LPS-induced Cell Injury Research Articles

Circ_0040994 depletion alleviates lipopolysaccharide-induced HK2 cell injury through miR-17-5p/TRPM7 axis.

Sepsis is a fatal systemic inflammatory disease that causes septic acute kidney injury (AKI). In this work, we explored the roles of circ_0040994 in lipopolysaccharide (LPS)-induced human kidney-2 (HK2) cell injury. Circ_0040994, miR-17-5p and transient receptor potential melastatin 7 (TRPM7) expression were detected by qRT-PCR. Cell functions were examined by MTT assay, flow cytometry assay, western blot, ELISA assay, and oxidative stress assay. The molecular association was detected by dual-luciferase reporter assay. Circ_0040994 was upregulated in the serum of septic AKI patients in comparison with the serum of healthy controls. Silencing circ_0040994 enhanced cell viability but inhibited cell apoptosis, cell inflammation and oxidative stress in LPS-triggered HK2 cells. Circ_0040994 acted as a miR-17-5p sponge to regulate the level of TRPM7. Moreover, miR-17-5p could alleviate LPS-induced HK2 cell injury by suppressing TRPM7. Circ_0040994 downregulation alleviated LPS-induced HK2 cell injury through the miR-17-5p/TRPM7 axis.

EXOSOMAL CIRCVMA21 DERIVED FROM ADIPOSE-DERIVED STEM CELLS ALLEVIATES SEPSIS-INDUCED ACUTE KIDNEY INJURY BY TARGETING MIR-16-5P.

Background: Exosome from adipose-derived stem cells (ADSCs-Exo) has been shown to inhibit the progression of human diseases, including sepsis-related acute kidney injury (AKI). CircVMA21 is considered to be an important regulator for sepsis-related AKI. However, whether ADSCs-Exo affected sepsis-induced AKI by delivering circVMA21 is not clear. Methods: Adipose-derived stem cells were identified by alizarin red staining, oil red O staining, and flow cytometry. Exosome from adipose-derived stem cells was authenticated by transmission electron microscopy, nanoparticle tracking analysis, western blot analysis, and immunofluorescence assay. Cell apoptosis was assessed by flow cytometry, and inflammation cytokine levels were determined by ELISA. Lactate production was assessed using Lactate Acid Content Assay Kit. The expression levels of aerobic glycolysis-related markers, circVMA21 and miR-16-5p, was evaluated by quantitative real time-polymerase chain reaction. Dual-luciferase reporter assay and RIP assay were employed to detect RNA interaction. Animal experiments were used to evaluate the role of ADSCs-Exo on renal function and cell injury in LPS-induced AKI mice model. Results: Exosome from adipose-derived stem cells inhibited LPS-induced HK-2 cell apoptosis, inflammation, and aerobic glycolysis. Knockdown of exosomal circVMA21 derived from ADSCs enhanced HK-2 cell injury induced by LPS. In terms of mechanism, circVMA21 could serve as sponge for miR-16-5p. Besides, miR-16-5p inhibitor reversed the promotion effect of Exo-sh-circVMA21 on LPS-induced cell injury. In addition, ADSCs-Exo protected LPS-induced AKI in mice by increasing circVMA21 expression and decreasing miR-16-5p expression. Conclusion: Exosomal circVMA21 derived by ADSCs relieved LPS-induced AKI through targeting miR-16-5p, which provided a potential molecular target for treating sepsis-related AKI.

Calycosin Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Mice through the miR-375-3p/ROCK2 Axis

Objective: Septic patients are especially vulnerable to acute lung injury (ALI). Calycosin (CAL) has various promising pharmacological activities. This paper aims to expound on the role of CAL in mice with sepsis-induced ALI and the associated mechanisms. Methods: Mouse models of sepsis-induced ALI were established using lipopolysaccharide (LPS). Pulmonary histopathological changes were observed by HE staining. Cell apoptosis was assessed by TUNEL staining. Pulmonary edema was evaluated by measuring wet/dry weight. Bronchoalveolar lavage fluid (BALF) was collected to count inflammatory cells. In vitro LPS models were established using MLE-12 cells. miR-375-3p expression was determined by RT-qPCR. Cell viability and apoptosis were assessed by MTT assay and flow cytometry. Levels of inflammatory cytokines were determined by ELISA. The target relationship between miR-375-3p and ROCK2 was analyzed by the dual-luciferase assay. ROCK2 protein level was determined by Western blot. Results: miR-375-3p was weakly-expressed in mice with sepsis-induced ALI, and CAL treatment elevated miR-375-3p expression. CAL treatment mitigated pulmonary tissue damage and edema, decreased apoptosis and inflammatory cells, downregulated levels of pro-inflammatory cytokines, and upregulated levels of anti-inflammatory cytokines in mice with sepsis-induced ALI. CAL treatment increased MLE-12 cell viability and decreased apoptosis and inflammation in MLE-12 cells. Inhibition of miR-375-3p partially abrogated CAL-mediated protective action on MLE-12 cells. miR-375-3p attenuated LPS-induced MLE-12 cell injury by targeting ROCK2. Conclusion: CAL upregulates miR-375-3p to target ROCK2, thus protecting against sepsis-induced ALI in mice.

ELAVL1 promotes LPS-induced endothelial cells injury through modulation of cytokine storm

Sepsis is a life-threatening systemic organ dysfunction caused by the host's unregulated response to a widespread bacterial infection. Endothelial injury is a major pathophysiologic symptom of sepsis and is considered a critical factor in promoting the progression of disease severity. ELAV like RNA binding protein 1(ELAVL1) is a ubiquitously expressed RNA-binding protein that may play an important role during sepsis. Nonetheless, the molecular mechanisms of ELAVL1 on endothelial cell damage in sepsis have not been well defined. Here, we aimed to confirm the role of ELAVL1 in sepsis-induced endothelial cell damage using lipopolysaccharide (LPS)-induced zebrafish and endothelial cells (ECs) models. We found that zebrafish larvae treated with LPS exhibited systemic endothelial cell damage, mostly manifested as pericardial edema, curved tail, and impaired angiogenesis. LPS treatments also significantly induced the expression levels of inflammatory cytokines (interleukin-6 (IL-6), IL-8, and tumor necrosis factor (TNF)-α) in vivo. In vitro, we observed the increase of ELAVL1 cytoplasmic translocation with LPS treatment. Mechanistically, targeted disruption of the ELAVL1 gene decreased the expression of TNF-α, IL-6, and IL-8 during induction of sepsis and alleviated LPS-induced blood vessel injury in zebrafish. Taken together, our study indicates that ELAVL1 knockdown may alleviate sepsis-induced endothelial cells injury by suppressing cytokine storm. Our research suggests that inhibition of ELAVL1 could reduce the level of inflammatory cytokine production induced by LPS and protect against endothelial cell injury. ELAVL1 might be a potential therapeutic target to block endothelial cells injury associated with sepsis.

CircSFMBT2-OA alleviates chondrocyte apoptosis and extracellular matrix degradation through repressing NF-κB/NLRP3 inflammasome activation

BackgroundIntra-articular inflammation and cartilage degradation are the major pathological characteristics of osteoarthritis (OA). Mounting studies have revealed that circular RNAs (circRNAs) act as an important regulatory role in inflammatory diseases and are frequently dys-expressed in OA cartilage tissues. ObjectiveHere, a dys-regulated cicrRNA (has_circ_0017636, termed circSFMBT2-OA) was identified, and its role in regulating lipopolysaccharide (LPS)-induced chondrocyte injury was next investigated. MethodsCHON-001 chondrocytes were treated with LPS, and then the levels of circSFMBT2-OA, cartilage-related genes, and pro-inflammatory cytokines were measured using quantitative real-time PCR (qRT-PCR) and Western blot analysis. CHON-001 cell viability, proliferation, and apoptosis were assayed using Cell Counting Kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EDU), and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, respectively. ResultsCircSFMBT2-OA level was significantly down-regulated in OA cartilage tissues and LPS-treated CHON-001 cells. Functionally, circSFMBT2-OA overexpression accelerated cell proliferation, and suppressed cell apoptosis, pro-inflammatory cytokines production, matrix-degrading enzymes expression, and ECM degradation in CHON-001 cells. Inversely, circSFMBT2-OA depletion decreased cell viability and increased matrix-degrading enzymes expression and ECM degradation. Mechanistically, circSFMBT2-OA inhibited LPS-induced NF-κB/NOD-like receptor family pyrin domain containing protein 3 (NLRP3) inflammasome activation in CHON-001 cells. Consequently, NLRP3 activator reversed the effect of circSFMBT2-OA on repressing LPS-induced CHON-001 cell injury. ConclusionThese data reveal a vital effect of a novel circSFMBT2-OA on repressing OA progression and provide a promising target to treat OA.

CIRC_0001818 TARGETS MIR-136-5P TO INCREASE LIPOPOLYSACCHARIDE-INDUCED HK2 CELL INJURIES BY ACTIVATING TXNIP/NLRP3 INFLAMMASOME PATHWAY.

Background: The implication of circular RNAs (circRNAs) in sepsis-related complications arouses much attention, which provides additional treatment options for sepsis-related complications. The purpose of this study is to unveil the function and functional mechanism of circ_0001818 in cell models of septic acute kidney injury (AKI). Methods: Septic AKI cell models were constructed using HK2 cells treated with lipopolysaccharide (LPS). The expression levels of circ_0001818, miR-136-5p, and thioredoxin interacting protein (TXNIP) mRNA were examined by quantitative real-time polymerase chain reaction. Cell viability and death were explored by CCK-8 and flow cytometry assays. The activity of oxidative stress-related markers was examined using commercial kits. The secretion of inflammatory factors was examined using ELISA kits. The binding between miR-136-5p and circ_0001818 or TXNIP was validated by dual-luciferase reporter test and pull-down assay. The receiver operating characteristic curve was depicted to assess the diagnostic value of circ_0001818, miR-136-5p, and TXNIP in serumal exosomes from patients with septic AKI. Results: Circ_0001818 expression was elevated in LPS-treated HK2 cells. Loss-of-function assays displayed that circ_0001818 downregulation alleviated LPS-induced HK2 cell death, oxidative stress, inflammatory release, and inflammasome activation. MiR-136-5p was targeted by circ_0001818, and inhibition of miR-136-5p attenuated the effects of circ_0001818 downregulation, thus recovering LPS-induced HK2 cell injuries. MiR-136-5p targeted the downstream TXNIP, and circ_0001818 dysregulation could affect TXNIP expression via targeting miR-136-5p. Overexpression of TXNIP overturned the effects of circ_0001818 downregulation. Moreover, circ_0001818, miR-136-5p, and TXNIP in serumal exosomes had diagnostic values. Conclusions: Circ_0001818 targets miR-136-5p to activate TXNIP expression, leading to the contribution of LPS-induced HK2 cell injury.

Human amniotic mesenchymal stem cell-conditioned medium inhibited LPS-induced cell injury and inflammation by modulating CD14/TLR4-mediated signaling pathway in monocytes

Human amniotic mesenchymal stem cells (hAMSCs) have attracted considerable attention as a promising regenerative therapy. Many studies reported that the conditioned medium of hAMSCs (AM-CM) exerted anti-inflammatory and immunomodulatory functions, while its underlying mechanism is poorly understood. In this study, we first confirmed that AM-CM (25%, 50%, 100%) was optimal for anti-inflammation at 24 h. Lipopolysaccharide (LPS)-induced alteration of cell morphology, the decrease of cell proliferation, and the upregulation of cell apoptosis were significantly reversed in AM-CM-treated THP-1 cells. 25% and 50% AM-CM significantly decreased LPS-induced intracellular reactive oxygen species (ROS) production and proinflammatory cytokines secretion. Mechanistically, we found that AM-CM treatment suppressed LPS-induced activation of MAPK and NF-κB pathways by inhibiting CD14/TLR4 in THP-1 cells. Meanwhile, activation of NLRP3 inflammasome was also dose-dependently attenuated by AM-CM treatment. Thus, AM-CM may exert positive influences on the inflammation microenvironment and provide a novel strategy for improving tissue regeneration.

Protective effect of the total flavonoids from Clinopodium chinense against LPS-induced mice endometritis by inhibiting NLRP3 inflammasome-mediated pyroptosis

Ethnopharmacological relevanceClinopodium chinense (Benth.) O. Kuntze (C. chinense) is a Chinese herbal medicine used in treating gynecological hemorrhagic diseases for hundreds of years. Flavonoids are one kind of the major components in C. chinense. The flavonoids of C. chinense (TFC) have a vital role in treating endometritis but the underlying therapeutic mechanisms of TFC against endometritis have been rarely reported. Aim of the studyTo elucidate the therapeutic effect and possible mechanisms of TFC against lipopolysaccharide (LPS)-induced endometritis in vivo and LPS-induced primary mouse endometrial epithelial cells (MEECs) injury in vitro. Materials and methodsThe holistic phytochemicals of the TFC and TFC-contained serum were screened and identified using UPLC-Q-TOF-MS. The model of endometritis was established by intrauterine injection of LPS (5 mg/mL) into female BALB/c mice, and the model mice were treated with TFC for 7 days. The value of MPO was measured by Myeloperoxidase assay kit, the pathological changes in the endometrium were evaluated using H&E staining and transmission electron microscope (TEM), the secretions of IL-18, IL-1β and TNF-α were determined by ELISA kits, the mRNA expressions of IL-18, IL-1β and TNF-α were determined by RT-PCR assay, and the protein levels of TLR4, IKBα, p-IKBα, p65, p-p65, caspase-1, ASC, NLRP3 and GSDMD were measured by Western blot. Subsequently, MEECs were isolated from the uterus of pregnant female mice, injured by LPS for 24 h and incubated with the TFC-contained serum. Finally, cell viability, LDH release, hoechst 33342/PI staining, immunofluorescence staining, scanning electron microscope observation, ELISA assay, RT-PCR detection and Western blot analysis were carried out to further validate the therapeutic effect and the underlying mechanisms of TFC. ResultsA total of 6 compounds in the plasma of mice after being intragastric administrated of TFC were identified. The results in vivo showed that TFC significantly reduced MPO value and alleviated pathological injury of the endometrium. Furthermore, TFC significantly decreased the serum IL-18, IL-1β and TNF-α levels, and the mRNA levels of IL-18, IL-1β and TNF-α. TFC also inhibited the expressions of TLR4, p-IKBα, p-p65, caspase-1, ASC, NLRP3 and GSDMD. Besides, compared with the model group in MEECs cells, TFC-contained serum prevented pyroptosis, decreased the levels of IL-18 and IL-1β, and inhibited the mRNA expressions of IL-18, IL-1β and GSDMD. TFC-contained serum also reversed the activation of NLRP3 inflammasome caused by nigericin, and restrainted the translocation of NF-κB into nuclear. ConclusionsTFC protects mice endometritis from the injury of LPS via suppressing the activation of NLRP3 inflammasome and pyroptosis, the underlying mechanisms of which were related to restraining the TLR4/NF-κB/NLRP3 pathway activation.

Downregulation of ROR2 attenuates LPS-induced A549 cell injury through JNK and ERK signaling pathways.

We aimed to determine whether receptor tyrosine kinase-like orphan receptor 2 (ROR2) is involved in the occurrence of acute lung injury(ALI) by an animal study and explore the effect of ROR2 downregulation on lipopolysaccharide (LPS)-treated human lung carcinoma A549 cells by a cytological study. Murine models of ALI were successfully constructed by intratracheal instillation of LPS. Meanwhile, A549 cell line stimulated with LPS was used for a cytological study. The expression of ROR2 and its effect on proliferation, cell cycle, apoptosis, and inflammation were detected. It was found that LPS administration markedly inhibited the cell proliferation, resulted in cell cycle arrest at G1 phage, elevated levels of pro-inflammatory cytokines and apoptosis rate of A549 cells. However, LPS-mediated adverse effects mentioned above were significantly ameliorated by downregulation of ROR2 in comparison with LPS treatment. In addition, administration of ROR2 siRNA notably decreased the phosphorylation level of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in LPS-challenged A549 cells. Thus, the present data indicate that downregulation of ROR2 may decrease LPS-induced inflammatory responses and cell apoptosis through inhibiting JNK and ERK signaling pathway, which attenuates ALI.

Downregulation of miR-527 alleviates sepsis-induced acute kidney injury via targeting Beclin1.

Sepsis-induced acute kidney injury (AKI) is known to result from the inflammatory responses. MiRNAs participate in the development of sepsis-induced AKI. Nevertheless, the function of miR-527 in sepsis-induced AKI remains unclear. Cell viability was evaluated by CCK8 assay, and TUNEL staining was applied to assess cell apoptosis. Pro-inflammatory cytokine (TNF-α, IL-6 and IL-1β) levels were evaluated by ELISA. Meanwhile, the relation among miR-527 and Beclin1 was detected by dual luciferase report assay. Western blot and RT-qPCR were used to examine the protein and mRNA levels, respectively. Furthermore, an in vivo model was constructed to assess the function of miR-527 in sepsis-induced AKI. MiR-527 downregulation significantly alleviated the symptoms of sepsis-induced AKI in mice. MiR-527 level in HK-2 cells was significantly upregulated by LPS, and downregulation of miR-527 notably reversed LPS-induced inhibition of HK-2 cell viability by inhibiting apoptosis. In addition, LPS greatly increased TNF-α, IL-6 and IL-1β levels in supernatant of HK-2 cells, while miR-527 inhibitor partially restored this phenomenon. Meanwhile, Beclin1 was found to be the downstream mRNA of miR-527, and miR-527 inhibitor notably upregulated the level of LC3. MiR-527 downregulation reversed LPS-induced HK-2 cell injury through suppression of TGF-β pathway. Downregulation of miR-527 alleviated sepsis-induced AKI via targeting Beclin1. Thus, miR-527 might act as a vital mediator in sepsis-induced AKI.

Inhibition of sepsis-induced acute kidney injury via the circITCH-miR-579-3p-ZEB2 axis.

Circular RNAs (circRNAs) are linked to the regulation of sepsis-induced acute kidney injury (AKI). However, the function of circITCH in the development of sepsis-induced AKI is still unclear. The levels of circITCH, miR-579-3p and ZEB2 were examined by real-time PCR and immunoblotting. Then, the roles of circITCH in cell viability, apoptosis, and inflammation in lipopolysaccharide (LPS)-treated HK-2 cells were evaluated. The further mechanism was investigated using rescue assays. CircITCH was downregulated in septic AKI patients and LPS-triggered HK-2 cells. CircITCH overexpression restored cell viability in LPS-treated HK-2 cells and restrained apoptosis and inflammatory cytokine production. CircITCH negatively regulated miR-579-3p, thereby upregulating ZEB2 expression. Taken together, circITCH alleviates LPS-induced HK-2 cell injury by regulating miR-579-3p/ZEB2 signal axis, which provides a theoretical basis for AKI therapy.

Apelin-36 alleviates LPS-induced trophoblast cell injury by inhibiting GRP78/ASK1/JNK signaling

Pre-eclampsia (PE) is a type of hypertensive disorder of pregnancy that poses a serious threat to the health of both mother and fetus. Inhibition of the inflammatory environment on trophoblast cells is of great significance to improve PE. Apelin-36 is an endogenous active peptide with strong anti-inflammatory activity. Therefore, this study aims to investigate the effects of Apelin-36 on lipopolysaccharide (LPS)-induced trophoblast cells and its potential mechanism. The levels of inflammatory factors (TNF-α, IL-8, IL-6 and MCP-1) were detected by reverse transcription-quantitative PCR (RT-qPCR). The proliferation, apoptosis, migration and invasion capacities in trophoblast cells were detected by CCK-8, TUNEL staining, wound healing and Transwell assays, respectively. GRP78 was overexpressed by cell transfection. Western blot was applied for the identification of protein levels. Apelin concentration-dependently decreased the expression of inflammatory cytokines and p-p65 protein level in trophoblast cells induced by LPS. Apelin treatment reduced LPS-induced apoptosis and improved the proliferation, invasion and migration capacities of LPS-mediated trophoblast cells. Additionally, Apelin down-regulated GRP78, p-ASK1 and p-JNK protein levels. The inhibition on LPS-induced trophoblast cell apoptosis and the promotion on invasion and migration by Apelin-36 was counteracted by GRP78 overexpression. To sum up, Apelin-36 could alleviate LPS-induced cell inflammation and apoptosis and improve the invasion and migration of trophoblasts by inhibiting the GRP78/ASK1/JNK signaling.

CIRCULAR RNA CIRC_0099188 CONTRIBUTES TO LPS-INDUCED HPAEpiC CELL INJURY BY TARGETING THE MIR-1236-3P/HMGB3 AXIS.

Purpose: This study is designed to explore the role and mechanism of circ_0099188 in LPS-engendered HPAEpiC cells. Methods: Circ_0099188, microRNA-1236-3p (miR-1236-3p), and high mobility group box 3 (HMGB3) levels were measured using real-time quantitative polymerase chain reaction. Cell viability and apoptosis were assessed using cell counting kit-8 (CCK-8) and flow cytometry assays. Protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), cleaved-caspase 3, cleaved-caspase 9, and HMGB3 were determined using Western blot assay. IL-6, IL-8, IL-1β, and TNF-α levels were analyzed using enzyme-linked immunosorbent assays. After predicting using Circinteractome and Targetscan, the binding between miR-1236-3p and circ_0099188 or HMGB3 was verified using a dual-luciferase reporter, RNA immunoprecipitation, and RNA pull-down assays. Results: Circ_0099188 and HMGB3 were highly expressed, and miR-1236-3p was decreased in LPS-stimulated HPAEpiC cells. Also, the downregulation of circ_0099188 might overturn LPS-triggered HPAEpiC cell proliferation, apoptosis, and inflammatory response. Mechanically, circ_0099188 is able to affect HMGB3 expression by sponging miR-1236-3p. Conclusion: Circ_0099188 knockdown might mitigate LPS-induced HPAEpiC cell injury by targeting the miR-1236-3p/HMGB3 axis, providing an underlying therapeutic strategy for pneumonia treatment.

CIRC_0008882 STIMULATES PDE7A TO SUPPRESS SEPTIC ACUTE KIDNEY INJURY PROGRESSION BY SPONGING MIR-155-5P.

Background: The importance of circular RNA (circRNA) in the progression of septic acute kidney injury (AKI) was gradually recognized. It has been confirmed that circ_0008882 expression was decreased in the blood of patients with AKI. However, the role of circ_0008882 in septic AKI progression remains unclear. Methods: Human kidney-2 (HK2) cells were stimulated with lipopolysaccharide (LPS) to establish a septic AKI cell model. The RNA and protein expression of circ_0008882, miR-155-5p, phosphodiesterase 7A (PDE7A), PCNA, Bax, and Bcl-2 were detected by quantitative real-time polymerase chain reaction and Western blot. Cell viability was investigated by cell counting kit-8 assay. Enzyme-linked immunosorbent assay (ELISA) was adopted to measure the levels of inflammatory factors (TNF-α, IL-1β, and IL-6). Flow cytometry was implemented to evaluate cell cycle and cell apoptosis. The Caspase3 activity was examined using Caspase3 Assay Kit. Dual-luciferase reporter assay and RNA immunoprecipitation assay were applied to verify the molecular target relations. Results: Septic AKI serum samples and LPS-induced HK2 cells displayed low expression of circ_0008882 and PDE7A, and high expression of miR-155-5p when compared with the controls. Overexpression of circ_0008882 relieved LPS-induced HK2 cell injury. MiR-155-5p was a target of circ_0008882, and miR-155-5p mimic restored circ_0008882 overexpression-mediated effects on LPS-treated HK2 cells. PDE7A was identified as a target gene of miR-155-5p, and PDE7A downregulation almost reverted the improvement impacts induced by the miR-155-5p inhibitor. Conclusions: Overexpression of circ_0008882 impeded LPS-induced HK2 cell injury by modulating miR-155-5p/PDE7A pathway, implying that circ_0008882 might be a possible circRNA-targeted therapy for septic AKI.

MiR-493-5p alleviates lipopolysaccharide-induced inflammation in ATDC5 chondrogenic cells by targeting TAB2

Purpose: To investigate the effect of miR-493-5p in lipopolysaccharide (LPS) -induced ATDC5 chondrogenic cells.
 Methods: The MTT assay was used to determine the viability of LPS-induced ATDC5 cells. ENCORI (starbase.sysu.edu.cn/) was used to predict the target of miR-493-5p. Quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) was used to determine miR-493-5p expression in ATDC5 controlled cells and cells treated with various combinations of LPS, negative control miRNA (NC), miR-493-5p mimic, TAB2 overexpression vector, and miR-493-5p inhibitor. qRT-PCR, while western blot was used to assess TAB2 mRNA and protein expression levels in control, LPS, LPS + NC, LPS + miR-493-5p mimic, and LPS + miR-493-5p inhibitor groups. The qRT-PCR and ELISA were used to evaluate TNF-α, IL-18, and IL-1β expressions in control, NC, LPS, LPS + NC, miR-493-5p mimic, and LPS + miR-493-5p mimic. Furthermore, the two techniques were also used determine LPS + miR-493-5p inhibitor, LPS + TAB2 overexpression, and LPS + TAB2 overexpression + miR-493-5p mimic groups.
 Results: MiR-493-5p expression was downregulated in LPS-induced ATDC5 cells (p < 0.001). Overexpression of miR-493-5p increased LPS-induced ATDC5 cell viability but decreased expression of inflammatory factors (p < 0.001). MiR-493-5p targeted and inhibited TAB2 expression in LPS-induced ATDC5 cells (p < 0.001). TAB2 overexpression reversed the suppression of TAB2 by miR-493-5p in LPS-induced ATDC5 cell injury (p < 0.001).
 Conclusion: MiR-493-5p alleviates LPS-induced inflammation of ATDC5 chondrogenic cells by targeting TAB2. Thus, miR-493-5p and TAB2 have potentials for use as clinical therapeutic targets for OA.

LncRNA RMRP aggravates LPS-induced HK-2 cell injury and AKI mice kidney injury by upregulating COX2 protein via targeting ELAVL1

ObjectivesThere is emerging evidence that long non-coding RNA component of mitochondrial RNA processing endoribonuclease (lncRNA RMRP) is involved in acute kidney injury (AKI) progression, but the specific mechanism of action still requires further investigation. MethodsThe lipopolysaccharide (LPS)-treated HK-2 cells were transfected with pcDNA-RMRP or si-RMRP, or transfected with pcDNA-ELAV like RNA binding protein 1 (ELAVL1) or si-ELAVL1, and cell viability, apoptosis, inflammatory factor secretion and oxidative stress were detected. The LPS-treated HK-2 cells were transfected with si-RMRP alone or together with pcDNA-ELAVL1, and cell behaviors were examined. The LPS-treated HK-2 cells were transfected with si-ELAVL1 alone or together with pcDNA- cyclooxygenase-2 (COX2), and the cellular changes were observed. The LPS-treated HK-2 cells were transfected with si-RMRP alone or together with pcDNA-ELAVL1, or together with pcDNA-ELAVL1 and si-COX2, and cell behaviors were examined. A mouse model of AKI was constructed using male C57BL/6 mice by the method of cecal ligation and puncture and intraperitoneal injection of LPS to explore the effect of RMRP silencing on renal injury in vivo. ResultsRMRP and ELAVL1 was upregulated in LPS-treated HK-2 cells, and RMRP or ELAVL1 overexpression inhibited cell viability and promoted cell apoptosis, inflammatory factor secretion and oxidative stress, and RMRP knockdown showed the opposite effects. ELAVL1 upregulated COX2 protein expression and overexpression of COX2 reversed the promoting effects of RMRP knockdown on cell viability, as well as the inhibitory effects on cell apoptosis, inflammatory factor secretion and oxidative stress. Mechanistic findings suggested that RMRP aggravates LPS induced cell injury by activating prostaglandin E (PGE)/janus kinase-2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. We observed that knockdown of RMRP expression significantly alleviated renal tissue apoptosis, inflammatory factor secretion, and oxidative stress with AKI mice. ConclusionsOur findings may provide a new reference for the treatment of AKI.

CIRC_0114428 INFLUENCES THE PROGRESSION OF SEPTIC ACUTE KIDNEY INJURY VIA REGULATING MIR-370-3P/TIMP2 AXIS.

Background: Septic acute kidney injury (AKI) is a serious complication of sepsis, which greatly threatened the life safety of critically ill patients. Recently, circular RNA is considered to be implicated in sepsis-induced renal cell damage. However, the role of circ_0114428 in sepsis AKI is still unclear. Methods: LPS was used to establish a sepsis-related AKI cell model. The expression of circ_0114428, microRNA (miR)-370-3p, tissue inhibitor of metalloproteinase-2 (TIMP2), Proliferating cell nuclear antigen, Bax, and Bcl-2 were detected by quantitative real-time polymerase chain reaction and Western blot. Cell counting kit 8 and enzyme-linked immunosorbent assay were used to measure cell proliferation ability and the secretion of inflammatory factors (tumor necrosis factor α, interleukin 1β, and interleukin 6), respectively. Cell cycle and apoptosis rate were analyzed by flow cytometry. Caspase-3 assay kits were used to detect Caspase-3 activity. Interaction between miR-370-3p and circ_0114428 or TIMP2 was analyzed by bioinformatics analysis, a dual-luciferase reporter assay, and RNA immunoprecipitation assay. Results: Circ_0114428 was upregulated in septic AKI serum samples and LPS-induced HK2 cells. The knockdown of circ_0114428 notably promoted cell proliferation and cycle, whereas it restrained cell inflammation and apoptosis in LPS-stimulated HK2 cells. Subsequent mechanism analysis revealed that miR-370-3p was a target of circ_0114428, and miR-370-3p inhibition could rescue the effects of circ_0114428 downregulation on LPS-induced cell injury. Meanwhile, TIMP2 was a target gene of miR-370-3p. miR-370-3p mimic could attenuate LPS-induced cell injury, whereas these impacts were overturned by overexpressed TIMP2. Furthermore, circ_0114428 enhanced TIMP2 protein expression by sponging miR-370-3p. Conclusion: Our data demonstrated that circ_0114428 contributed to septic AKI progression by regulating miR-370-3p-mediated TIMP2 expression, which provided a promising target for septic AKI treatment.

Long noncoding RNA MALAT1 inhibition attenuates sepsis-induced acute lung injury through modulating the miR-129-5p/PAX6/ZEB2 axis.

This research aimed to investigate the role of the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/microRNA-129-5p (miR-129-5p)/paired box gene 6 (PAX6) axis in sepsis-induced acute lung injury (ALI). MLE-12 cells and C57BL/6 mice were induced by LPS to establish lung injury in in vitro and in vivo models. Cell viability and apoptosis were measured by cell counting kit-8 assay and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining, respectively. Levels of inflammatory cytokines in cell supernatants and bronchoalveolar lavage fluid (BALF) were detected by ELISA. Lung injury was evaluated by lung wet weight-to-dry weight ratio and hematoxylin-eosin staining. MALAT1, PAX6, and zinc finger E-box-binding homeobox 2 (ZEB2) expression was elevated and miR-129-5p expression was reduced in the serum of patients with sepsis-induced ALI, LPS-induced MLE-12 cells, and lung tissues of ALI mice. MALAT1 interference delayed the LPS-induced cell proliferation decrease, apoptosis increase, and inflammatory factor increase. miR-129-5p inhibition could reverse the delaying effect of MALAT1 interference on LPS-induced lung cell injury. PAX6 overexpression (oe) reversed the inhibitory effect of miR-129-5p oe on LPS-induced lung cell injury. Downregulating MALAT1 reduced pulmonary edema, inflammatory cytokine levels, lung injury, and apoptosis in ALI mice. Moreover, miR-129-5p suppression or PAX6 oe reversed the delaying effect of MALAT1 interference on sepsis-induced ALI. MALAT1 aggravates sepsis-induced ALI via the miR-129-5p/PAX6/ZEB2 axis.

Circ_0044411 silencing protects infantile pneumonia from lipopolysaccharide-induced cell injury by sponging miR-141-3p to inhibit CCL16 expression.

Circular RNA (circRNA) has been found to play an important role in the progression of many diseases, including infantile pneumonia. However, the role of circ_0044411 in infantile pneumonia progression is still unclear. MRC-5 cells were incubated with lipopolysaccharide (LPS) for 12h to establish the in vitro cellular model for infantile pneumonia. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of circ_0044411, miR-141-3p (micoRNA-141-3p) and CCL16 (CC motif chemokine ligand 16). Cell viability and proliferation was assessed by 3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyl tetrazolium bromide (MTT) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. The levels of inflammatory factors IL-1β, IL-6 and TNF-α were determined by enzyme-linked immunosorbent assay kits. Cell apoptosis and caspase-3 activity were detected by flow cytometry analysis and caspase-3 activity assay kit. The target interaction was confirmed by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA pull-down assay. Circ_0044411 was highly repressed in the serum of infantile pneumonia patients and LPS-induced MRC-5 cells. Circ_0044411 could promote the cell viability and proliferation, inhibit inflammatory response and apoptosis in LPS-induced MRC-5 cells. Circ_0044411 could serve as a sponge of miR-141-3p, and miR-141-3p could reverse the function of circ_0044411 on LPS-induced MRC-5 cell injury. In addition, miR-141-3p could target CCL16, and miR-141-3p could protect MRC-5 cells from LPS-induced cell injury by targeting CCL16. Furthermore, circ_0044411 sponged miR-141-3p to positively regulate CCL16 expression. Circ_0044411 knockdown promoted cell viability and proliferation, inhibited inflammatory response and apoptosis by regulating miR-141-3p/CCL16 axis, indicating that circ_0044411 might be a potential therapeutic target for IP.

Overexpression of FTO alleviates osteoarthritis by regulating the processing of miR-515-5p and the TLR4/MyD88/NF-κB axis.

Osteoarthritis (OA) is regarded as the most prevalent chronic joint disease. Fat-mass and obesity-associated gene (FTO) is involved in OA alleviation. This study elucidated the role of FTO in OA and the associated mechanism. We established a cell injury model by stimulating human normal chondrocytes (C28/I2) with lipopolysaccharide (LPS), and measured cell viability, apoptosis, and inflammatory cytokines using CCK-8, flow cytometry, Western blot, and ELISA. TLR4, MyD88, p/t-p65, and p/t-IκBα levels, FTO, COX-2, and iNOS mRNA levels, and m6A methylation levels were measured by Western blot, RT-qPCR, and colorimetry. RNA immunoprecipitation and co-immunoprecipitation were conducted to confirm the interaction between FTO and DGCR8. pri-miR-515-5p process was regulated in an m6A-dependent manner. After predicting the presence of several binding sites between miR-515-5p and TLR4 on Targetscan, we further confirmed their relationship by dual-luciferase assay. OA rat models were established by monosodium iodoacetate injection. The pathological changes in knee joint were observed by HE staining. FTO was diminished in LPS-induced C28/I2 cells. With the increase of LPS concentration, cell viability was repressed, apoptosis rate was increased, and inflammatory markers were promoted, which were annulled by FTO overexpression. FTO interacted with DGCR8 and modulated the pri-miR-515-5p processing in an m6A-dependent manner. miR-515-5p silencing partially averted the inhibitory effect of FTO on LPS-induced cell injury. Given that TLR4 was a direct target of miR-515-5p, miR-515-5p inactivated the MyD88/NF-κB pathway by targeting TLR4. FTO overexpression improved cartilage structure in OA rats, reduced apoptosis, inhibited inflammation in synovial fluid, and repressed the TLR4/MyD88/NF-κB axis. FTO alleviated OA in an m6A-dependent manner via the miR-515-5p/TLR4/MyD88/NF-κB axis.