Oridonin Promotes Apoptosis in Rheumatoid Arthritis Fibroblast-like Synoviocytes Through PERK/eIF2α/CHOP of Endoplasmic Reticulum Stress Pathway.

Oridonin (ORI), derived from Chinese herbs Rabdosia rubescens, has anti-inflammatory, proapoptotic, anticancer effects. Previous studies have found that ORI induces apoptosis in rheumatoid arthritis fibroblast synovial cells (RA-FLSs), but this mechanism is not clear. We will investigate the apoptosis mechanism of ORI on RA-FLSs. RA-FLSs were treated with various concentrations of ORI (0, 5, 10, 15, 20, 25, and 30 µM) for 24 h. CCK8, LDH, and hochest/PI assay determined the viability, cytotoxicity, and death of ORI on RA-FLSs. The endoplasmic reticulum probe was used to observe structural changes of endoplasmic reticulum in RA-FLSs. RNA expression was detected with RNA sequencing analysis and quantitative real-time PCR. The PERK/eIF2α/CHOP pathway protein of the endoplasmic reticulum was verified with Western Blot. Our results show that ORI induced the apoptosis of RA-FLSs from CCK8, LDH, and Hochest/PI. The endoplasmic reticulum distribution was altered in RA-FLSs after being treated with ORI. Bioinformatics analysis of RNA sequencing data found that 1453 genes were elevated. The PERK/eIF2α/CHOP pathway of the endoplasmic reticulum was regulated from the Gene ontology and KEGG analysis. The results of quantitative real-time PCR and Western blot analysis verified the regulation of PERK/eIF2α/CHOP pathway in RA-FLSs. Our data imply that the endoplasmic reticulum's PERK/eIF2α/CHOP signaling pathway is certainly implicated in the induction of RA-FLS apoptosis by ORI. This study has important implications for the pharmacological effects of ORI and the treatment of RA.

Analysis of circRNAs profile in TNF-α treated DPSC.

BACKGROUND: Pulpitis often are characterized as sustained inflammation and impaired pulp self-repair. Circular RNAs (circRNAs) have been reported to be involved in the development of inflammation, but their influence in pulpitis is still unidentified, which was examined in our research. METHODS: In this study, TNF-α (20 ng/mL) was used to treat DPSCs, then MTS identified cell proliferation. The circRNAs profile in DPSCs with or without TNF-α treatment was evaluated using RNA sequencing and subsequently by bioinformatics analysis. After that, the circular structure was assessed using agarose gel electrophoresis, followed by Sanger sequencing. And the circRNAs expression was ratified using quantitative real-time polymerase chain reaction in cell and tissues samples. Additionally, the plausible mechanism of circRNAs was envisaged, and the circRNA-miRNA-mRNA linkage was plotted using Cytoscape. RESULTS: The treatment of TNF-α inhibited cell proliferation capabilities in DPSCs, which also made 1195 circRNA expressions undergo significant alterations. Among these changes, 11 circRNAs associated with inflammation were chosen for circular structure verification, and only seven circRNAs (hsa_circ_0001658, hsa_circ_0001978, hsa_circ_0003910, hsa_circ_0004314, hsa_circ_0004417, hsa_circ_0035915, and hsa_circ_0002545) had circular structure. Additionally, five circRNAs expressions (hsa_circ_0001978, hsa_circ_0003910, hsa_circ_0004314, hsa_circ_0004417, and hsa_circ_0035915) had significantly altered between with or without TNF-α treated DPSCs. Furthermore, hsa_circ_0001978 and hsa_circ_0004417 were increased in patients suffering from pulpitis. Furthermore, their ceRNA linkage and Kyoto Encyclopedia of Genes and Genomes analysis suggested that these two circRNAs may participate in the inflammation development of pulpitis via mitogen-activated protein kinase and the Wnt signaling pathway. CONCLUSION: This study revealed that the circRNAs profile was altered in TNF-α treated DPSCs. Also, hsa_circ_0001978 and hsa_circ_0004417 may be involved in the inflammation progress of pulpitis. These outcomes provided the latest information for additional research on pulpitis.

Baicalin attenuates adriamycin-induced nephrotic syndrome by regulating fibrosis procession and inflammatory reaction.

BACKGROUND: Baicalin has anti-inflammatory, antibacterial, blood platelet aggregation-inhibiting, free oxygen radical-clearing, and endotoxin-decreasing properties. However, its molecular mechanism involved in the treatment of Adriamycin-induced nephrotic syndrome (NS) is still unclear. OBJECTIVE: This study aimed to explore the effects of baicalin on Adriamycin-induced nephrotic syndrome (NS) and to characterize the genes involved in this progression. METHODS: We established Adriamycin-induced NS model in 32 rats and used six rats in Sham group. Urinary total protein content and creatinine serum were assessed as physiological indicators. H&E staining was used to observe the pathological changes. We determined gene expression profiles using transcriptome sequencing in the rat kidney tissues from Sham, Adriamycin, and Adriamycin + baicalin groups. KEGG was carried out to analyze the enriched pathways of differentially expressed genes among these groups. RESULTS: Baicalin treatment relieved renal injury in NS rats. Expression of 363 genes was significantly different between the Adriamycin and Adriamycin + baicalin M groups. Most of the differentially expressed genes were enriched in pathways involved in epithelial-mesenchymal transition (EMT), fibrosis, apoptosis, and inflammation. CONCLUSIONS: Overall, these data suggest that Adriamycin-induced NS can be attenuated by baicalin through the suppression of fibrosis-related genes and inflammatory reactions. Baicalin is a potential drug candidate for the treatment of NS, and the identified genes represent potential therapeutic targets.

Dexamethasone can attenuate the pulmonary inflammatory response via regulation of the lncH19/miR-324-3p cascade.

OBJECTIVE: To investigate lncRNAs and their roles in regulating the pulmonary inflammatory response under dexamethasone (Dex) treatment. METHODS: IL-1ß (10 ng/mL) and LPS (1 µg/mL) was used to construct inflammatory cell models with A549 cells; IL-1ß performed better against LPS. Different concentrations of Dex were used to attenuate the inflammation induced by IL-1ß, and its effect was assessed via RT-PCR to detect inflammatory cytokine-related mRNA levels, including those of IKß-α, IKKß, IL-6, IL-8, and TNF-α. Furthermore, ELISA was used to detect the levels of the inflammatory cytokines TNF-α, IL-6, and IL-8. RT-PCR was used to quantify the levels of lncRNAs, including lncMALAT1, lncHotair, lncH19, and lncNeat1. LncH19 was most closely associated with the inflammatory response, which was induced by IL-1ß and attenuated by Dex. Among the lncRNAs, the level of lncH19 showed the highest increase following treatment with 1 and 10 µM Dex. Therefore, lncH19 was selected for further functional studies. LncH19 expression was inhibited by shRNA transduced with lentivirus. Cell assays for cell proliferation and apoptosis as well as RT-PCR, western blot, and ELISA for inflammatory genes were conducted to confirm the functions of lncH19. The predicted target miRNAs of lncH19 were hsa-miR-346, hsa-miR-324-3p, hsa-miR-18a-3p, hsa-miR-18b-5p, hsa-miR-146b-3p, hsa-miR-19b-3p, and hsa-miR-19a-3p. Following estimation via RT-PCR, hsa-miR-346, hsa-miR-18a-3p, and hsa-miR-324-3p showed consistent patterns in A549 NC and A549 shlncH19. An miRNA inhibitor was transfected into A549 NC and A549 shlncH19 cells, and the expression levels were determined via RT-PCR. hsa-miR-324-3p was inhibited the most compared with hsa-miR-346 and hsa-miR-18a-3p and was subjected to further functional studies. RT-PCR, ELISA, and western blotting for inflammatory gene detection were conducted to validate the functions of the target hsa-miR-324-3p. RESULTS: Treatment with 1 and 10 µM Dex could effectively attenuate the inflammatory response. During this process, lncH19 expression significantly increased (P < 0.05). Therefore, treatment with 1 µM Dex was used for further study. Under IL-1ß treatment with or without Dex, lncH19 inhibition led to an increase in cell proliferation; a decrease in cell apoptosis; an increase in the protein levels of inflammatory genes; phosphorylation of P65, ICAM-1, and VCAM-1; and increase inflammatory cytokines. Prediction of the targets of lncH19 and validation via RT-PCR revealed that miR-346, miR-18a-3p, and miR-324-3p negatively correlate with lncH19. Additionally, Dex increased the lncH19 expression but reduced that of the miRNAs. Among the miRNAs, miR-324-3p was the most markedly downregulated miRNA following treatment of miRNA inhibitors. The MTS assay and cell apoptosis assay showed that the miR-324-3p inhibitor inhibited cell proliferation and induced cell apoptosis, thereby significantly attenuating the inflammatory response, which reversed the effect of lncH19 in regulating cell proliferation and the secretion of inflammatory cytokines (P < 0.05). Therefore, lncH19 might regulate miR-324-3p in pulmonary inflammatory response under Dex treatment. CONCLUSION: Dex can attenuate the pulmonary inflammatory response by regulating the lncH19/miR-324-3p cascade.

Long noncoding RNA atlas of the inflammation caused by asthma in mice.

There is little evidence regarding the roles of long noncoding RNAs (lncRNAs) in inflammation caused by asthma. In this study, we successfully generated an asthma mouse model that was induced by ovalbumin (OVA). The effects of dexamethasone (Dex) treatment on lung tissue were investigated using pathological and biochemical methods, including Diff-Quik staining, enzyme-linked immunosorbent assay (ELISA), hematoxylin-eosin (H&E) staining, and western blotting (WB). The inflammation was effectively relieved with Dex treatment. High-throughput sequencing revealed that a total of 1490 lncRNAs were detected in lung tissue samples. Differential expression analysis revealed that the Dex group had 20 upregulated and 15 downregulated lncRNAs compared with those in the Model group. Moreover, nine differentially expressed and inflammation-related lncRNAs were verified by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Furthermore, the regulation networks of these nine lncRNAs, their potential binding microRNA (miRNAs), and the putative target genes showed that these lncRNAs play important roles in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. We further identified the expression levels of three potential binding miRNAs by qRT-PCR. The results of this study contribute to a better understanding of the functions of lncRNAs in inflammation caused by asthma.

Oridonin suppresses autophagy and survival in rheumatoid arthritis fibroblast-like synoviocytes.

Context: Oridonin exhibits various pharmacological and physiological activities, including antioxidant, antibacterial, anti-inflammatory, pro-apoptotic, anticancer and neurological effects. However, its role in rheumatoid arthritis (RA) is yet to be revealed.Objective: We evaluated the effects of oridonin on the survival and autophagy of RA-fibroblast-like synoviocytes (FLSs).Materials and methods: RA-FLSs were treated with oridonin at serial concentrations of 0, 2, 4, 6, 8 and 10 µg/mL for 24, 48 and 72 h. Then, cell proliferation and apoptosis were measured. A GFP-LC3 plasmid was transfected into the cells to determine autophagy.Results: Oridonin suppressed RA-FLS proliferation in a dose-dependent manner. The half maximal inhibitory concentrations (IC50) of oridonin at 24, 48 and 72 h were 8.28, 7.88 and 8.35 µg/mL, respectively. Treatment with oridonin for 24 h increased apoptosis by 4.1%, and increased the protein levels of Bax and cleaved caspase-3 but significantly decreased the levels of IL-1ß in the culture supernatant (p < 0.05). In addition, 6 h of oridonin treatment significantly decreased the number of GFP-LC3 punctate dots and inhibited the protein levels of ATG5 and Beclin1 by 80.01% and 42.12%, respectively. Chloroquine (CQ) significantly reinforced the effects of oridonin on inhibition of autophagy, suppression of proliferation, and induction of apoptosis in RA-FLSs (p < 0.05).Conclusions: Our results indicate that treatment with oridonin in combination with CQ inhibits autophagy and cell proliferation and induces apoptosis in RA-FLSs more effectively than treatment oridonin alone. This finding indicates that oridonin is a potential therapeutic agent for RA.

Treatment with Melittin Induces Apoptosis and Autophagy of Fibroblastlike Synoviocytes in Patients with Rheumatoid Arthritis.

BACKGROUND: Melittin, the major medicinal component of honeybee venom, exerts antiinflammatory, analgesic, and anti-arthritic effects in patients with Rheumatoid Arthritis (RA). RA is an inflammatory autoimmune joint disease that leads to irreversible joint destruction and functional loss. Fibroblast-Like Synoviocytes (FLS) are dominant, special mesenchymal cells characterized by the structure of the synovial intima, playing a crucial role in both the initiation and progression of RA. OBJECTIVE: In this study, we evaluated the effects of melittin on the viability and apoptosis of FLS isolated from patients with RA. METHODS: Cell viability was determined using CCK-8 assays; apoptosis was evaluated by flow cytometry, and the expression levels of apoptosis-related proteins (caspase-3, caspase-9, BAX, and Bcl-2) were also determined. To explore whether melittin alters inflammatory processes in RA-FLS, IL-1ß levels were determined using an enzyme-linked immunosorbent assay (ELISA). Furthermore, we performed GFP-LC3 punctate fluorescence dot assays and western blotting (for LC3, ATG5, p62, and Beclin 1) to assess autophagy in RA-FLS. RESULTS: Our results show that melittin can significantly impair viability, promote apoptosis and autophagy, and inhibit IL-1ß secretion in RA-FLS. CONCLUSION: Melittin may be useful in preventing damage to the joints during accidental local stimulation.