Polyphenolic Compounds Inhibit Osteoclast Differentiation While Reducing Autophagy through Limiting ROS and the Mitochondrial Membrane Potential

Laha, Dipranjan; Sarkar, Jaganmay; Maity, Jyotirindra; Pramanik, Asmita; Howlader, Md Sariful Islam; Barthels, Derek; Das, Hiranmoy

doi:10.3390/biom12091220

Cited by 12 publications

(8 citation statements)

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“…As described previously ( 13 , 15 ), ECSIT deleted macrophages had lower Δψm when compared to control cells. RANKL is known to increase Δψm by stimulating OXPHOS ( 18 , 19 ). Accordingly, RANKL increased Δψm in control cells while this response was abrogated in cells lacking ECSIT ( Figure 4C ).…”

Section: Resultsmentioning

confidence: 99%

ECSIT is essential for RANKL-induced stimulation of mitochondria in osteoclasts and a target for the anti-osteoclastogenic effects of estrogens

et al. 2023

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IntroductionEstrogens inhibit bone resorption and preserve bone mass, at least in part, via direct effects on osteoclasts. The binding of RANKL, the critical cytokine for osteoclast differentiation, to its receptor in osteoclast precursor cells of the monocyte lineage recruits the adaptor protein TRAF6 and activates multiple signaling pathways. Early effects of RANKL include stimulation of mitochondria. 17β-estradiol (E2) prevents the effects of RANKL on mitochondria and promotes mitochondria mediated apoptotic cell death. However, the molecular mechanisms responsible for the actions of RANKL and estrogens on mitochondria remain unknown. Evolutionarily Conserved Signaling Intermediate in Toll Pathway (ECSIT) is a complex I-associated protein that regulates immune responses in macrophages following the engagement of Toll-like receptors, which also recruit TRAF6. Here, we examined whether ECSIT could be implicated in the rapid effects of RANKL and E2 on osteoclast progenitors.MethodsBone marrow-derived macrophages (BMMs) from C57BL/6 mice were cultured with RANKL (30 ng/ml) with or without E2 (10-8 M). ECSIT-TRAF6 interaction was evaluated by co-immunoprecipitation and ECSIT levels in mitochondria and cytosolic fractions by Western blot. ShRNA lentivirus particles were used to knockdown ECSIT. Osteoclasts were enumerated after tartrate-resistant acid phosphatase staining. Oxygen consumption and extracellular acidification rates were measured with Seahorse XFe96 Analyzer. ATP, lactate, and NAD/NADH were measured with commercial assay kits. NADH oxidation to NAD was used to evaluate Complex I activity. Total and mitochondrial ROS, and mitochondrial membrane potential were measured with H2DCFDA, MitoSOX, and TMRM probes, respectively. Degradation of DEVD-AFC was used to measure Caspase-3 activity.ResultsWe found that RANKL promoted ECSIT-TRAF6 interaction and increased the levels of ECSIT in mitochondria. E2 abrogated these effects of RANKL. Silencing of ECSIT decreased osteoclast differentiation and abrogated the inhibitory effects of E2 on osteoclastogenesis. Loss of ECSIT decreased complex I activity, oxygen consumption, NAD+/NADH redox ratio, and ATP production and increased mitochondrial ROS. In the absence of ECSIT, the stimulatory actions of RANKL on complex I activity and all other markers of oxidative phosphorylation, as well as their inhibition by E2, were prevented. Instead, RANKL stimulated apoptosis of osteoclast progenitors.DiscussionThese findings suggest that dysregulated mitochondria cause a switch in RANKL signaling from pro-survival to pro-apoptotic. In addition, our results indicate that ECSIT represents a central node for the early effects of RANKL on mitochondria and that inhibition of ECSIT-mediated mitochondria stimulation might contribute to the bone protective actions of estrogens.

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Section: Resultsmentioning

confidence: 99%

ECSIT is essential for RANKL-induced stimulation of mitochondria in osteoclasts and a target for the anti-osteoclastogenic effects of estrogens

et al. 2023

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“…The results showed that TRIM45 regulated NLRP3 in an Atg5-dependent manner. Considering the correlation between autophagy, ROS and mitochondrial membrane potential [ 54 , 55 ], we also examined the changes in these indices in the different groups. The results showed that knocking down TRIM45 could improve mitochondrial membrane potential and reduce ROS production.…”

Section: Discussionmentioning

confidence: 99%

TRIM45 aggravates microglia pyroptosis via Atg5/NLRP3 axis in septic encephalopathy

Huang,

Ye,

Zhao

et al. 2023

J Neuroinflammation

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Background Neuroinflammation mediated by microglial pyroptosis is an important pathogenic mechanism of septic encephalopathy (SAE). It has been reported that TRIM45 is associated with tumours and inflammatory diseases. However, the role of TRIM45 in SAE and the relationship between TRIM45 and microglial pyroptosis are unknown. In this study, we found that TRIM45 played an important role in regulating microglial pyroptosis and the molecular mechanism. Methods SAE was induced by intraperitoneal injection of LPS in WT and AAV-shTRIM45 mice. BV2 cells were treated with LPS/ATP in vitro. Cognitive function was assessed by the Morris water maze. Nissl staining was used to evaluate histological and structural lesions. ELISA was used to dectect neuroinflammation. qPCR was used to detect the mRNA levels of inflammatory cytokines, NLRP3, and autophagy genes. Western blotting and immunofluorescence analysis were used to analyse the expression of the proteins. Changes in reactive oxygen species (ROS) in cells were observed by flow cytometry. Changes in mitochondrial membrane potential in BV2 cells were detected by JC-1 staining. Peripheral blood mononuclear cells were extracted from blood by density gradient centrifugation and then used for qPCR, western blotting and flow detection. To further explore the mechanism, we used the overexpression plasmids TRIM45 and Atg5 as well as siRNA-TRIM45 and siRNA-Atg5 to analyse the downstream pathway of NLRP3. The protein and mRNA levels of TRIM45 in peripheral blood mononuclear cells from sepsis patients were examined. Results Knocking down TRIM45 protected against neuronal damage and cognitive impairment in septic mice. TRIM45 knockdown inhibited microglial pyroptosis and the secretion of inflammatory cytokines in vivo and in vitro, which was mediated by NLRP3/Gsdmd-N activation. Overexpression of TRIM45 could activate NLRP3 and downstream proteins. Further examination showed that TRIM45 regulated the activation of NLRP3 by altering Atg5 and regulating autophagic flux. It was also found that overexpression and knockdown of TRIM45 affected the changes in ROS and mitochondrial membrane potential. Thus, knocking down TRIM45 could reduce microglial pyroptosis, the secretion of proinflammatory cytokines, and neuronal damage and improve cognitive function. In addition, the level of TRIM45 protein in septic patients was increased. There was a positive linear correlation between APACHE II score and TRIM45, between SOFA score and TRIM45. Compared to group GCS > 9, level of TRIM45 were increased in group GCS ≤ 8. Conclusion TRIM45 plays a key role in neuroinflammation caused by LPS, and the mechanism may involve TRIM45-mediated exacerbation of microglial pyroptosis via the Atg5/NLRP3 axis. Graphical Abstract

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“…The mitochondrial membrane potentials (MMP) were determined by JC-1 staining as described previously [ 29 , 30 ] ; experiments were set as described above. The DPSC ℗ -treated cells and untreated cells were incubated in 5 μM of JC-1 dye for 15 min at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Secretome of Dental Pulp-Derived Stem Cells Reduces Inflammation and Proliferation of Glioblastoma Cells by Deactivating Mapk-Akt Pathway

Prateeksha¹,

Howlader²,

Hansda³

et al. 2023

jbyyj

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Background: Dental pulp-derived stem cells (DPSC) is a promising therapy as they modulate the immune response, so we evaluated the inhibitory effect of DPSC secretome (DPSC ℗ ) on the proliferation and inflammation in human glioblastoma (GBM) cells (U-87 MG) and elucidated the concomitant mechanisms involved. Methods: The U87-MG cells were cultured with DPSC ℗ for 24 h and assessed the expression of inflammatory molecules using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), generation of reactive oxygen species (ROS), and mitochondrial functionality using a seahorse flux analyzer. MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay and cell cycle analysis were performed to evaluate the proliferation and cell cycle. Finally, the protein levels were determined by western blot. Results: DPSC ℗ reduced the inflammation and proliferation of U-87 MG cells by down-regulating the pro-inflammatory markers and up-regulating anti-inflammatory markers expressions through ROS-mediated signaling. Moreover, DPSC ℗ significantly reduced the mitochondrial membrane potential (MMP) in the cells. The cellular bioenergetics revealed that all the parameters of oxygen consumption rate (OCAR) and the extracellular acidification rate (ECAR) were significantly decreased in the GBM cells after the addition of DPSC ℗ . Additionally, DPSC ℗ decreased the GBM cell proliferation by arresting the cell cycle at the G1 phase through activation (phosphorylation) of checkpoint molecule CHK1. Furthermore, mechanistically, we found that the DPSC ℗ impedes the phosphorylation of the mitogen-activated protein kinases (P38 MAPK) and protein kinase B (AKT) pathway. Conclusion: Our findings lend the first evidence of the inhibitory effects of DPSC ℗ on proliferation and inflammation in GBM cells by altering the P38 MAPK-AKT pathway.

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Polyphenolic Compounds Inhibit Osteoclast Differentiation While Reducing Autophagy through Limiting ROS and the Mitochondrial Membrane Potential

Cited by 12 publications

References 55 publications

ECSIT is essential for RANKL-induced stimulation of mitochondria in osteoclasts and a target for the anti-osteoclastogenic effects of estrogens

ECSIT is essential for RANKL-induced stimulation of mitochondria in osteoclasts and a target for the anti-osteoclastogenic effects of estrogens

TRIM45 aggravates microglia pyroptosis via Atg5/NLRP3 axis in septic encephalopathy

Secretome of Dental Pulp-Derived Stem Cells Reduces Inflammation and Proliferation of Glioblastoma Cells by Deactivating Mapk-Akt Pathway

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