Xinfeng Capsule Inhibits Pyroptosis and Ameliorates Myocardial Injury in Rats with Adjuvant Arthritis via the GAS5/miR-21/TLR4 Axis.

Purpose: This study probed the mechanism of action of Xinfeng Capsule (XFC) in myocardial injury in rats with adjuvant arthritis (AA) via the growth arrest-specific transcript 5 (GAS5)/microRNA-21 (miR-21)/Toll-like receptor 4 (TLR4) axis. Methods: Rats were injected with Freund's complete adjuvant to establish a rat model of AA. Then, some modeled rats were given normal saline or drugs only, and some modeled rats were injected with adeno-associated viruses or necrosulfonamide (NSA; a pyroptosis inhibitor) before drug administration. Toe swelling and arthritis index (AI) were calculated. Pathological and morphological changes in synovial and myocardial tissues were analyzed with hematoxylin-eosin staining, and pyroptotic vesicles and the ultrastructural changes of myocardial tissues were observed with transmission electron microscopy. The serum levels of interleukin (IL)-1ß, IL-18, IL-6, and tumor necrosis factor (TNF)-α were detected, and lactate dehydrogenase (LDH) release was measured in myocardial tissues, accompanied by the examination of GAS5, miR-21, TLR4, nuclear factor-kB (NF-κB) p65, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), Caspase-1, and Gasdermin D (GSDMD) expression in myocardial tissues. Results: After AA modeling, rats presented with significantly increased toe swelling and AI scores, synovial and myocardial tissue damage, elevated pyroptotic vesicles, and markedly enhanced serum levels of IL-1ß, IL-18, IL-6, and TNF-α, accompanied by significantly diminished GAS5 expression, substantially augmented miR-21, TLR4, NF-κB p65, NLRP3, Caspase-1, and GSDMD expression, greatly increased LDH release in myocardial tissues. XFC treatment significantly declined toe swelling, AI scores, synovial and myocardial tissue damage, and the serum levels of IL-1ß, IL-18, IL-6, and TNF-α in AA rats. Additionally, XFC treatment markedly elevated GAS5 expression and substantially lowered LDH release and miR-21, TLR4, NF-κB p65, NLRP3, Caspase-1, and GSDMD expression in myocardial tissues of AA rats. Moreover, the above effects of XFC in AA rats were further promoted by GAS5 overexpression or NSA treatment. Conclusion: XFC alleviated myocardial injury in AA rats by regulating the GAS5/miR-21/TLR4 axis and inhibiting pyroptosis and pro-inflammatory cytokine secretion.

Propofol rescues LPS-induced toxicity in HRT-8/SVneo cells via miR-216a-5p/TLR4 axis.

OBJECTIVE: The purpose of this study was to evaluate the effect of propofol on lipopolysaccharide (LPS)-induced toxicity in HTR-8/SVneo cells. METHODS: In this study, HTR-8/SVneo cells were induced by LPS. The cells were treated with different concentrations of propofol. Cell proliferation, apoptosis, invasion, and wound healing rate were measured by MTT, flow cytometry, Transwell, and wound-healing assay. The relative mRNA expression levels of miR-216a-5p, TLR, MyD88, and NF-κB(p65) were measured by qRT-PCR. The protein expression levels of TLR, MyD88, and p-NF-κB(p65) were detected by western blot. The p-NF-κB(p65) nuclear volume was evaluated by cell immunofluorescence. RESULTS: Compared with control group, the cell proliferation, invasion, and wound healing rate were significantly decreased and the cell apoptosis rate was significantly increased in LPS- induced HTR-8/SVneo cells (P < 0.01). With propofol supplement, the cell proliferation, migration, and invasion abilities were significantly recovered and apoptosis rate was significantly inhibited (P < 0.05). The expression levels of miR-216a-5p, TLR4, MyD88, NF-κB(p65), and p-NF-κB(p65), and p-NF-κB(p65) nuclear volume were significantly different between propofol group and model group (P < 0.05). However, after knockdown of miR-216a-5p expression by si-miR-216a-5p transfection, the cell proliferation, migration, and invasion abilities were significantly inhibited and apoptosis rate was notably increased (P < 0.05). CONCLUSION: Propofol improves LPS-induced toxicity in HTR-8/SVneo cells via regulation miR-216a-5p/TLR4 axis.

Small extracellular vesicles from hypoxic mesenchymal stem cells alleviate intervertebral disc degeneration by delivering miR-17-5p.

Minimally invasive repair strategies are a very promising approach for the treatment of intervertebral disc degeneration (IDD). In recent years, small extracellular vesicles (sEVs) secreted from mesenchymal stem cells (MSCs) have been shown great potential in alleviating IDD. However, in vitro experiments, MSCs are usually exposed to a normoxic micro-environment, which differs greatly from the hypoxic micro-environment in vivo. The primary purpose of our research was to determine whether sEVs isolated from MSCs under hypoxic status (H-sEVs) exhibit a more beneficial effect on protecting IDD compared with sEVs derived from MSCs under normoxic status (N-sEVs). A tail IDD rat model and a series of experiments in vitro were conducted to compare the beneficial effects of PBS, N-sEVs, and H-sEVs treatment. Then, to validate the role of sEVs miRNAs in IDD, a miRNA microarray sequencing analysis and a series of rescue experiments were conducted. Luciferase activity, RNA-ChIP and western blot were performed to explore the potential mechanisms. The results indicate that sEVs alleviate IDD by ameliorating the homeostatic imbalance between anabolism and catabolism in vivo and in vitro. Microarray sequencing result shows that miR-17-5p is maximally enriched in H-sEVs. Toll-like receptor 4 (TLR4) was determined to be a target downstream gene of miR-17-5p. Finally, it was found that H-sEVs miR-17-5p may modulate proliferation and synthesis of human nucleus pulposus cells (HNPCs) matrix via TLR4 pathway. In conclusion, H-sEVs miR-17-5p alleviate IDD via promoting HNPCs matrix proliferation and synthesis, providing new therapeutic targets for IDD. STATEMENT OF SIGNIFICANCE: Intervertebral disc degeneration (IDD) is the primary cause of low back pain (LBP), which is a huge burden to society. Our research demonstrates for the first time that hypoxic pretreatment of small extracellular vesicles (H-sEVs) effectively alleviated the progress of IDD. In short, in the present research, we found that H-sEVs miR-17-5p could modulate proliferation and synthesis of nucleus pulposus cells (NPCs) matrix via TLR4/PI3K/AKT pathway. Therefore, hypoxic pre-treatment is a prospective and efficient method to optimize the therapeutic effect of MSCs-derived sEVs. miRNA and MSCs-derived sEVs combination may be a promising therapeutic approach for IDD.

LncRNA UCA1 remits LPS-engendered inflammatory damage through deactivation of miR-499b-5p/TLR4 axis.

Neonatal pneumonia is a high neonatal mortality disease. The current research was designed to elucidate the modulatory function and feasible molecular mechanism of UCA1 in LPS-induced injury in pneumonia. Herein, LPS was applied to induce WI-38 cell inflammatory damage. We displayed that UCA1 was elevated in LPS-injured WI-38 cells. In the functional aspect, intervention of UCA1 evidently aggrandized cell viability in LPS-triggered WI-38 cells. In the meanwhile, elimination of UCA1 distinctly assuaged cell apoptosis concomitant with declined levels of proapoptotic proteins Bax and C-caspase-3, and ascended the expression of antiapoptotic protein Bcl-2. Subsequently, disruption of UCA1 manifestly restrained inflammatory damage as characterized by declination of multiple pro-inflammatory factors IL-1ß, IL-6, and TNF-α in WI-38 cells under LPS circumstance. More importantly, we predicted and verified that UCA1 functioned as a ceRNA by efficaciously binding to miR-499b-5p thereby inversely adjusting miR-499b-5p expression. Interesting, TLR4 was identified as direct target of miR-499b-5p, and positively regulated by UCA1 through sponging miR-499b-5p. Mechanistically, absence of miR-499b-5p or restoration of TLR4 impeded the beneficial effects of UCA1 ablation on LPS-stimulated apoptosis and inflammatory response. Collectively, these observations illuminated that UCA1 inhibition protected WI-38 cells against LPS-managed inflammatory injury and apoptosis process via miR-499b-5p/TLR4 crosstalk, which ultimately influencing the development of pneumonia.

Chicory ameliorates hyperuricemia via modulating gut microbiota and alleviating LPS/TLR4 axis in quail.

BACKGROUND: High-purine diet can cause gut microbiota disorder, which is closely related to the occurrence of hyperuricemia (HUA). At the same time, the development of HUA is often accompanied by renal impairment. Chicory, a natural medicine, has a significant effect on lowering uric acid. However, whether its concrete mechanism is associated with the regulation of gut microbiota and renal damage is still unclear. METHODS: Hyperuricemic quails induced by high-purine diet were used, and quails were divided into control (CON), model (MOD), and model plus high, middle, low doses of chicory. The uricosuric effect was evaluated by detecting the uric acid levels in serum and feces. Meanwhile, the morphology of intestine and kidney were observed by hematoxylin and eosin (HE) staining, and the expression of intestinal barrier junction proteins Occludin, Claudin-1 were detected by quantitative real-time polymerase chain reaction (qPCR) and western blotting. Furthermore, the latent mechanism was clarified by analyzing 16S rRNA amplicon of gut microbiota and measuring the changes of LPS/TLR4 axis inflammatory response of the kidney by western blotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: The results showed that serum uric acid levels were significantly decreased, and the feces uric acid levels were noticeably increased after the intervention of chicory. In addition, chicory could repair the damage of intestinal mucosa and improve the permeability of intestinal barrier. Moreover, the 16S rRNA sequencing analysis uncovered that chicory restored gut microbiota by increasing the probiotics flora (Bifidobacterium, Erysipelotrichaceae) and reducing the pathogenic bacteria group (Helicobacteraceae). Furthermore, it was found that chicory reduced the LPS/TLR4 axis inflammatory response by down regulating the serum LPS and TLR4/NF-κB inflammatory pathway in kidney, thus promoting the excretion of uric acid in kidney. CONCLUSION: Chicory intervention ameliorated HUA via modulating the imbalance of gut microbiota and suppressing LPS/TLR4 axis inflammatory reaction in quail model, which may be a promising candidate for hyperuricemia-relieving properties.

Inflammatory extracellular vesicles prompt heart dysfunction via TRL4-dependent NF-κB activation.

Background: After myocardial infarction, necrotic cardiomyocytes release damage-associated proteins that stimulate innate immune pathways and macrophage tissue infiltration, which drives inflammation and myocardial remodeling. Circulating inflammatory extracellular vesicles play a crucial role in the acute and chronic phases of ischemia, in terms of inflammatory progression. In this study, we hypothesize that the paracrine effect mediated by these vesicles induces direct cytotoxicity in cardiomyocytes. Thus, we examined whether reducing the generation of inflammatory vesicles within the first few hours after the ischemic event ameliorates cardiac outcome at short and long time points. Methods: Myocardial infarction was induced in rats that were previously injected intraperitoneally with a chemical inhibitor of extracellular-vesicle biogenesis. Heart global function was assessed by echocardiography performed at 7, 14 and 28 days after MI. Cardiac outcome was also evaluated by hemodynamic analysis at sacrifice. Cytotoxic effects of circulating EV were evaluated ex-vivo in a Langendorff, system by measuring the level of cardiac troponin I (cTnI) in the perfusate. Mechanisms undergoing cytotoxic effects of EV derived from pro-inflammatory macrophages (M1) were studied in-vitro in primary rat neonatal cardiomyocytes. Results: Inflammatory response following myocardial infarction dramatically increased the number of circulating extracellular vesicles carrying alarmins such as IL-1α, IL-1ß and Rantes. Reducing the boost in inflammatory vesicles during the acute phase of ischemia resulted in preserved left ventricular ejection fraction in vivo. Hemodynamic analysis confirmed functional recovery by displaying higher velocity of left ventricular relaxation and improved contractility. When added to the perfusate of isolated hearts, post-infarction circulating vesicles induced significantly more cell death in adult cardiomyocytes, as assessed by cTnI release, comparing to circulating vesicles isolated from healthy (non-infarcted) rats. In vitro inflammatory extracellular vesicles induce cell death by driving nuclear translocation of NF-κB into nuclei of cardiomyocytes. Conclusion: Our data suggest that targeting circulating extracellular vesicles during the acute phase of myocardial infarction may offer an effective therapeutic approach to preserve function of ischemic heart.

Exosome-shuttled miR-216a-5p from hypoxic preconditioned mesenchymal stem cells repair traumatic spinal cord injury by shifting microglial M1/M2 polarization.

BACKGROUND: Spinal cord injury (SCI) can lead to severe motor and sensory dysfunction with high disability and mortality. In recent years, mesenchymal stem cell (MSC)-secreted nano-sized exosomes have shown great potential for promoting functional behavioral recovery following SCI. However, MSCs are usually exposed to normoxia in vitro, which differs greatly from the hypoxic micro-environment in vivo. Thus, the main purpose of this study was to determine whether exosomes derived from MSCs under hypoxia (HExos) exhibit greater effects on functional behavioral recovery than those under normoxia (Exos) following SCI in mice and to seek the underlying mechanism. METHODS: Electron microscope, nanoparticle tracking analysis (NTA), and western blot were applied to characterize differences between Exos and HExos group. A SCI model in vivo and a series of in vitro experiments were performed to compare the therapeutic effects between the two groups. Next, a miRNA microarray analysis was performed and a series of rescue experiments were conducted to verify the role of hypoxic exosomal miRNA in SCI. Western blot, luciferase activity, and RNA-ChIP were used to investigate the underlying mechanisms. RESULTS: Our results indicate that HExos promote functional behavioral recovery by shifting microglial polarization from M1 to M2 phenotype in vivo and in vitro. A miRNA array showed miR-216a-5p to be the most enriched in HExos and potentially involved in HExos-mediated microglial polarization. TLR4 was identified as the target downstream gene of miR-216a-5p and the miR-216a-5p/TLR4 axis was confirmed by a series of gain- and loss-of-function experiments. Finally, we found that TLR4/NF-κB/PI3K/AKT signaling cascades may be involved in the modulation of microglial polarization by hypoxic exosomal miR-216a-5p. CONCLUSION: Hypoxia preconditioning represents a promising and effective approach to optimize the therapeutic actions of MSC-derived exosomes and a combination of MSC-derived exosomes and miRNAs may present a minimally invasive method for treating SCI.

Upregulation of lncRNA VDR/CASC15 induced by facilitates cardiac hypertrophy through modulating miR-432-5p/TLR4 axis.

Sustained cardiac hypertrophy has threatened human health. With the development of human genome project, non-coding RNAs (ncRNAs) have attracted more and more attentions of researchers. As a subgroup of ncRNAs, long non-coding RNAs (lncRNAs) has been widely studied in human diseases, including cardiac hypertrophy. According to search results of bioinformatics website, lncRNA CASC15 potentially participates in the course of cardiac hypertrophy. According to the result of qRT-PCR, CASC15 expression was upregulated when cardiomyocytes were treated with Ang-II. Moreover, CASC15 was highly expressed in cardiac hypertrophic model. Upregulation of CASC15 was accompanied with some hypertrophic responses. To explore the specific biological function of CASC15 in cardiac hypertrophy, loss-of-function experiments were conducted in Ang-II-induced cardiomyocytes. Results of immunofluorence staining revealed that cell surface area enlarged by Ang-II was decreased when CASC15 was silenced. The expression levels of hypertrophic factors were attenuated by knockdown of CASC15. To detect the molecular mechanism by which CASC15 regulates the progression of cardiac hypertrophy, mechanism experiments were designed and carried out. It was found that CASC15 was activated by the transcription factor VDR. Furthermore, CASC15 can upregulate TLR4 by competitively binding miR-432-5p. In conclusion, Upregulation of lncRNA CASC15 induced by VDR facilitates cardiac hypertrophy via miR-432-5p/TLR4 axis.