Apoptosis In Fibroblasts Research Articles

GSK-3β-mediated activation of NLRP3 inflammasome leads to pyroptosis and apoptosis of rat cardiomyocytes and fibroblasts

We previously demonstrated that GSK-3β mediates NLRP3 inflammasome activation and IL-1β production in cardiac fibroblasts (CFs) after myocardial infarction (MI). In this study, we show how GSK-3β-mediated activation of the NLRP3 inflammasome/caspase-1/IL-1β pathway leads to apoptosis and pyroptosis of cardiomyocytes (CMs) and CFs. Administration of lipopolysaccharide (LPS)/ATP to primary newborn rat cardiac fibroblasts (RCFs) led to increase in proteins of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), caspase-1, IL-1β, and IL-18. Additionally, the expression of caspase-3 and N-terminal fragments of gasdermin D (N-GSDMD) and the Bax/Bcl-2 ratio increased. Administration of the GSK-3β inhibitor SB216763 reduced the levels of apoptosis- and pyroptosis-related proteins regulated by NLRP3 inflammasome activation in RCFs. Next, we transferred the culture supernatant of LPS/ATP-treated RCFs to in vitro primary newborn rat cardiomyocytes (RCMs). The results showed that SB216763 attenuate the upregulation of the ratios of Bax/Bcl-2 and the expression of caspase-3 and N-GSDMD in RCMs. Direct stimulation of RCMs and H9c2 cells with recombinant rat IL-1β increased the p-GSK-3β/GSK-3β and Bax/Bcl-2 ratios and the expression of caspase-3 and N-GSDMD, while both SB216763 and TLR1 (an IL-1β receptor inhibitor) markedly reduced these effects, as assessed using propidium iodide positive staining and the lactate dehydrogenase release assay. The caspase-11 inhibitor wedelolactone decreased the expression level of N-GSDMD but did not alter the p-GSK-3β/GSK-3β ratio. Lastly, we established a Sprague–Dawley rat MI model to confirm that SB216763 diminished the increase in caspase-3 and N-GSDMD expression and the Bax/Bcl-2 ratio in the ischemic area. These data demonstrate that GSK-3β regulates apoptosis and pyroptosis of RCMs and RCFs due to NLRP3 inflammasome activation in RCFs.

Loss of p19Arf promotes fibroblast survival during leucine deprivation

ABSTRACTFibroblasts are quiescent and tumor suppressive in nature but become activated in wound healing and cancer. The response of fibroblasts to cellular stress has not been extensively investigated, however the p53 tumor suppressor has been shown to be activated in fibroblasts during nutrient deprivation. Since the p19 Alternative reading frame (p19Arf) tumor suppressor is a key regulator of p53 activation during oncogenic stress, we investigated the role of p19Arf in fibroblasts during nutrient deprivation. Here, we show that prolonged leucine deprivation results in increased expression and nuclear localization of p19Arf, triggering apoptosis in primary murine adult lung fibroblasts (ALFs). In contrast, the absence of p19Arf during long-term leucine deprivation resulted in increased ALF proliferation, migration and survival through upregulation of the Integrated Stress Response pathway and increased autophagic flux. Our data implicates a new role for p19Arf in response to nutrient deprivation. This article has an associated First Person interview with the first author of the paper.

LncRNA OSER1-AS1 regulates the inflammation and apoptosis of rheumatoid arthritis fibroblast like synoviocytes via regulating miR-1298-5p/E2F1 axis

ABSTRACT It has been reported that long noncoding RNAs (LncRNAs) take part in the progression and occurrence of rheumatoid arthritis (RA). The current work aimed to dig the effect of lncRNA OSER1-AS1 on RA and the associated mechanism. Quantitative real-time polymerase chain reaction (qRT-PCR) was made to decide that OSER1-AS1 was significantly lowly expressed in synovial tissue and serum of RA patients, which was consistent in RA-FLSs cell lines. The result of ROC curve indicated that OSER1-AS1 could be a diagnostic biomarker for RA patients. Cell Counting Kit-8 assay (CCK-8), EdU staining and flow cytometry were performed to explore the effect of OSER1-AS1 on RA-FLSs in vitro. Relative levels of interleukin-1 (IL-1), interleukin-6 (IL-6), matrix metalloproteinases-3 (MMP-3) were detected by ELISA and the result displayed that overexpression of OSER1-AS1 inhibited RA-induced inflammatory production of IL-1, IL-6 and MMP3. Bioinformatics analysis, luciferase reporter, RNA immunoprecipitation assays (RIP) and RNA pull-down assay were conducted to confirm the binding between microRNA-1298-5p (miR-1298-5p) and OSER1-AS1 or E2F transcription factor 1 (E2F1). Mechanistically, OSER1-AS1 serves as a competing endogenous (ceRNA) in RA-FLSs through the sponge of miR-1298-5p and increase in the expression of E2F1. Further restoration experiments revealed that miR-1298-5p mimics and E2F1 silencing could partially reverse the inhibiting effect of OSER1-AS1 overexpression on propagation and apoptosis in RA-FLSs. The results illustrated the biological mechanism of OSER1-AS1/miR-1298-59/E2F1 axis in RA progression. The outcomes indicated that OSER1-AS1 might be adopted as a hopeful diagnostic and therapeutic objective for RA.

A Review Into the Effects of Pamidronic Acid and Zoledronic Acid on the Oral Mucosa in Medication-Related Osteonecrosis of the Jaw.

Medication-related osteonecrosis of the jaw (MRONJ) is a growing problem without an effective treatment, presenting as necrotic bone sections exposed via lesions in the overlying soft tissue. There is currently a lack of clarity on how the factors involved in MRONJ development and progression contribute to disease prognosis and outcomes. Bisphosphonates (BPs), the most common cause of MRONJ, affect bone remodeling, angiogenesis, infection, inflammation and soft tissue toxicity, all of which contribute to MRONJ development. This article reviews the cellular mechanisms through which BPs contribute to MRONJ pathology, with a focus on the effects on cells of the oral mucosa. BPs have been shown to reduce cell viability, reduce proliferation, and increase apoptosis in oral keratinocytes and fibroblasts. BPs have also been demonstrated to reduce epithelial thickness and prevent epithelial formation in three-dimensional tissue engineered models of the oral mucosa. This combination of factors demonstrates how BPs lead to the reduced wound healing seen in MRONJ and begins to uncover the mechanisms through which these effects occur. The evidence presented here supports identification of targets which can be used to develop novel treatment strategies to promote soft tissue wound healing and restore mucosal coverage of exposed bone in MRONJ.

The Effects of Rapamycin on the Proliferation, Migration, and Apoptosis of Human Tracheal Fibroblasts (HTrF) and Human Tracheal Epithelial Cells (HTEpiC).

Background: Restenosis after airway stenting needs to be addressed urgently. Rapamycin has been proven to inhibit restenosis elsewhere. This study aimed at observing its effects on the respiratory tract. Methods: CCK-8, wound healing, Transwell and apoptosis assays were performed to detect the effects of rapamycin on the survival, migration, and apoptosis, respectively, of human tracheal fibroblasts (HTrF) and human tracheal epithelial cells (HTEpiC). Results: The effective concentrations of paclitaxel, mitomycin C and rapamycin on HTrF were 10−7–10−4 mol/L, 10−6–10−4 mol/L, and 10−5–10−4 mol/L, respectively. At the effective concentrations, the inhibition rates of paclitaxel on HTEpiC were (43.03 ± 1.12)%, (49.49 ± 0.86)%, (55.22 ± 1.43)%, and (93.19 ± 0.45)%; the inhibition rates of mitomycin C on HTEpiC were (88.11 ± 0.69)%, (93.82 ± 0.96)%, and (94.94 ± 0.54)%; the inhibition rates of rapamycin on HTEpiC were (10.19 ± 0.35)% and (94.55 ± 0.71)%. At the concentration of (1–4) × 10−5 mol/L, the inhibition rate of rapamycin on HTrF was more than 50%, and that on HTEpiC was less than 20% (p < 0.05). Conclusions: Compared to paclitaxel and mitomycin C, rapamycin had the least effect on HTEpiC while effectively inhibiting HTrF. The optimum concentration range was (1–4) × 10−5 mol/L.

Silencing circular RNAPTPN12 promoted the growth of keloid fibroblasts by activating Wnt signaling pathway via targeting microRNA-21-5p

ABSTRACT Keloid is a skin disease marked by fibroplasia, and fibroblasts viability plays a considerable part in keloid. Our research was devoted to assessing the involvement and mechanism of circPTPN12 in keloid. The level of circPTPN12 and miR-21-5p was estimated by qRT-PCR in keloid tissues and cells. MTT analysis was devoted to evaluating the multiplication of keloid fibroblasts. Additionally, transwell assay was dedicated to verifying cell migration and invasion. Furthermore, keloid fibroblasts apoptosis level was assessed adopting flow cytometry, and the relevancy between miR-21-5p and circPTPN12, miR-21-5p, and SMAD7 was assessed by dual luciferase assay. Similarly, RIP and RNA pull-down assay verified the relevance between genes. Moreover, levels of SMAD7 and proteins concerned in Wnt signaling pathway were appraised by Western blot. The level of circPTPN12 declined in keloid. circPTPN12 knockout could enhance the multiplication, migration, invasion, and decline apoptosis of keloid fibroblasts. Indeed, miR-21-5p could be packed with circPTPN12 sponge, SMAD7 was downstream effect factor of miR-21-5p, and miR-21-5p inhibitors partially reversed the promoting effect of silencing circPTPN12 on keloid formation. Otherwise, the level of SMAD7 was adjusted by circPTPN12 and miR-21-5p. Silencing circPTPN12 targeted miR-21-5p and activated Wnt pathway to accelerate keloid fibroblasts growth. Taken together, silencing circPTPN12 promotes the growth of keloid fibroblasts by activating Wnt pathway targeting miR-21-5p. CircPTPN12 may play a considerable part in keloid formation, which supplies a reference for molecularly targeted therapy keloid.

CD30-targeted therapy induces apoptosis of inflammatory cytokine-stimulated synovial fibroblasts and ameliorates collagen antibody-induced arthritis in mice.

It has been reported that levels of soluble CD30 in serum and joint fluid are significantly elevated in patients with rheumatoid arthritis (RA). This study aimed to investigate whether CD30 could be a therapeutic target for RA. The expression and localization of CD30 were examined by immunohistochemical and double immunofluorescence staining on synovial tissue samples obtained from patients with RA or osteoarthritis (OA) during surgery. Changes in CD30 expression of fibroblast-like synoviocytes (FLS) from RA patients with or without TNFα and IL-1β stimulation were examined by the polymerase chain reaction (PCR) and flow cytometry. Collagen antibody-induced arthritis (CAIA) was created in DBA/1 mice, and the therapeutic effect of brentuximab vedotin (BV) was examined by clinical score, histological findings and measurement of serum levels of SAA, IL-6, and TNFα. CD30 expression was significantly higher in samples from patients with RA than from those with OA. Double immunofluorescence showed a low rate of co-localization of CD30 with CD20 or CD90, but a high rate of co-localization of CD30 and CD138. CD30 mRNA expression was upregulated 11.7-fold in FLS following stimulation by inflammatory cytokines. The clinical scores of CAIA mice were significantly lower following both BV treatments, however, the histological scores of CAIA mice were significantly lower only following treatment with high dose BV (70mg/kg). CD30 was expressed on immunocompetent cells in synovial tissue from RA patients and in cytokine-stimulated FLS in vitro. High dose BV (70mg/kg) showed significant therapeutic effects in ameliorating inflammation and joint destruction in CAIA mice, but low dose BV (30mg/kg) was insufficient.

Vanillic acid alleviates lipopolysaccharides-induced endoplasmic reticulum stress and inflammation in human lung fibroblasts by `inhibiting MAPK and NF-κB pathways

Persistent endoplasmic reticulum stress promotes aberrant inflammation and induces cell death, and inflammation is implicated in the pathogenesis of pneumonia. Vanillic acid exerts pharmacological activities, such as anti-inflammatory, antimicrobial, and antioxidant effects. However, the role of vanillic acid in pneumonia has not been elucidated yet. Human lung fibroblasts (WI-38 and MRC-5) were incubated with different concentrations of lipopolysaccharides to mimic the cell model of pneumonia. Lipopolysaccharides-treated lung fibroblasts were then incubated with different concentrations of vanillic acid. Cell viability and apoptosis were detected by MTT assay and flow cytometry, respectively. Quantitative real-time polymerase chain reaction and enzyme-linked-immunosorbent serologic assay were used to measure the levels of inflammatory factors. Western blot assay was used to detect endoplasmic reticulum stress and downstream pathway. Lipopolysaccharides induced decrease of cell viability in WI-38 and MRC-5 whereas vanillic acid increased cell viability of lipopolysaccharides-treated lung fibroblasts. Lipopolysaccharides-induced increase of cell apoptosis in lung fibroblasts was suppressed by vanillic acid through up-regulation of BCL2, and down-regulation of BCL2 associated X (BAX) and cleaved caspase-3. Vanillic acid reduced levels of tumor necrosis factor-α (TNF-α), Interleukin 6 (IL-6), and IL-1β in lipopolysaccharides-treated lung fibroblasts. Protein expression of glucose-regulated protein 78 (GRP78), X-box binding protein 1 (XBP-1), activating transcription factor-6 (ATF-6), ATF-4, and C/EBP homologous protein (CHOP) in lung fibroblasts were up-regulated by lipopolysaccharides while reduced by vanillic acid. Vanillic acid attenuated lipopolysaccharides-induced decrease of IκBα and increase of p-IκBα, p-p65, p-ERK, and p-JNK in fibroblasts. Vanillic acid exerted anti-inflammatory effect against lipopolysaccharides-induced human lung fibroblasts by inhibiting mitogen-activated protein kinase and nuclear factor kappa B pathways.

Nrg1/ErbB signaling-mediated regulation of fibrosis after myocardial infarction.

Appropriate fibrotic tissue formation after myocardial infarction (MI) is crucial to the maintenance of the heart's structure. M2-like macrophages play a vital role in post-MI fibrosis by activating cardiac fibroblasts. Because the mechanism by which post-MI cardiac fibrosis is regulated is not fully understood, we investigated, in vitro and in vivo, the cellular and molecular mechanisms of post-MI fibrotic tissue formation, especially those related to the regulation of cellular senescence and apoptosis. CD206+ F4/80+ CD11b+ M2-like macrophages collected from mouse hearts on post-MI day 7showed increased expression of neuregulin 1 (Nrg1). Nrg1 receptor epidermal growth factor receptors ErbB2 and ErbB4 were expressed on cardiac fibroblasts in the infarct area. M2-like macrophage-derived Nrg1suppressed both hydrogen peroxide-induced senescence and apoptosis of fibroblasts, whereas blockade of ErbB function significantly accelerated both processes. M2-like macrophage-derived Nrg1/ErbB/PI3K/Akt signaling, shown to be related to anti-senescence, was activated in damaged cardiac fibroblasts. Interestingly, systemic blockade of ErbB function in MI model mice enhanced senescence and apoptosis of cardiac fibroblasts and exacerbated inflammation. Further, increased accumulation of M2-like macrophages resulted in excessive post-MI progression of fibrosis in mice hearts. The molecular mechanism underlying the regulation of fibrotic tissue formation in the infarcted myocardium was shown in part to be attenuation of apoptosis and senescence of cardiac fibroblasts by the activation of Nrg1/ErbB/PI3K/Akt signaling. M2-like macrophage-mediated regulation of Nrg1/ErbB signaling has a substantial effect on fibrotic tissue formation in the infarcted adult mouse heart and is critical for suppressing the progression of senescence and apoptosis of cardiac fibroblasts.

CircCOL5A1 inhibits proliferation, migration, invasion, and extracellular matrix production of keloid fibroblasts by regulating the miR-877-5p/EGR1 axis

BackgroundCircular RNA (circRNA) has been proved to mediate the biological functions of fibroblasts to participate in the regulation of keloid formation. However, the role of circCOL5A1 in keloid formation remains to be further confirmed. MethodsPrimary keloid fibroblasts were isolated form keloid tissues. The expression of circCOL5A1, microRNA (miR)− 877–5p, and early growth response 1 (EGR1) were determined by quantitative real-time PCR. Transfection experiments were carried out to explore the effects of circCOL5A1, miR-877–5p, and EGR1 on cell functions. Cell proliferation, migration, invasion and apoptosis were detected using cell counting kit 8 assay, colony formation assay, transwell assay and flow cytometry. The protein levels of apoptosis markers, extracellular matrix (ECM) markers and EGR1 were measured by western blot analysis. The mechanism of circCOL5A1 was confirmed by RNA pull-down assay, dual-luciferase reporter assay and RIP assay. ResultsOur data showed that circCOL5A1 was upregulated in keloid tissues and fibroblasts. Silencing of circCOL5A1 had an inhibition effect on proliferation, migration, invasion and ECM production, while had a promotion effect on apoptosis in keloid fibroblasts. MiR-877–5p could be sponged by circCOL5A1, and its overexpression could repress the biological functions of keloid fibroblasts. The rescue experiments showed that miR-877–5p inhibitor could reverse the suppressive effect of circCOL5A1 knockdown on the biological functions of keloid fibroblasts. In addition, EGR1 was a target of miR-877–5p, and its expression was positively regulated by circCOL5A1. The inhibition effect of miR-877–5p on the biological functions of keloid fibroblasts could be abolished by EGR1 overexpression. ConclusionIn summary, circCOL5A1 facilitates keloid fibroblast proliferation, migration, invasion and ECM production through the miR-877–5p/EGR1 axis, thereby potentially promoting keloid formation.

Development and Characterization of Curcumin-Silver Nanoparticles as a Promising Formulation to Test on Human Pterygium-Derived Keratinocytes.

Pterygium is a progressive disease of the human eye arising from sub-conjunctival tissue and extending onto the cornea. Due to its invasive growth, pterygium can reach the pupil compromising visual function. Currently available medical treatments have limited success in suppressing efficiently the disease. Previous studies have demonstrated that curcumin, polyphenol isolated from the rhizome of Curcuma longa, induces apoptosis of human pterygium fibroblasts in a dose- and time-dependent manner showing promising activity in the treatment of this ophthalmic disease. However, this molecule is not very soluble in water in either neutral or acidic pH and is only slightly more soluble in alkaline conditions, while its dissolving in organic solvents drastically reduces its potential use for biomedical applications. A nanoformulation of curcumin stabilized silver nanoparticles (Cur-AgNPs) seems an effective strategy to increase the bioavailability of curcumin without inducing toxic effects. In fact, silver nitrates have been used safely for the treatment of many ophthalmic conditions and diseases for a long time and the concentration of AgNPs in this formulation is quite low. The synthesis of this new compound was achieved through a modified Bettini’s method adapted to improve the quality of the product intended for human use. Indeed, the pH of the reaction was changed to 9, the temperature of the reaction was increased from 90 °C to 100 °C and after the synthesis the Cur-AgNPs were dispersed in Borax buffer using a dialysis step to improve the biocompatibility of the formulation. This new compound will be able to deliver both components (curcumin and silver) at the same time to the affected tissue, representing an alternative and a more sophisticated strategy for the treatment of human pterygium. Further in vitro and in vivo assays will be required to validate this formulation.

Effect of Interleukin 6 on Scleral Fibroblast Proliferation, Differentiation, and Apoptosis Involved in Myopic Scleral Remodeling

Introduction: Scleral hypoxia (HO) is present in myopic eyes, and interleukin (IL)-6 is increased in the aqueous humor of patients with high myopia. The aim of this study was to investigate the effects of IL-6 on scleral fibroblast proliferation, differentiation, and apoptosis under conditions of HO and the possible role of IL-6 in myopic scleral remodeling. Methods: Primary human scleral fibroblasts (HSFs) were cultured using a tissue mass adherent method. First, cells were cultured under conditions of HO (2% O2) or normoxia (NO, 20% O2) for different times. A quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence were used to detect the expression of IL-6 in HSFs. Next, cells were divided into five groups: NO, HO, HO plus IL-6, HO plus IL-6 receptor inhibitor (IL6RI), and HO plus IL-6 and IL6RI. The groups were treated separately for 72 h. Cell counting kit-8 assay and flow cytometry were used to detect cell proliferation and apoptosis, respectively. Western blotting and qRT-PCR were used to detect the expression of various genes in the transforming growth factor-β1/Smad2/matrix metalloproteinase-2 pathway; these methods and immunofluorescence were also used to detect transdifferentiation of HSFs. Results: HO resulted in upregulation of IL-6 expression in HSFs. Compared with NO, HO resulted in diminished cell proliferation and increased apoptosis and differentiation in HSFs; the above trend was further enhanced by the addition of IL6RI. Compared with the HO group, the addition of IL-6 led to a decrease in cell proliferation and an increase in apoptosis and differentiation of HSFs; the above trends showed opposite changes after the addition of both IL-6 and IL6RI. Additionally, IL-6 and IL6RI exerted opposite regulatory effects on the transforming growth factor-β1/Smad2/matrix metalloproteinase-2 pathway under conditions of HO. Conclusion: HO caused HSFs to overexpress IL-6. IL-6 has a role in scleral remodeling in myopic eyes through affecting the proliferation, differentiation, and apoptosis of HSFs.

The Inhibition of Adipose-Derived Stem Cells on the Invasion of Keloid Fibroblasts.

Background: Keloids represent the dysregulation of cutaneous wound healing caused by aberrant fibroblast activities. Adipose-derived stem cells have been recognized as a promising treatment for keloids. However, the molecular mechanisms have not been fully elucidated.Objectives: to explicitly demonstrate the relationship between adipose-derived stem cells alleviating keloids and alterations of Col-1, Col-3, CTGF, and P-4-HB.Methods: Skin biopsies were obtained from 10 keloid patients and 9 healthy volunteers. Fibroblasts isolated from all samples were divided into two groups, one co-cultured with adipose-derived stem cells and the other grown independently. We compared the wound-healing rates, fibroblast survival rates, apoptosis rates, mRNA expressions, and protein levels of Col-1, Col-3, CTGF, and P-4-HB between separated groups.Results: We found no significant differences between normal fibroblasts and keloid fibroblasts in terms of wound-healing rate, survival rate, or apoptosis rate at the baseline. With adipose-derived stem cells, wound-healing rate and survival rate of normal fibroblasts were promoted, whereas in keloid fibroblasts, they were reduced. The apoptosis rate of normal fibroblasts and keloid fibroblasts were restrained, with the restraint in keloid fibroblasts being more evident. The protein levels of Col-3, CTGF, and P-4-HB were lower in keloid fibroblasts co-cultured with adipose-derived stem cells than in normal fibroblasts under similar conditions.Conclusions: Adipose-derived stem cells strongly suppressed keloid fibroblasts' proliferative and invasive behavior. However, adipose-derived stem cells negatively regulated keloid fibroblast apoptosis. Adipose-derived stem cells can be a potential keloid therapy worth further investigation.

Long Noncoding RNA CASC2 Facilitated Wound Healing through miRNA-155/HIF-1α in Diabetic Foot Ulcers.

Diabetic foot ulcers (DFU) are among the serious complications which are closely linked to diabetes mellitus. However, there is still a lack of accurate and effective standard prevention and treatment programs for DFU. In this manuscript, we have investigated the function of lncRNA cancer susceptibility candidate 2 (CASC2)/miR-155/hypoxia-inducible factor 1-alpha (HIF-1α) in the wound healing of DFU. We have analyzed lncRNA CASC2`s expression in the marginal tissues of ulcers in patients and mice with DFU. Additionally, the interaction relationship and mechanism between lncRNA CASC2, miR-155, and HIF-1α were determined, which proved the effects of lncRNA CASC2/miR-155/HIF-1α on fibroblasts apoptosis, proliferation, and migration. According to our study, the lncRNA CASC2's expression was low in the tissues of ulcers of DFU mice and patients. lncRNA CASC2's overexpression promoted fibroblasts migration, proliferation, and inhibited apoptosis and was beneficial for the healing of wounds, preferably in the DFU mice. In addition, lncRNA CASC2 directly targets miR-155 and HIF-1α functions as miR-155's target gene. Overexpression of miR-155 abrogated the function of lncRNA CASC2. Similarly, HIF-1α's inhibition has reversed the effect of miR-155 downregulation on fibroblasts. In general, overexpression of lncRNA CASC2 facilitated wound healing through miR-155/HIF-1α in DFU.

Quantification of Caspase 3 Levels in Patients with Periodontitis with or without Diabetes Mellitus

Background: Diabetes mellitus is a debilitating systemic disease with several major complications affecting the quality and length of life. Periodontal disease has been considered another diabetic complication in addition to cardiovascular disease, nephropathy, neuropathy, peripheral vascular disease. Caspase-3 plays an important role in intracellular signaling pathways that regulate apoptosis. High levels of glucose could induce human periodontal ligament fibroblast apoptosis.&#x0D; Aim: The aim of the study is to compare caspase 3 levels in periodontitis patients with or without diabetes mellitus.&#x0D; Materials and Methods: 30 patients were included in the study and they are divided into 3 groups: Group a- Periodontal health; Group b- Periodontitis with diabetes mellitus and Group c- Periodontitis patients without diabetes mellitus. Whole unstimulated saliva was collected from 30 patients using expectoration into sterile bulbs. Caspase 3 levels in saliva samples were measured in duplicate using a commercially available Human Caspase-3 (CASP3) enzyme linked immunosorbent assay (ELISA) Kit. Results were analyzed statistically by a one-way analysis of variance (ANOVA) and significant differences between the mean values were measured using Newman-Keuls multiple comparison test using Graph Pad Prism version 5. The results with the p &lt; 0.05 level were considered to be statistically significant.&#x0D; Results: From this study, it was observed that there was a significant increase of caspase 3 levels in periodontitis patients with diabetes mellitus (86.29±24.25 pmol/L) when compared to periodontitis patients without diabetes (55.06±12.90 pmol/L). The results showed a positive correlation and high level of significance when compared between periodontally healthy patients and periodontitis patients along with diabetes mellitus (86.29±24.25 pmol/L). Also, relatively significant results were observed in comparison between periodontally healthy patients (43.37±15.35 pmol/L) and patients with periodontitis without diabetes mellitus (55.06±12.90 pmol/L). Please add the most important statistical values.&#x0D; Conclusion: The present study showed that caspase-3 concentrations in saliva increases in patients with periodontitis complicated along with diabetes mellitus. Moreover, saliva concentrations of caspase-3 increase with periodontal disease and caspase-3 plays a role as a biomarker of periodontal disease and its progression.

Lucanthone, Autophagy Inhibitor, Enhances the Apoptotic Effects of TRAIL through miR-216a-5p-Mediated DR5 Upregulation and DUB3-Mediated Mcl-1 Downregulation.

A lucanthone, one of the family of thioxanthenones, has been reported for its inhibitory effects of apurinic endonuclease-1 and autophagy. In this study, we investigated whether lucanthone could enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in various cancer cells. Combined treatment with lucanthone and TRAIL significantly induced apoptosis in human renal carcinoma (Caki and ACHN), prostate carcinoma (PC3), and lung carcinoma (A549) cells. However, combined treatment did not induce apoptosis in normal mouse kidney cells (TCMK-1) and normal human skin fibroblast (HSF). Lucanthone downregulated protein expression of deubiquitinase DUB3, and a decreased expression level of DUB3 markedly led to enhance TRAIL-induced apoptosis. Ectopic expression of DUB3 inhibited combined treatment with lucanthone and TRAIL-induced apoptosis. Moreover, lucanthone increased expression level of DR5 mRNA via downregulation of miR-216a-5p. Transfection of miR-216a-5p mimics suppressed the lucanthone-induced DR5 upregulation. Taken together, these results provide the first evidence that lucanthone enhances TRAIL-induced apoptosis through DR5 upregulation by downregulation of miR-216a-5p and DUB3-dependent Mcl-1 downregulation in human renal carcinoma cells.

M. tuberculosis CRISPR/Cas proteins are secreted virulence factors that trigger cellular immune responses

ABSTRACT The role of prokaryotic CRISPR/Cas system proteins as a defensive shield against invasive nucleic acids has been studied extensively. Non-canonical roles in pathogenesis involving intracellular targeting of certain virulence-associated endogenous mRNA have also been reported for some Type I and Type II CRISPR/Cas proteins, but no such roles have yet been established for Type III system proteins. Here, we demonstrate that M. tuberculosis (Type III-A system) CRISPR/Cas proteins Csm1, Csm3, Csm5, Csm6, and Cas6 are secreted and induce host immune responses. Using cell and animal experiments, we show that Cas6, in particular, provokes IFN-γ release from PBMCs from active tuberculosis (TB) patients, and its deletion markedly attenuates virulence in a murine M. tuberculosis challenge model. Recombinant MTBCas6 induces apoptosis of macrophages and lung fibroblasts, and interacts with the surface of cells in a caspase and TLR-2 independent manner. Transcriptomic and signal pathway studies using THP-1 macrophages stimulated with MTBCas6 indicated that MTBCas6 upregulates expression of genes associated with the NF-κB pathway leading to higher levels of IL-6, IL-1β, and TNF-α release, cytokines known to activate immune system cells in response to M. tuberculosis infection. Our findings suggest that, in addition to their intracellular shielding role, M. tuberculosis CRISPR/Cas proteins have non-canonical extracellular roles, functioning like a virulent sword, and activating host immune responses.

Competing Endogenous RNA Network in Non-Keloid-Prone Individuals During Wound Healing.

To study the interaction between differentially expressed long non-coding RNAs (lncRNAs), microRNAs, and messenger RNAs during wound healing in normal individuals. The GSE113621 dataset was downloaded from gene expression matrix, specimens regarding non-keloid-prone individuals were selected, including items before and 6 weeks after injury. A Pearson correlation coefficient of > 0.95 was selected as the index to screen targeting relationships among different RNAs. Cytoscape was used to construct a network diagram. The expression of 2547 lncRNAs was changed during the wound healing process-1479 were upregulated and 1068 were downregulated. After analyzing competitive endogenous RNA network, 4 upregulated (MEG8, MEG3, MIR181A1HG, MIR4435-2HG) lncRNAs were found expressed during wound healing. MEG8/MEG3 may regulate fibroblast proliferation, differentiation, and apoptosis through hsa-miR-296-3p/miR-6763-5p. In-depth mining of gene expression matrix data indicated that lncRNAs and a competitive endogenous RNA regulatory network participate in the wound healing process, possibly providing novel intervention targets and treatment options for delayed wound healing.

Xinfeng Capsules promotes apoptosis of synovial fibroblasts and attenuates inflammation in rheumatoid arthritis by regulating lncRNA MAPKAPK5-AS1

To explore the regulatory effects of Xinfeng Capsules(XFC) on the apoptosis of synovial fibroblasts(FLS) and inflammation in rheumatoid arthritis(RA) via lncRNA MAPKAPK5-AS1(MK5-AS1). Thirty healthy people and 30 patients with RA due to spleen deficiency and dampness exuberance were collected for extracting the peripheral blood mononuclear cells(PBMCs) before and after XFC treatment, which were used to observe the correlation between MK5-AS1 and clinical indicators as well as MK5-AS1 expression before and after XFC treatment. Following the establishment of RA-FLS cell line and the preparation of XFC-containing serum, MK5-AS1-overexpression plasmid was constructed and transfected into RA-FLS for investigating the efficacy of XFC-containing serum in regulating inflammation and apoptosis of RA-FLS via MK5-AS1. The expression of MK5-AS1 in PBMCs of patients with RA due to spleen deficiency and dampness exuberance was decreased(P&lt;0.001). The ROC curve analysis revealed the AUC of 83.9%. Correlation analysis showed that MK5-AS1 was negatively correlated with ESR, CRP, RF, CCP, and spleen deficiency and dampness exuberance syndrome score. The expression of MK5-AS1 increased significantly after XFC treatment(P&lt;0.001). As demonstrated by association analysis, XFC decreased MK5-AS1, ESR, CRP, RF, and spleen deficiency and dampness exuberance syndrome score, with the degree of support all greater than 83%, confidence greater than 80%, and lift greater than 1. The results of RT-qPCR showed that the MK5-AS1 RNA expression significantly decreased after TNF-α stimulation(P&lt;0.01), which, however, increased significantly after the intervention with XFC-containing serum(P&lt;0.05). Such expression rose again after the transfection of pcDNA3.1-MK5-AS1(P&lt;0.01). ELISA results showed that TNF-α stimulation elevated the expression of pro-inflammatory factor IL-17 but lowered the expression of anti-inflammatory factor IL-4(P&lt;0.01). After intervention with XFC-containing serum, the expression of IL-17 decreased while that of IL-4 increased(P&lt;0.01). The transfection of pcDNA3.1-MK5-AS1 contributed to the reduction in IL-17 expression but the elevation in IL-4 expression(P&lt;0.01). The immunofluorescence(IF) findings demonstrated that the expression of pro-apoptotic protein Bax was down-regulated, whereas that of the anti-apoptotic protein Bcl-2 was up-regulated after TNF-α stimulation(P&lt;0.01). After the intervention with XFC-containing serum, the Bax expression was increased, while Bcl-2 expression was decreased(P&lt;0.01), which were remarkably collaborated by the transfection of pcDNA3.1-MK5-AS1(P&lt;0.05). The expression of MK5-AS1 is significantly decreased in both RA-PBMCs and RA-FLS, implying that XFC inhibits inflammatory reaction and promotes the apoptosis in RA by regulating the expression of MK5-AS1.

Role of haematopoietic cell-specific protein 1-associated protein X-1 gene in lipopolysaccharide-induced apoptosis of human dermal fibroblasts.

Wound healing may be disrupted by lipopolysaccharide (LPS)-induced mitochondrial dysfunction, inflammation, and excessive oxidative stress, which can lead to undesirable consequences. The haematopoietic cell-specific protein 1-associated protein X-1 (HAX-1) is a mitochondrial matrix protein that regulates mitochondrial function. This study aimed to comprehensively identify the role of HAX-1 in the inhibition of LPS-induced mitochondrial dysfunction and apoptosis in human dermal fibroblasts (HDFs). HAX-1 expression was assessed in the HDF-a cell line using real-time polymerase chain reaction, western blotting, and immunohistochemical staining. The viability, migration, and apoptosis of HDF-a cells were evaluated using the water-soluble tetrazolium-1 assay, transwell assay, and flow cytometry analysis, respectively. Mitochondrial function was evaluated based on reactive oxygen species (ROS) generation and mitochondrial membrane potential (ΔΨm). Our results demonstrated that LPS stimulation markedly repressed HAX-1 expression in HDFs and silencing of HAX-1 led to mitochondrial ROS accumulation, ΔΨm disruption, and abnormal mitochondrial morphology. Accordingly, overexpression of HAX-1 or administration of metformin enhanced mitochondrial fusion and normalized mitochondrial dynamics, thereby reversing LPS-induced mitochondrial dysfunction, fibroblast apoptosis, and viability and migration inhibition in HDF-a cells. These data support a mechanism wherein HAX-1 plays a crucial role in LPS-induced fibroblast apoptosis in a mitochondria-dependent manner.