The interaction of p38 with its upstream kinase MKK6

Mitogen‐activated protein kinase (MAPK; p38, ERK, and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full‐length MKK6 and p38. It was shown that p38 engages MKK6 not only via its hydrophobic docking groove, but also influences helix αF, a secondary structural element that plays a key role in organizing the kinase core. It was also shown that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, it was demonstrated that these interactions with p38 are conserved independent of the MKK6 activation state. Together, the results revealed differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.     

METTL3/N6‐methyladenosine/ miR‐21‐5p promotes obstructive renal fibrosis by regulating inflammation through SPRY1/ERK/NF‐κB pathway activation

Renal fibrosis induced by urinary tract obstruction is a common clinical occurrence; however, effective treatment is lacking, and a deeper understanding of the mechanism of renal fibrosis is needed. Previous studies have revealed that miR‐21 impacts liver and lung fibrosis progression by activating the SPRY1/ERK/NF‐kB signalling pathway. However, whether miR‐21 mediates obstructive renal fibrosis through the same signalling pathway has not been determined. Additionally, studies have shown that N6‐methyladenosine (m⁶A) modification‐dependent primary microRNA (pri‐microRNA) processing is essential for maturation of microRNAs, but its role in the maturation of miR‐21 in obstructive renal fibrosis has not yet been investigated in detail. To address these issues, we employed a mouse model of unilateral ureteral obstruction (UUO) in which the left ureters were ligated for 3, 7 and 14 days to simulate the fibrotic process. In vitro, human renal proximal tubular epithelial (HK‐2) cells were transfected with plasmids containing the corresponding sequence of METTL3, miR‐21‐5p mimic or miR‐21‐5p inhibitor. We found that the levels of miR‐21‐5p and m⁶A modification in the UUO model groups increased significantly, and as predicted, the SPRY1/ERK/NF‐kB pathway was activated by miR‐21‐5p, confirming that miR‐21‐5p plays an important role in obstructive renal fibrosis by enhancing inflammation. METTL3 was found to play a major catalytic role in m⁶A modification in UUO mice and drove obstructive renal fibrosis development by promoting miR‐21‐5p maturation. Our research is the first to demonstrate the role of the METTL3‐m⁶A‐miR‐21‐5p‐SPRY1/ERK/NF‐kB axis in obstructive renal fibrosis and provides a deeper understanding of renal fibrosis.     

N‐acetylcysteine improves diabetic associated erectile dysfunction in streptozotocin‐induced diabetic mice by inhibiting oxidative stress

Oxidative stress appears to play a role in the pathogenesis of diabetes mellitus erectile dysfunction (DMED). This study aimed to investigate the effect of N‐acetylcysteine (NAC) on DMED in streptozotocin‐induced diabetic mice and to explore potential mechanisms. In the present study, we show that an erectile dysfunction is present in the streptozotocin‐induced mouse model of diabetes as indicated by decreases in intracavernous pressure responses to electro‐stimulation as well as from results of the apomorphine test of erectile function. After treatment of NAC, the intracavernous pressure was increased. In these DMED mice, oxidative stress and inflammatory responses were significantly reduced within the cavernous microenvironment, while activity of antioxidant enzymes in this cavernous tissue was enhanced after NAC treatment. These changes protected mitochondrial stress damage and a significant decreased in apoptosis within the cavernous tissue of DMED mice. This appears to involve activation of the nuclear factor erythroid 2‐like‐2 (Nrf2) signalling pathway, as well as suppression of the mitogen‐activated protein kinase (MAPK) p38/ NF‐κB pathway within cavernous tissue. In conclusion, NAC can improve erectile function through inhibiting oxidative stress via activating Nrf2 pathways and reducing apoptosis in streptozotocin‐induced diabetic mice. NAC might provide a promising therapeutic strategy for individuals with DMED.     

Genetic signature of human longevity in PKC and NF‐κB signaling

Gene variants associated with longevity are also associated with protection against cognitive decline, dementia and Alzheimer''s disease, suggesting that common physiologic pathways act at the interface of longevity and cognitive function. To test the hypothesis that variants in genes implicated in cognitive function may promote exceptional longevity, we performed a comprehensive 3‐stage study to identify functional longevity‐associated variants in ~700 candidate genes in up to 450 centenarians and 500 controls by target capture sequencing analysis. We found an enrichment of longevity‐associated genes in the nPKC and NF‐κB signaling pathways by gene‐based association analyses. Functional analysis of the top three gene variants (NFKBIA, CLU, PRKCH) suggests that non‐coding variants modulate the expression of cognate genes, thereby reducing signaling through the nPKC and NF‐κB. This matches genetic studies in multiple model organisms, suggesting that the evolutionary conservation of reduced PKC and NF‐κB signaling pathways in exceptional longevity may include humans.     

Inhibition of PKC‐δ reduce rhabdomyolysis‐induced acute kidney injury

Despite extensive research, the mechanisms underlying rhabdomyolysis‐induced acute kidney injury (AKI) remain largely elusive. In this study, we established both cell and murine models of rhabdomyolysis‐induced AKI by using myoglobin and glycerin, respectively, and provided evidence that protein kinase Cδ (PKC‐δ) was activated in both models and subsequently promoted cell apoptosis. Moreover, we found that this detrimental effect of PKC‐δ activation can be reversed by its pharmaceutical inhibitor rottlerin. Furthermore, we detected and confirmed the existence of PKC‐δ‐mediated myoglobin‐induced cell apoptosis and the expression of TNF‐α and IL1‐β via regulation of the p38MAPK and ERK1/2 signalling pathways. In summary, our research revealed the role of PKC‐δ in renal cell apoptosis and suggests that PKC‐δ is a viable therapeutic target for rhabdomyolysis‐induced AKI.     

MiR‐223‐3p inhibits rTp17‐induced inflammasome activation and pyroptosis by targeting NLRP3

The incidence of syphilis caused by Treponema pallidum subsp pallidum (T pallidum) infection is accompanied by inflammatory injuries of vascular endothelial cells. Studies have revealed that T pallidum infection could induce inflammasome activation and pyroptosis in macrophages. MicroRNA‐223‐3p (miR‐223‐3p) was reported to be a negative regulator in inflammatory diseases. The present study aimed to explore whether miR‐223‐3p regulates T pallidum‐induced inflammasome activation and pyroptosis in vascular endothelial cells, and determine the mechanisms which underlie this process. MiR‐223‐3p levels in syphilis and control samples were determined. The biological function of miR‐223‐3p in the NLRP3 inflammasome and pyroptosis was evaluated in T pallidum‐infected human umbilical vein endothelial cells (HUVECs). We observed a dramatic decrease in miR‐223‐3p levels in syphilis patients (n = 20) when compared to healthy controls (n = 20). Moreover, miR‐223‐3p showed a notable inhibitory effect on recombinant Tp17 (rTP17)‐induced caspase‐1 activation, resulting in decrease in IL‐1β production and pyroptosis, which was accompanied by the release of lactate dehydrogenase (LDH) in HUVECs. Additionally, the dual‐luciferase assay confirmed that NLRP3 is a direct target of miR‐223‐3p. Moreover, NLRP3 overexpression or knockdown largely blocked the effects of miR‐223‐3p on T pallidum‐induced inflammasome activation and pyroptosis in HUVECs. Most importantly, a notable negative correlation was observed between miR‐223‐3p and NLRP3, caspase‐1, and IL‐1β, respectively, in the serum of syphilis patients and healthy controls. Taken together, our results reveal that miR‐223‐3p targets NLRP3 to suppress inflammasome activation and pyroptosis in T pallidum‐infected endothelial cells, implying that miR‐223‐3p could be a potential target for syphilis patients.     

Pseudolaric acid B ameliorates synovial inflammation and vessel formation by stabilizing PPARγ to inhibit NF‐κB signalling pathway

Synovial macrophage polarization and inflammation are essential for osteoarthritis (OA) development, yet the molecular mechanisms and regulation responsible for the pathogenesis are still poorly understood. Here, we report that pseudolaric acid B (PAB) attenuated articular cartilage degeneration and synovitis during OA. PAB, a diterpene acid, specifically inhibited NF‐κB signalling and reduced the production of pro‐inflammatory cytokines, which further decreased M1 polarization and vessel formation. We further provide in vivo and in vitro evidences that PAB suppressed NF‐κB signalling by stabilizing PPARγ. Using PPARγ antagonist could abolish anti‐inflammatory effect of PAB and rescue the activation of NF‐κB signalling during OA. Our findings identify a previously unrecognized role of PAB in the regulation of OA and provide mechanisms by which PAB regulates NF‐κB signalling through PPARγ, which further suggest targeting synovial inflammation or inhibiting vessel formation at early stage could be an effective preventive strategy for OA.     

MicroRNA‐411‐3p inhibits bleomycin‐induced skin fibrosis by regulating transforming growth factor‐β/Smad ubiquitin regulatory factor‐2 signalling

Skin fibrosis, which is characterized by fibroblast proliferation and increased extracellular matrix, has no effective treatment. An increasing number of studies have shown that microRNAs (miRNAs/miRs) participate in the mechanism of skin fibrosis, such as in limited cutaneous systemic sclerosis and pathological scarring. The objective of the present study was to determine the role of miR‐411‐3p in bleomycin (BLM)‐induced skin fibrosis and skin fibroblast transformation. Using Western blot analysis and real‐time quantitative polymerase chain reaction assess the expression levels of miR‐411‐3p, collagen (COLI) and transforming growth factor (TGF)‐β/Smad ubiquitin regulatory factor (Smurf)‐2/Smad signalling factors both in vitro and in vivo with or without BLM. To explore the regulatory relationship between miR‐411‐3p and Smurf2, we used the luciferase reporter assay. Furthermore, miR‐411‐3p overexpression was identified in vitro and in vivo via transfection with Lipofectamine 2000 reagent and injection. Finally, we tested the dermal layer of the skin using haematoxylin and eosin and Van Gieson''s staining. We found that miR‐411‐3p expression was decreased in bleomycin (BLM)‐induced skin fibrosis and fibroblasts. However, BLM accelerated transforming growth factor (TGF)‐β signalling and collagen production. Overexpression of miR‐411‐3p inhibited the expression of collagen, F‐actin and the TGF‐β/Smad signalling pathway factors in BLM‐induced skin fibrosis and fibroblasts. In addition, miR‐411‐3p inhibited the target Smad ubiquitin regulatory factor (Smurf)‐2. Furthermore, Smurf2 was silenced, which attenuated the expression of collagen via suppression of the TGF‐β/Smad signalling pathway. We demonstrated that miR‐411‐3p exerts antifibrotic effects by inhibiting the TGF‐β/Smad signalling pathway via targeting of Smurf2 in skin fibrosis.     

DEK‐targeting aptamer DTA‐64 attenuates bronchial EMT‐mediated airway remodelling by suppressing TGF‐β1/Smad,MAPK and PI3K signalling pathway in asthma

This study is to investigate the inhibitory effects and mechanisms of DEK‐targeting aptamer (DTA‐64) on epithelial mesenchymaltransition (EMT)‐mediated airway remodelling in mice and human bronchial epithelial cell line BEAS‐2B. In the ovalbumin (OVA)‐induced asthmatic mice, DTA‐64 significantly reduced the infiltration of eosinophils and neutrophils in lung tissue, attenuated the airway resistance and the proliferation of goblet cells. In addition, DTA‐64 reduced collagen deposition, transforming growth factor 1 (TGF‐β1) level in BALF and IgE levels in serum, balanced Th1/Th2/Th17 ratio, and decreased mesenchymal proteins (vimentin and α‐SMA), as well as weekend matrix metalloproteinases (MMP‐2 and MMP‐9) and NF‐κB p65 activity. In the in vitro experiments, we used TGF‐β1 to induce EMT in the human epithelial cell line BEAS‐2B. DEK overexpression (ovDEK) or silencing (shDEK) up‐regulated or down‐regulated TGF‐β1 expression, respectively, on the contrary, TGF‐β1 exposure had no effect on DEK expression. Furthermore, ovDEK and TGF‐β1 synergistically promoted EMT, whereas shDEK significantly reduced mesenchymal markers and increased epithelial markers, thus inhibiting EMT. Additionally, shDEK inhibited key proteins in TGF‐β1‐mediated signalling pathways, including Smad2/3, Smad4, p38 MAPK, ERK1/2, JNK and PI3K/AKT/mTOR. In conclusion, the effects of DTA‐64 against EMT of asthmatic mice and BEAS‐2B might partially be achieved through suppressing TGF‐β1/Smad, MAPK and PI3K signalling pathways. DTA‐64 may be a new therapeutic option for the management of airway remodelling in asthma patients.     

Vitamin B6 prevents excessive inflammation by reducing accumulation of sphingosine‐1‐phosphate in a sphingosine‐1‐phosphate lyase–dependent manner

Vitamin B6 is necessary to maintain normal metabolism and immune response, especially the anti‐inflammatory immune response. However, the exact mechanism by which vitamin B6 plays the anti‐inflammatory role is still unclear. Here, we report a novel mechanism of preventing excessive inflammation by vitamin B6 via reduction in the accumulation of sphingosine‐1‐phosphate (S1P) in a S1P lyase (SPL)‐dependent manner in macrophages. Vitamin B6 supplementation decreased the expression of pro‐inflammatory cytokines by suppressing nuclear factor‐κB and mitogen‐activated protein kinases signalling pathways. Furthermore, vitamin B6–reduced accumulation of S1P by promoting SPL activity. The anti‐inflammatory effects of vitamin B6 were inhibited by S1P supplementation or SPL deficiency. Importantly, vitamin B6 supplementation protected mice from lethal endotoxic shock and attenuated experimental autoimmune encephalomyelitis progression. Collectively, these findings revealed a novel anti‐inflammatory mechanism of vitamin B6 and provided guidance on its clinical use.     

circ‐AKT3 aggravates renal ischaemia‐reperfusion injury via regulating miR‐144‐5p /Wnt/β‐catenin pathway and oxidative stress

Renal ischaemia‐reperfusion (RI/R) injury is one major pathological state of acute kidney injury (AKI) with a mortality rate ranking 50% to 80%. MiR‐144‐5p acts as a molecular trigger in various diseases. We presumed that miR‐144‐5p might be involved RI/R injury progression. We found that RI/R injury decreased miR‐144‐5p expression in rat models. MiR‐144‐5p downregulation promoted cell apoptosis rate and activated Wnt/β‐catenin signal in RI/R injury rats. By performing bioinformatic analysis, RIP, RNA pull‐down, luciferase reporter experiments, we found that circ‐AKT3 sponged to miR‐144‐5p and decreased its expression in RI/R injury rats. Moreover, we found that circ‐AKT3 promoted cell apoptosis rate and activated Wnt/β‐catenin signal, and miR‐144‐5p mimic reversed the promotive effect of circ‐AKT3 in rat models. We also found that circ‐AKT3 increased the oxidative stress level in rat models. In conclusion, our study suggests that the circAKT3 is involved RI/R injury progression through regulating miR‐144‐5p/Wnt/β‐catenin pathway and oxidative stress.     

Aneuploid senescent cells activate NF‐κB to promote their immune clearance by NK cells

The immune system plays a major role in the protection against cancer. Identifying and characterizing the pathways mediating this immune surveillance are thus critical for understanding how cancer cells are recognized and eliminated. Aneuploidy is a hallmark of cancer, and we previously found that untransformed cells that had undergone senescence due to highly abnormal karyotypes are eliminated by natural killer (NK) cells in vitro. However, the mechanisms underlying this process remained elusive. Here, using an in vitro NK cell killing system, we show that non‐cell‐autonomous mechanisms in aneuploid cells predominantly mediate their clearance by NK cells. Our data indicate that in untransformed aneuploid cells, NF‐κB signaling upregulation is central to elicit this immune response. Inactivating NF‐κB abolishes NK cell‐mediated clearance of untransformed aneuploid cells. In cancer cell lines, NF‐κB upregulation also correlates with the degree of aneuploidy. However, such upregulation in cancer cells is not sufficient to trigger NK cell‐mediated clearance, suggesting that additional mechanisms might be at play during cancer evolution to counteract NF‐κB‐mediated immunogenicity.     

MicroRNA‐495‐3p diminishes doxorubicin‐induced cardiotoxicity through activating AKT

Doxorubicin (Dox) is a broad‐spectrum antitumour agent; however, its clinical application is impeded due to the cumulative cardiotoxicity. The present study aims to investigate the role and underlying mechanisms of microRNA‐495‐3p (miR‐495‐3p) in Dox‐induced cardiotoxicity. Herein, we found that cardiac miR‐495‐3p expression was significantly decreased in Dox‐treated hearts, and that the miR‐495‐3p agomir could prevent oxidative stress, cell apoptosis, cardiac mass loss, fibrosis and cardiac dysfunction upon Dox stimulation. In contrast, the miR‐495‐3p antagomir dramatically aggravated Dox‐induced cardiotoxicity in mice. Besides, we found that the miR‐495‐3p agomir attenuated, while the miR‐495‐3p antagomir exacerbated Dox‐induced oxidative stress and cellular injury in vitro. Mechanistically, we demonstrated that miR‐495‐3p directly bound to the 3′‐untranslational region of phosphate and tension homology deleted on chromosome ten (PTEN), downregulated PTEN expression and subsequently activated protein kinase B (PKB/AKT) pathway, and that PTEN overexpression or AKT inhibition completely abolished the cardioprotective effects of the miR‐495‐3p agomir. Our study for the first time identify miR‐495‐3p as an endogenous protectant against Dox‐induced cardiotoxicity through activating AKT pathway in vivo and in vitro.     

METTL3‐mediated maturation of miR‐589‐5p promotes the malignant development of liver cancer

MiR‐589‐5p could promote liver cancer, but the specific mechanisms are largely unknown. This study examined the role and mechanisms of miR‐589‐5p in liver cancer. The expressions of miR‐589‐5p, METTL3 and m6A in liver cancers were determined by RT‐qPCR. The relationship between miR‐589‐5p and METTL3‐mediated m6A methylation was examined by m6A RNA immunoprecipitation. After transfection, the viability, migration, invasion and expressions of METTL3 and miR‐589‐5p in liver cancer cells were detected by CCK‐8, wound‐healing, transwell and RT‐qPCR. After the xenograft tumour was established in mice, the tumour volume was determined and the expressions of METTL3, miR‐589‐5p, MMP‐2, TIMP‐2, E‐cadherin, N‐cadherin and Vimentin in tumour tissue were detected by RT‐qPCR and Western blotting. In vitro study showed that miR‐589‐5p and METTL3 were highly expressed in liver cancer. METTL3 was positively correlated with miR‐589‐5p. METTL3 up‐regulated the expression of miR‐589‐5p and promoted the maturation of miR‐589‐5p. Overexpressed miR‐589‐5p and METTL3 promoted the viability, migration and invasion of liver cancer cells, while the effects of silencing miR‐589‐5p and METTL3 on the cells were the opposite. The effects of METTL3 overexpression and silencing were reversed by miR‐589‐5p inhibitor and mimic, respectively. In vivo study showed that METLL3 silencing inhibited the growth of xenograft tumour and the expressions of METTL3, MMP‐2, N‐cadherin and Vimentin, promoted the expressions of TIMP‐2 and E‐cadherin, while miR‐589‐5p mimic caused the opposite results and further reversed the effects of METLL3 silencing. In summary, this study found that METTL3‐mediated maturation of miR‐589‐5p promoted the malignant development of liver cancer.     

Glycyrrhizin regulates rat TMJOA progression by inhibiting the HMGB1‐RAGE/TLR4‐NF‐κB/AKT pathway

To investigate the role of glycyrrhizin on the progression of temporomandibular joint osteoarthritis (TMJOA) and the underlying mechanism by regulation of the high‐mobility group box 1 (HMGB1) receptor for advanced glycation end products (RAGE)/toll‐like receptor 4 (TLR4)‐nuclear factor kappa B (NF‐κB)/protein kinase B (AKT) pathway. After a rat model of TMJOA was built by intra‐articular injection of monosodium iodoacetate, glycyrrhizin was intragastrically administered at low concentration (20 mg/kg) or high concentration (50 mg/kg). Micro‐computed tomography, histological and immunohistochemical analysis were used to reveal the progression of TMJOA. Rat TMJ chondrocytes and disc cells were cultured in inflammatory condition with different doses of glycyrrhizin. Western blot was used to evaluate the effect of glycyrrhizin on the HMGB1‐RAGE/TLR4‐NF‐κB/AKT pathway. Administration of glycyrrhizin alleviated cartilage degeneration, lowered the levels of inflammatory and catabolic mediators and reduced the production of HMGB1, RAGE and TLR4 in TMJOA animal model. Increased production of RAGE and TLR4, and activated intracellular NF‐κB and/or AKT signalling pathways in chondrocytes and disc cells were found in inflammatory condition. Upon activation, matrix metalloprotease‐3 and interleukin‐6 were upregulated. Glycyrrhizin inhibited not only HMGB1 release but also RAGE and TLR4 in inflammatory condition. Glycyrrhizin alleviated the pathological changes of TMJOA by regulating the HMGB1‐RAGE/TLR4‐NF‐kB/AKT signalling pathway. This study revealed the potential of glycyrrhizin as a novel therapeutic drug to suppress TMJ cartilage degradation.