened overall survival (OS) and progression-free survival (PFS) (log rank p<0.05). Multivariate Cox proportional hazards model also indicated significantly shorter OS (HR, 1.709; 95 % CI, 1.127-2.591) and PFS (HR, 1.767; 95 % CI, 1.168-2.974) were observed in the low-methylation-level group compared to the high-methylation-level group. Furthermore, S100A8 overexpression in Huh7 and MHCC-97H hepatoma cell lines led to increased cell proliferation, migration, invasion, and tumor growth. These findings suggested S100A8 methylation to be served as potential diagnosis and prognosis marker for HCC. S100A8 also may play as a tumor promoter in HCC.

Impact of CAG repeat length in the androgen receptor gene on male infertility - a meta-analysis.

CAG repeats are polymorphic nucleotide repeats present in the androgen receptor gene. Many studies have estimated the association between CAG repeat length and male infertility, but the conclusions are controversial. Previous meta-analyses have come to different conclusions; however, new studies have been published. An updated meta-analysis was conducted. PubMed, CBM, CNKI and Web of Science databases were systematically searched for studies published from 1 January 2000 to 1 October 2015. Case-control studies on the association between CAG repeat length and male infertility using appropriate methodology were included. Forty studies were selected, including 3858 cases and 3161 controls. Results showed statistically significantly longer CAG repeat length among cases compared with controls (SMD = 0.14; 95% CI, 0.02-0.26). Shorter repeat length was associated with a lower risk of male infertility compared with a longer repeat length in the overall analysis (OR = 0.79, 95% CI: 0.66-0.95). Moreover, CAG repeat length was associated with male infertility in Caucasian populations, but not Asian or Egyptian populations. Subgroup analysis revealed no significant difference in German populations, but CAG repeat length was associated with male infertility in China and the USA. There were no significant differences between cases and controls in azoospermia and severe oligozoospermia.

Serum quantitative proteomic analysis reveals potential zinc-associated biomarkers for nonbacterial prostatitis.

BACKGROUND: Prostatitis is one of the most common urological problems afflicting adult men. The etiology and pathogenesis of nonbacterial prostatitis, which accounts for 90-95% of cases, is largely unknown. As serum proteins often indicate the overall pathologic status of patients, we hypothesized that protein biomarkers of prostatitis might be identified by comparing the serum proteomes of patients with and without nonbacterial prostatitis. METHODS: All untreated samples were collected from subjects attending the Fangchenggang Area Male Health and Examination Survey (FAMHES). We profiled pooled serum samples from four carefully selected groups of patients (n = 10/group) representing the various categories of nonbacterial prostatitis (IIIa, IIIb, and IV) and matched healthy controls using a mass spectrometry-based 4-plex iTRAQ proteomic approach. More than 160 samples were validated by ELISA. RESULTS: Overall, 69 proteins were identified. Among them, 42, 52, and 37 proteins were identified with differential expression in Category IIIa, IIIb, and IV prostatitis, respectively. The 19 common proteins were related to immunity and defense, ion binding, transport, and proteolysis. Two zinc-binding proteins, superoxide dismutase 3 (SOD3), and carbonic anhydrase I (CA1), were significantly higher in all types of prostatitis than in the control. A receiver operating characteristic curve estimated sensitivities of 50.4 and 68.1% and specificities of 92.1 and 83.8% for CA1 and SOD3, respectively, in detecting nonbacterial prostatitis. The serum CA1 concentration was inversely correlated to the zinc concentration in expressed-prostatic secretions. CONCLUSIONS: Our findings suggest that SOD3 and CA1 are potential diagnostic markers of nonbacterial prostatitis, although further large-scale studies are required. The molecular profiles of nonbacterial prostatitis pathogenesis may lay a foundation for discovery of new therapies.

Impact of caspase-8 (CASP8) -652 6N del and D302H polymorphisms on prostate cancer in different ethnic groups.

BACKGROUND: Despite evidence suggesting roles for caspase-8 (CASP8) -652 6N del and D302H polymorphisms in prostate cancer (PCa), the association of these polymorphisms with PCa risk remains inconclusive. Therefore, a meta-analysis was performed to more precisely estimate the association of CASP8 -652 6N del and D302H polymorphisms with PCa susceptibility. MATERIALS AND METHODS: A comprehensive literature search was conducted to identify all case-control studies of CASP8 D302H and -652 6N del polymorphisms and PCa risk. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to assess the strength of the association and the precision of the estimate, respectively. RESULTS: Nine -625 6N del studies and 4 D302H studies were included. CASP8 -652 6N del and D302H polymorphisms were not significantly associated with PCa risk in the overall analyses. However, in the subgroup analysis stratified by ethnicity, -625 6N del was significantly associated with PCa risk in the East Asian and Indian populations under the recessive model. Furthermore, the subgroup analysis strongly suggested that D302H was associated with lower PCa risk in the Non-Indian population under the dominant model. CONCLUSIONS: In our meta-analysis, ethnic-specific differences were evident in the association of CASP8 -625 6N del and D302H polymorphisms with PCa risk.