Exosomes derived from smooth muscle cells ameliorate diabetes‐induced erectile dysfunction by inhibiting fibrosis and modulating the NO/cGMP pathway

Erectile dysfunction (ED) is a major health issue among men with diabetes, and ED induced by diabetes mellitus (DMED) is particularly difficult to treat. Therefore, novel therapeutic approaches for the treatment of DMED are urgently needed. Exosomes, nanosized particles involved in many physiological and pathological processes, may become a promising tool for DMED treatment. In this study, we investigated the therapeutic effect of exosomes derived from corpus cavernosum smooth muscle cells (CCSMC‐EXOs) on erectile function in a rat model of diabetes and compared their effect with that of exosomes derived from mesenchymal stem cells (MSC‐EXOs). We incubated labelled CCSMC‐EXOs and MSC‐EXOs with CCSMCs and then observed uptake of the exosomes at different time points using laser confocal microscopy. CCSMC‐EXOs were more easily taken up by CCSMCs. The peak concentration and retention time of labelled CCSMC‐EXOs and MSC‐EXOs in the corpus cavernosum of DMED rats after intracavernous injection were compared by in vivo imaging techniques. Intracavernous injection of CCSMC‐EXOs was associated with a relatively high peak concentration and long retention time. Our data showed that CCSMC‐EXOs could improve erectile function in DMED rats. Meanwhile, CCSMC‐EXOs could exert antifibrotic effects by increasing the smooth muscle content and reducing collagen deposition. CCSMC‐EXOs also increased the expression of eNOS and nNOS, followed by increased levels of NO and cGMP. These findings initially identify the possible role of CCSMC‐EXOs in ameliorating DMED through inhibiting corporal fibrosis and modulating the NO/cGMP signalling pathway, providing a theoretical basis for a breakthrough in the treatment of DMED.     

Antrodia camphorata polysaccharide improves inflammatory response in liver injury via the ROS/TLR4/NF‐κB signal

Antrodia Camphorata Polysaccharide (ACP) refers to a kind of polysaccharide extracted from the natural porous fungus Antrodia camphorata. This study investigated the mechanism of action of ACP in protecting the liver. The results showed that ACP suppressed the LPS‐induced KC cell activation, reduced the expression of inflammatory factors, increased the SOD level and suppressed ROS expression. In addition, N‐acetylcysteine (NAC) was adopted for pre‐treatment to suppress ROS. The results indicated that NAC synergistically exerted its effect with ACP, suggesting that ACP played its role through suppressing ROS. Further detection revealed that ACP activated the Nrf2 signal. It was discovered in the mouse model that, ACP effectively improved liver injury in mice, decreased ALT and AST levels, and suppressed the expression of inflammatory factors. This study suggests that ACP can exert its effect against oxidative stress via the Nrf2‐ARE signalling, which further improves the production of ROS and the activation of TLR4‐NF‐κB signalling, and protects the liver against liver injury.     

MOAP‐1‐mediated dissociation of p62/SQSTM1 bodies releases Keap1 and suppresses Nrf2 signaling

Nrf2 signaling is vital for protecting cells against oxidative stress. However, its hyperactivation is frequently found in liver cancer through excessive build‐up of p62/SQSTM1 bodies that sequester Keap1, an adaptor of the E3‐ubiquitin ligase complex for Nrf2. Here, we report that the Bax‐binding protein MOAP‐1 regulates p62‐Keap1‐Nrf2 signaling through disruption of p62 bodies. Upon induction of cellular stresses that stimulate formation of p62 bodies, MOAP‐1 is recruited to p62 bodies and reduces their levels independent of the autophagy pathway. MOAP‐1 interacts with the PB1‐ZZ domains of p62 and interferes with its self‐oligomerization and liquid–liquid phase separation, thereby disassembling the p62 bodies. Loss of MOAP‐1 can lead to marked upregulation of p62 bodies, enhanced sequestration of Keap1 by p62 and hyperactivation of Nrf2 antioxidant target genes. MOAP‐1‐deficient mice exhibit an elevated tumor burden with excessive levels of p62 bodies and Nrf2 signaling in a diethylnitrosamine (DEN)‐induced hepatocarcinogenesis model. Together, our data define MOAP‐1 as a negative regulator of Nrf2 signaling via dissociation of p62 bodies.     

Fibroblast growth factor 21 reverses high‐fat diet‐induced impairment of vascular function via the anti‐oxidative pathway in ApoE knockout mice

Circulating endothelial progenitor cells (EPCs), which function in vascular repair, are the markers of endothelial dysfunction and vascular health. Fibroblast growth factor 21 (FGF21), a liver‐secreted protein, plays a crucial role in glucose homeostasis and lipid metabolism. FGF21 has been reported to attenuate the progression of atherosclerosis, but its impact on EPCs under high oxidative stress conditions remains unclear. In vitro studies showed that the β‐klotho protein was expressed in cultured EPCs and that its expression was upregulated by FGF21 treatment. Hydrogen peroxide (H₂O₂)‐induced oxidative stress impaired EPC function, including cell viability, migration and tube formation. Pretreatment with FGF21 restored the functions of EPCs after the exposure to H₂O₂. Administration of N(ω)‐nitro‐L‐arginine methyl ester (L‐NAME), an inhibitor of nitric oxide synthase, inhibited the effects of FGF21 in alleviating oxidative injury by suppressing endothelial nitric oxide synthase (eNOS). In an in vivo study, the administration of FGF21 significantly reduced total cholesterol (TC) and blood glucose levels in apolipoprotein E (ApoE)‐deficient mice that were fed a high‐fat diet (HFD). Endothelial function, as reflected by acetylcholine‐stimulated aortic relaxation, was improved after FGF21 treatment in ApoE‐deficient mice. Analysis of mRNA levels in the aorta indicated that FGF21 increased the mRNA expression of eNOS and upregulated the expression of the antioxidant genes superoxide dismutase (SOD)1 and SOD2 in ApoE‐deficient mice. These data suggest that FGF21 improves EPC functions via the Akt/eNOS/nitric oxide (NO) pathway and reverses endothelial dysfunction under oxidative stress. Therefore, administration of FGF21 may ameliorate a HFD‐induced vascular injury in ApoE‐deficient mice.     

Enhancing fractalkine/CX3CR1 signalling pathway can reduce neuroinflammation by attenuating microglia activation in experimental diabetic retinopathy

The concept of diabetic retinopathy (DR) has been extended from microvascular disease to neurovascular disease in which microglia activation plays a remarkable role. Fractalkine (FKN)/CX3CR1 is reported to regulate microglia activation in central nervous system diseases. To characterize the effect of FKN on microglia activation in DR, we employed streptozotocin‐induced diabetic rats, glyoxal‐treated R28 cells and hypoxia‐treated BV2 cells to mimic diabetic conditions and explored retinal neuronal apoptosis, reactive oxygen species (ROS), as well as the expressions of FKN, Iba‐1, TSPO, NF‐κB, Nrf2 and inflammation‐related cytokines. The results showed that FKN expression declined with diabetes progression and in glyoxal‐treated R28 cells. Compared with normal control, retinal microglia activation and inflammatory factors surged in both diabetic rat retinas and hypoxia‐treated microglia, which was largely dampened by FKN. The NF‐κB and Nrf2 expressions and intracellular ROS were up‐regulated in hypoxia‐treated microglia compared with that in normoxia control, and FKN significantly inhibited NF‐κB activation, activated Nrf2 pathway and decreased intracellular ROS. In conclusion, the results demonstrated that FKN deactivated microglia via inhibiting NF‐κB pathway and activating Nrf2 pathway, thus to reduce the production of inflammation‐related cytokines and ROS, and protect the retina from diabetes insult.     

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.     

The anti‐apoptotic Bcl‐2 protein regulates hair follicle stem cell function

Maintaining the architecture, size and composition of an intact stem cell (SC) compartment is crucial for tissue homeostasis and regeneration throughout life. In mammalian skin, elevated expression of the anti‐apoptotic Bcl‐2 protein has been reported in hair follicle (HF) bulge SCs (BSCs), but its impact on SC function is unknown. Here, we show that systemic exposure of mice to the Bcl‐2 antagonist ABT‐199/venetoclax leads to the selective loss of suprabasal BSCs (sbBSCs), thereby disrupting cyclic HF regeneration. RNAseq analysis shows that the pro‐apoptotic BH3‐only proteins BIM and Bmf are upregulated in sbBSCs, explaining their addiction to Bcl‐2 and the marked susceptibility to Bcl‐2 antagonism. In line with these observations, conditional knockout of Bcl‐2 in mouse epidermis elevates apoptosis in BSCs. In contrast, ectopic Bcl‐2 expression blocks apoptosis during HF regression, resulting in the accumulation of quiescent SCs and delaying HF growth in mice. Strikingly, Bcl‐2‐induced changes in size and composition of the HF bulge accelerate tumour formation. Our study identifies a niche‐instructive mechanism of Bcl‐2‐regulated apoptosis response that is required for SC homeostasis and tissue regeneration, and may suppress carcinogenesis.     

4‐octyl Itaconate inhibits lipopolysaccharide (LPS)‐induced osteoarthritis via activating Nrf2 signalling pathway

Small molecule drug intervention for chondrocytes is a valuable method for the treatment of osteoarthritis (OA). The 4‐octyl itaconate (OI) is a cellular derivative of itaconate with sound cell permeability and transformation rate. We attempted to confirm the protective role of OI in chondrocytes and its regulatory mechanism. We used lipopolysaccharide (LPS) to induce chondrocyte inflammation injury. After the OI treatment, the secretion and mRNA expression of Il‐6, Il‐10, Mcp‐1 and Tnf‐α were detected by ELISA and qPCR. The protective effect of OI on articular cartilage was further verified in surgical destabilization of the medial meniscus model of OA. Cell death and apoptosis were evaluated based on CCK8, LDH, Typan blue staining, Annexin V and TUNEL analyses. The small interfering RNAs were used to knockout the Nrf2 gene of chondrocytes to verify the OI‐mediated Nrf2 signalling pathway. The results revealed that OI protects cells from LPS‐induced inflammatory injury and attenuates cell death and apoptosis induced by LPS. Similar protective effects were also observed on articular cartilage in mice. The OI activated Nrf2 signalling pathway and promoted the stable expression and translocation of Nrf2 into the nucleus. When the Nrf2 signalling pathway was blocked, the protective effect of OI was significantly counteracted in chondrocytes and a mouse arthritis model. Both itaconate and its derivative (i.e., OI) showed important medical effects in the treatment of OA.     

Kcnh2 mediates FAK/AKT‐FOXO3A pathway to attenuate sepsis‐induced cardiac dysfunction

ObjectivesMyocardial dysfunction is a significant manifestation in sepsis, which results in high mortality. Even Kcnh2 has been hinted to associate with the pathological process, its involved signalling is still elusive.Materials and methodsThe caecal ligation puncture (CLP) surgery or lipopolysaccharide (LPS) injection was performed to induce septic cardiac dysfunction. Western blotting was used to determine KCNH2 expression. Cardiac function was examined by echocardiography 6 hours after CLP and LPS injection in Kcnh2 knockout (Kcnh2^+/‐) and NS1643 injection rats (n ≥ 6/group). Survival was monitored following CLP‐induced sepsis (n ≥ 8/group).ResultsSepsis could downregulate KCNH2 level in the rat heart, as well as in LPS‐stimulated cardiomyocytes but not cardiac fibroblast. Defect of Kcnh2 (Kcnh2^+/‐) significantly aggravated septic cardiac dysfunction, exacerbated tissue damage and increased apoptosis under LPS challenge. Fractional shortening and ejection fraction values were significantly decreased in Kcnh2^+/‐ group than Kcnh2^+/+ group. Survival outcome in Kcnh2^+/‐ septic rats was markedly deteriorated, compared with Kcnh2^+/+ rats. Activated Kcnh2 with NS1643, however, resulted in opposite effects. Lack of Kcnh2 caused inhibition of FAK/AKT signalling, reflecting in an upregulation for FOXO3A and its downstream targets, which eventually induced cardiomyocyte apoptosis and heart tissue damage. Either activation of AKT by activator or knockdown of FOXO3A with si‐RNA remarkably attenuated the pathological manifestations that Kcnh2 defect mediated.ConclusionKcnh2 plays a protection role in sepsis‐induced cardiac dysfunction (SCID) via regulating FAK/AKT‐FOXO3A to block LPS‐induced myocardium apoptosis, indicating a potential effect of the potassium channels in pathophysiology of SCID.     

Downregulation of p300/CBP‐associated factor inhibits cardiomyocyte apoptosis via suppression of NF‐κB pathway in ischaemia/reperfusion injury rats

Cardiomyocyte apoptosis is the main reason of cardiac injury after myocardial ischaemia‐reperfusion (I/R) injury (MIRI), but the role of p300/CBP‐associated factor (PCAF) on myocardial apoptosis in MIRI is unknown. The aim of this study was to investigate the main mechanism of PCAF modulating cardiomyocyte apoptosis in MIRI. The MIRI model was constructed by ligation of the rat left anterior descending coronary vessel for 30 min and reperfusion for 24 h in vivo. H9c2 cells were harvested after induced by hypoxia for 6 h and then reoxygenation for 24 h (H/R) in vitro. The RNA interference PCAF expression adenovirus was transfected into rat myocardium and H9c2 cells. The area of myocardial infarction, cardiac function, myocardial injury marker levels, apoptosis, inflammation and oxidative stress were detected respectively. Both I/R and H/R remarkably upregulated the expression of PCAF, and downregulation of PCAF significantly attenuated myocardial apoptosis, inflammation and oxidative stress caused by I/R and H/R. In addition, downregulation of PCAF inhibited the activation of NF‐κB signalling pathway in cardiomyocytes undergoing H/R. Pretreatment of lipopolysaccharide, a NF‐κB pathway activator, could blunt these protective effects of PCAF downregulation on myocardial apoptosis in MIRI. These results highlight that downregulation of PCAF could reduce cardiomyocyte apoptosis by inhibiting the NF‐κB pathway, thereby providing protection for MIRI. Therefore, PCAF might be a promising target for protecting against cardiac dysfunction induced by MIRI.     

NAD+ ameliorates endotoxin‐induced acute kidney injury in a sirtuin1–dependent manner via GSK‐3β/Nrf2 signalling pathway

Acute kidney injury (AKI) is a substantial worldwide public health concern with no specific and effective therapies in clinic. NAD⁺ is a pivotal determinant of cellular energy metabolism involved in the progression of AKI; however, its mechanism in kidney injury remains poorly understood. Sirtuin 1 (SIRT1) is an NAD⁺‐dependent deacetylase associated with renal protection and acute stress resistance. In this study, we have investigated the role of NAD⁺ in AKI and the potential mechanism(s) involved in its renoprotective effect. NAD⁺ was notably decreased and negatively correlated with kidney dysfunction in AKI, restoring NAD⁺ with NMN significantly ameliorates LPS‐induced oxidative stress and apoptosis and attenuates renal damage. We also found that the protection of NAD⁺ is associated with SIRT1 expressions and performs in a SIRT1‐dependent manner. Inhibition of SIRT1 blunted the protective effect of NAD⁺ and up‐regulated the activity of glycogen synthase kinase‐3β (GSK‐3β) that was concomitant with mitigated Nrf2 nuclear accumulation, thereby exacerbates AKI. These findings suggest that NAD⁺/SIRT1/GSK‐3β/Nrf2 axis is an important mechanism that can protect against AKI which might be a potential therapeutic target for the treatment of AKI.     

LRG‐1 promotes fat graft survival through the RAB31‐mediated inhibition of hypoxia‐induced apoptosis

Autologous adipose tissue is an ideal soft tissue filling material, and its biocompatibility is better than that of artificial tissue substitutes, foreign bodies and heterogeneous materials. Although autologous fat transplantation has many advantages, the low retention rate of adipose tissue limits its clinical application. Here, we identified a secretory glycoprotein, leucine‐rich‐alpha‐2‐glycoprotein 1 (LRG‐1), that could promote fat graft survival through RAB31‐mediated inhibition of hypoxia‐induced apoptosis. We showed that LRG‐1 injection significantly increased the maintenance of fat volume and weight compared with the control. In addition, higher fat integrity, more viable adipocytes and fewer apoptotic cells were observed in the LRG‐1‐treated groups. Furthermore, we discovered that LRG‐1 could reduce the ADSC apoptosis induced by hypoxic conditions. The mechanism underlying the LRG‐1‐mediated suppression of the ADSC apoptosis induced by hypoxia was mediated by the upregulation of RAB31 expression. Using LRG‐1 for fat grafts may prove to be clinically successful for increasing the retention rate of transplanted fat.     

Involvement of kindlin‐2 in irisin’s protection against ischaemia reperfusion‐induced liver injury in high‐fat diet‐fed mice

Liver steatosis is associated with increased ischaemia reperfusion (I/R) injury. Our previous studies have shown that irisin, an exercise‐induced hormone, mitigates I/R injury via binding to αVβ5 integrin. However, the effect of irisin on I/R injury in steatotic liver remains unknown. Kindlin‐2 directly interacts with β integrin. We therefore suggest that irisin protects against I/R injury in steatotic liver via a kindlin‐2 dependent mechanism. To study this, hepatic steatosis was induced in male adult mice by feeding them with a 60% high‐fat diet (HFD). At 12 weeks after HFD feeding, the mice were subjected to liver ischaemia by occluding partial (70%) hepatic arterial/portal venous blood for 60 minutes, which was followed by 24 hours reperfusion. Our results showed HFD exaggerated I/R‐induced liver injury. Irisin (250 μg/kg) administration at the beginning of reperfusion attenuated liver injury, improved mitochondrial function, and reduced oxidative and endoplasmic reticulum stress in HFD‐fed mice. However, kindlin‐2 inhibition by RNAi eliminated irisin''s direct effects on cultured hepatocytes. In conclusion, irisin attenuates I/R injury in steatotic liver via a kindlin‐2 dependent mechanism.     

MicroRNA‐205 is associated with diabetes mellitus‐induced erectile dysfunction via down‐regulating the androgen receptor

As a major class of regulatory genes in majority metazoans, microRNAs (miRs) play an important role in various diseases including diabetes mellitus (DM). Lack of androgens has previously been associated with DM‐induced erectile dysfunction (DMED). In addition, the biological functioning of androgen is mediated by androgen receptor (AR). Herein, we sought to investigate whether miRs participate in AR‐associated DMED. Sprague‐Dawlay rats were employed to establish DMED models. After modelling, levels of miR‐205 and AR in their cavernous bodies were measured. The relationship between miR‐205 and AR was verified using a dual‐luciferase reporter gene assay. The underlying regulatory mechanisms of miR‐205 were investigated in concert with the treatment of mimics or inhibitors of miR‐205, or AR overexpression in the cavernous smooth muscle cells (CSMCs) isolated from rats with DMED. Meanwhile, the effects of miR‐205 and AR on cell proliferation and apoptosis were evaluated using MTT assay and flow cytometry respectively. Rats with DMED presented with increased miR‐205 and decreased AR levels in the cavernous bodies. AR was identified as a target gene of miR‐205. Down‐regulation of miR‐205 or up‐regulation of AR could increase proliferation and inhibits apoptosis of CSMCs in addition to improvements in the erectile functioning of rats with DMED. In summary, miR‐205 may contribute to the pathogenesis of DMED via down‐regulation of AR expressions.     

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.     

CircSAMD4A aggravates H/R‐induced cardiomyocyte apoptosis and inflammatory response by sponging miR‐138‐5p

Hypoxia/reoxygenation (H/R)‐induced myocardial cell injury is the main cause of acute myocardial infarction (AMI). Many proofs show that circular RNA plays an important role in the development of AMI. The purpose of this study was to investigate the role of circSAMD4A in H/R‐induced myocardial injury. The levels of circular SAMD4A (circSAMD4A) were detected in the heart tissues of AMI mice and H/R‐induced H9C2 cells, and the circSAMD4A was suppressed in AMI mice and H/R‐induced H9C2 cells to investigate its’ function in AMI. The levels of circSAMD4A and miR‐138‐5p were detected by real‐time quantitative PCR, and MTT assay was used to detect cell viability. TUNEL analysis and Annexin V‐FITC were used to determine apoptosis. The expression of Bcl‐2 and Bax proteins was detected by Western blot. IL‐1β, TNF‐α and IL‐6 were detected by ELISA kits. The study found that the levels of circSAMD4A were up‐regulated after H/R induction and inhibition of circSAMD4A expression would reduce the H/R‐induced apoptosis and inflammation. MiR‐138‐5p was down‐regulated in H/R‐induced H9C2 cells. circSAMD4A was a targeted regulator of miR‐138‐5p. CircSAMD4A inhibited the expression of miR‐138‐5p to promote H/R‐induced myocardial cell injury in vitro and vivo. In conclusion, CircSAMD4A can sponge miR‐138‐5p to promote H/R‐induced apoptosis and inflammatory response.     

Ipriflavone as a non‐steroidal glucocorticoid receptor antagonist ameliorates diabetic cognitive impairment in mice

Diabetic cognitive impairment (DCI) is a common diabetic complication with hallmarks of loss of learning ability and disorders of memory and behavior. Glucocorticoid receptor (GR) dysfunction is a main reason for neuronal impairment in brain of diabetic patients. Here, we determined that ipriflavone (IP) a clinical anti‐osteoporosis drug functioned as a non‐steroidal GR antagonist and efficiently ameliorated learning and memory dysfunction in both type 1 and 2 diabetic mice. The underlying mechanism has been intensively investigated by assay against the diabetic mice with GR‐specific knockdown in the brain by injection of adeno‐associated virus (AAV)‐ePHP‐si‐GR. IP suppressed tau hyperphosphorylation through GR/PI3K/AKT/GSK3β pathway, alleviated neuronal inflammation through GR/NF‐κB/NLRP3/ASC/Caspase‐1 pathway, and protected against synaptic impairment through GR/CREB/BDNF pathway. To our knowledge, our work might be the first to expound the detailed mechanism underlying the amelioration of non‐steroidal GR antagonist on DCI‐like pathology in mice and report the potential of IP in treatment of DCI.