Quercitrin attenuates the progression of osteoarthritis via inhibiting NF-κB signaling pathways and enhance glucose transport capacity.

Osteoarthritis (OA) is a common musculoskeletal disorder that impairs function and reduces the quality of life. Extracellular matrix (ECM) degradation and inflammatory mechanisms are crucial to the progression of OA. In this study, we aimed to investigate the anti-inflammatory activity, anti-ECM degradation property, and glucose transport capacity of quercitrin (QCT) on IL-1ß-treated rat primary chondrocytes. Rat primary chondrocytes were treated with IL-1ß to simulate inflammatory environmental conditions and OA in vitro. We examined the effects of QCT at concentrations ranging from 0 to 200 µM on the viability of rat chondrocytes and selected 5 µM for further study. Using qRT-PCR, immunofluorescent, immunocytochemistry, and western blotting techniques, we identified the potential molecular mechanisms and signaling pathways that are responsible for these effects. We established an OA rat model through anterior cruciate ligament transection (ACLT). The animals were then periodically injected with QCT into the knee articular cavity. Our in vivo and in vitro study showed that QCT could inhibit IL-1ß-activated inflammation and ECM degradation in chondrocyte. Furthermore, QCT could inhibit the NF-κB signal pathway and enhance glucose transport capacity in the IL-1ß-stimulated chondrocytes. In vivo study proved that QCT attenuates OA progression in rats. Overall, QCT inhibited the activation of NF-κB and enhanced glucose transport capacity to alleviate the progression of OA.

Gardenoside ameliorates inflammation and inhibits ECM degradation in IL-1ß-treated rat chondrocytes via suppressing NF-κB signaling pathways.

Osteoarthritis (OA) places a significant burden on society and finance, and there is presently no effective treatment beside late replacement surgery and symptomatic relief. The therapy of OA requires additional research. Gardenoside is a naturally compound extracted from Gardenia jasminoides Ellis, which has a variety of anti-inflammatory effects. However, few studies have been conducted to determine the role of gardenoside in OA. This study aimed to explore whether gardenoside has effect in OA treatment. Rat primary chondrocytes were treated with IL-1ß to simulate inflammatory environmental conditions and OA in vitro. We examined the effects of gardenoside at concentrations ranging from 0 to 200 µM on the viability of rat chondrocytes and selected 10 µM for further study. Via in vitro experiments, our study found that gardenoside lowers the gene expression of COX-2, iNOS, IL-6, and reduced the ROS production of chondrocytes induced by IL-1ß. Moreover, it effectively alleviates ECM degradation caused by IL-1ß and promotes the ECM synthesis in chondrocytes by upregulating collagen-II and the ACAN expression, downregulating the expression of MMP-3, MMP-13, and ADAMTS-5 expression. Further, our study showed that gardenoside inhibits NF-κB signaling pathway activated by IL-1ß in chondrocytes. We established an OA rat model by anterior cruciate ligament transection (ACLT). The animals were then periodically injected with gardenoside into the knee articular cavity. In vivo study suggested that gardenoside attenuates OA progression in rats. As a whole, in vitro and in vivo results highlight gardenoside is a promising OA treatment agent.

Self-Assemble Silk Fibroin Microcapsules for Cartilage Regeneration through Gene Delivery and Immune Regulation.

Effective treatments for cartilage defects are currently lacking. Gene delivery using proper delivery systems has shown great potential in cartilage regeneration. However, the inflammatory microenvironment generated by the defected cartilage severely affects the system's delivery efficiency. Therefore, this study reports a silk fibroin microcapsule (SFM) structure based on layer-by-layer self-assembly, in which interleukin-4 (IL-4) is modified on silk by click chemistry and loaded with lysyl oxidase plasmid DNA (LOX pDNA). The silk microcapsules display good biocompatibility and the release rate of genes can be adjusted by controlling the number of self-assembled layers. Moreover, the functionalized SFMs mixed with methacrylated gelatin (GelMA) exhibit good injectability. The IL-4 on the outer layer of the SFM can regulate macrophages to polarize toward the M2 type, thereby promoting cartilage matrix repair and inhibiting inflammation. The LOX pDNA loaded inside can be effectively delivered into cells to promote extracellular matrix generation, significantly promoting cartilage regeneration. The results of this study provide a promising biomaterial for cartilage repair, and this novel silk-based microcapsule delivery system can also provide strategies for the treatment of other diseases.

DPP4 inhibition mitigates ANG II-mediated kidney immune activation and injury in male mice.

Recent evidence suggests that dipeptidyl peptidase-4 (DPP4) inhibition with saxagliptin (Saxa) is renoprotective under comorbid conditions associated with activation of the renin-angiotensin-aldosterone system (RAAS), such as diabetes, obesity, and hypertension, which confer a high cardiovascular risk. Immune system activation is now recognized as a contributor to RAAS-mediated tissue injury, and, importantly, immunomodulatory effects of DPP4 have been reported. Accordingly, we examined the hypothesis that DPP4 inhibition with Saxa attenuates angiotensin II (ANG II)-induced kidney injury and albuminuria via attenuation of immune activation in the kidney. To this end, male mice were infused with either vehicle or ANG II (1,000 ng/kg/min, s.c.) for 3 wk and received either placebo or Saxa (10 mg/kg/day, p.o.) during the final 2 wk. ANG II infusion increased kidney, but not plasma, DPP4 activity in vivo as well as DPP4 activity in cultured proximal tubule cells. The latter was prevented by angiotensin receptor blockade with olmesartan. Further, ANG II induced hypertension and kidney injury characterized by mesangial expansion, mitochondrial damage, reduced brush border megalin expression, and albuminuria. Saxa inhibited DPP4 activity ∼50% in vivo and attenuated ANG II-mediated kidney injury, independent of blood pressure. Further mechanistic experiments revealed mitigation by Saxa of proinflammatory and profibrotic mediators activated by ANG II in the kidney, including CD8+ T cells, resident macrophages (CD11bhiF4/80loLy6C-), and neutrophils. In addition, Saxa improved ANG II suppressed anti-inflammatory regulatory T cell and T helper 2 lymphocyte activity. Taken together, these results demonstrate, for the first time, blood pressure-independent involvement of renal DPP4 activation contributing to RAAS-dependent kidney injury and immune activation.NEW & NOTEWORTHY This work highlights the role of dipeptidyl peptidase-4 (DPP4) in promoting ANG II-mediated kidney inflammation and injury. Specifically, ANG II infusion in mice led to increases in blood pressure and kidney DPP4 activity, which then led to activation of CD8+ T cells, Ly6C- macrophages, and neutrophils and suppression of anti-inflammatory T helper 2 lymphocytes and regulatory T cells. Collectively, this led to kidney injury, characterized by mesangial expansion, mitochondrial damage, and albuminuria, which were mitigated by DPP4 inhibition independent of blood pressure reduction.

Necrostatin-1 Analog DIMO Exerts Cardioprotective Effect against Ischemia Reperfusion Injury by Suppressing Necroptosis via Autophagic Pathway in Rats.

AIM: It has been reported that necrostatin-1 (Nec-1) is a specific necroptosis inhibitor that could attenuate programmed cell death induced by myocardial ischemia/reperfusion (I/R) injury. This study aimed to observe the effect and mechanism of novel Nec-1 analog (Z)-5-(3,5-dimethoxybenzyl)-2-imine-1-methylimidazolin-4-1 (DIMO) on myocardial I/R injury. METHODS: Male SD rats underwent I/R injury with or without different doses of DIMO (1, 2, or 4 mg/kg) treatment. Isolated neonatal rat cardiomyocytes were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment with or without DIMO (0.1, 1, 10, or 100 µM). Myocardial infarction was measured by TTC staining. Cardiomyocyte injury was assessed by lactate dehydrogenase assay (LDH) and flow cytometry. Receptor-interacting protein 1 kinase (RIP1K) and autophagic markers were detected by co-immunoprecipitation and Western blotting analysis. Molecular docking of DIMO into the ATP binding site of RIP1K was performed using GLIDE. RESULTS: DIMO at doses of 1 or 2 mg/kg improved myocardial infarct size. However, the DIMO 4 mg/kg dose was ineffective. DIMO at the dose of 0.1 µM decreased LDH leakage and the ratio of PI-positive cells followed by OGD/R treatment. I/R or OGD/R increased RIP1K expression and in its interaction with RIP3K, as well as impaired myocardial autophagic flux evidenced by an increase in LC3-II/I ratio, upregulated P62 and Beclin-1, and activated cathepsin B and L. In contrast, DIMO treatment reduced myocardial cell death and reversed the above mentioned changes in RIP1K and autophagic flux caused by I/R and OGD/R. DIMO binds to RIP1K and inhibits RIP1K expression in a homology modeling and ligand docking. CONCLUSION: DIMO exerts cardioprotection against I/R- or OGD/R-induced injury, and its mechanisms may be associated with the reduction in RIP1K activation and restoration impaired autophagic flux.

Sevoflurane Preconditioning Confers Delayed Cardioprotection by Upregulating AMP-Activated Protein Kinase Levels to Restore Autophagic Flux in Ischemia-Reperfusion Rat Hearts.

BACKGROUND Volatile anesthetic preconditioning confers delayed cardioprotection against ischemia/reperfusion injury (I/R). AMP-activated protein kinase (AMPK) takes part in autophagy activation. Furthermore, autophagic flux is thought to be impaired after I/R. We hypothesized that delayed cardioprotection can restore autophagic flux by activating AMPK. MATERIAL AND METHODS All male rat hearts underwent 30-min ischemia and 120-min reperfusion with or without sevoflurane exposure. AMPK inhibitor compound C (250 µg/kg, iv) was given at the reperfusion period. Autophagic flux blocker chloroquine (10 mg/kg, ip) was administrated 1 h before the experiment. Myocardial infarction, nicotinamide adenine dinucleotide (NAD⁺) content, and cytochrome c were measured. To evaluate autophagic flux, the markers of microtubule-associated protein 1 light chain 3 (LC3) I and II, P62 and Beclin 1, and lysosome-associated membrane protein-2 (LAMP 2) were analyzed. RESULTS The delayed cardioprotection enhanced post-ischemic AMPK activation, reduced infarction, CK-MB level, NAD⁺ content loss and cytochrome c release, and compound C blocked these effects. Sevoflurane restored impaired autophagic flux through a lower ratio of LC3II/LC3I, downregulation of P62 and Beclin 1, and higher expression in LAMP 2. Consistently, compound C inhibited these changes of autophagy flux. Moreover, chloroquine pretreatment abolished sevoflurane-induced infarct size reduction, CK-MB level, NAD⁺ content loss, and cytochrome c release, with concomitant increase the ratios of LC3II/LC3I and levels of P62 and Beclin 1, but p-AMPK expression was not downregulated by chloroquine. CONCLUSIONS Sevoflurane exerts a delayed cardioprotective effects against myocardial injury in rats by activation of AMPK and restoration of I/R-impaired autophagic flux.

A preoperative whey protein and glucose drink before hip fracture surgery in the aged improves symptomatic and metabolic recovery.

BACKGROUND AND OBJECTIVES: We investigated the effects of a carbohydrate-whey protein solution on aged patients undergoing hip fracture surgery. METHODS AND STUDY DESIGN: Forty patients were randomly assigned to the carbohydrate-whey protein (CHP) group or the control group (CTL). In the CHP group, a mixed solution of CHP was orally administered to patients before surgery: 400 mL was administered on the day before surgery, and 200 mL was administered 3 h before surgery. The size of the liquid dark area in the gastric antrum was measured by ultrasound, and the bleeding volume during surgery was assayed. The incidence of nausea, vomiting, thirst, hunger, and days of hospitalization and the levels of blood glucose, C-reactive protein (CRP) and serum albumin were assessed. RESULTS: There was no obvious liquid dark space in the gastric antrum. CHP administration improved postoperative thirst and hunger and resulted in increased albumin levels and decreased CRP concentrations and blood glucose fluctuations. CONCLUSIONS: Oral CHP before hip fracture surgery reduces the incidence of postoperative thirst and hunger and improves recovery in the aged.

Sevoflurane postconditioning protects against myocardial ischemia/reperfusion injury by restoring autophagic flux via an NO-dependent mechanism.

Volatile anesthetics improve postischemic cardiac function and reduce infarction even when administered for only a brief time at the onset of reperfusion. A recent study showed that sevoflurane postconditioning (SPC) attenuated myocardial reperfusion injury, but the underlying mechanisms remain unclear. In this study, we examined the effects of sevoflurane on nitric oxide (NO) release and autophagic flux during the myocardial ischemia/reperfusion (I/R) injury in rats in vivo and ex vivo. Male rats were subjected to 30 min ischemia and 2 h reperfusion in the presence or absence of sevoflurane (1.0 minimum alveolar concentration) during the first 15 min of reperfusion. We found that SPC significantly improved hemodynamic performance after reperfusion, alleviated postischemic myocardial infarction, reduced nicotinamide adenine dinucleotide content loss, and cytochrome c release in heart tissues. Furthermore, SPC significantly increased the phosphorylation of endothelial nitric oxide synthase (NOS) and neuronal nitric oxide synthase, and elevated myocardial NOS activity and NO production. All these effects were abolished by treatment with an NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg, i.v.). We also observed myocardial I/R-induced accumulation of autophagosomes in heart tissues, as evidenced by increased ratios of microtubule-associated protein 1 light chain 3 II/I, up-regulation of Beclin 1 and P62, and reduced lysosome-associated membrane protein-2 expression. SPC significantly attenuated I/R-impaired autophagic flux, which were blocked by L-NAME. Moreover, pretreatment with the autophagic flux blocker chloroquine (10 mg/kg, i.p.) increased autophagosome accumulation in SPC-treated heart following I/R and blocked SPC-induced cardioprotection. The same results were also observed in a rat model of myocardial I/R injury ex vivo, suggesting that SPC protects rat hearts against myocardial reperfusion injury by restoring I/R-impaired autophagic flux via an NO-dependent mechanism.

Heparin rescues factor V Leiden-associated placental failure independent of anticoagulation in a murine high-risk pregnancy model.

Low molecular weight heparin (LMWH) is being tested as an experimental drug for improving pregnancy outcome in women with inherited thrombophilia and placenta-mediated pregnancy complications, such as recurrent pregnancy loss. The role of thrombotic processes in these disorders remains unproven, and the issue of antithrombotic prophylaxis is intensely debated. Using a murine model of factor V Leiden-associated placental failure, we show that treatment of the mother with LMWH allows placental development to proceed and affords significant protection from fetal loss. Nonetheless, the therapeutic effect of LMWH is not replicated by anticoagulation; fondaparinux and a direct Xa inhibitor, C921-78, achieve anticoagulation similar to LMWH but produce little or no improvement in pregnancy outcome. Genetic attenuation of maternal platelet aggregation is similarly ineffective. In contrast, even a partial loss of thrombin sensitivity of maternal platelets protects pregnancies. Neonates born from these pregnancies are growth retarded, suggesting that placental function is only partially restored. The placentae are smaller but do not reveal any evidence of thrombosis. Our data demonstrate an anticoagulation-independent role of LMWH in protecting pregnancies and provide evidence against the involvement of thrombotic processes in thrombophilia-associated placental failure. Importantly, thrombin-mediated maternal platelet activation remains central in the mechanism of placental failure.

The influence of COMT and ABCB1 gene polymorphisms on sufentanil analgesic effect for postoperative pain in children with fracture.

The aim of this observational study was to investigate the effects of catechol-O-methyltransferase (COMT) and ATP-binding cassette transporter B1 (ABCB1) gene polymorphisms on the postoperative analgesic effect of sufentanil in Chinese Han pediatric patients with fractures. A total of 185 pediatric patients who underwent fracture surgery were included. Polymerase chain reaction-restriction fragment length polymorphism was used to detect the polymorphisms of COMT and ABCB1 genes. Sufentanil was used for postoperative analgesia. The pain level of the patients was evaluated using the face, legs, activity, cry, and consolability scale before surgery, during awakening, at 2, 6, 12, and 24 hours after surgery. The postoperative Ramsay sedation score, sufentanil consumption, and incidence of adverse reactions were also recorded. Pediatric patients with different genotypes of ABCB1 and COMT showed no statistically significant differences in general data such as age, gender, weight, height, surgical duration, and American Society of Anesthesiologists classification (P > .05). There were no statistically significant differences in sedation scores after surgery between different genotypes of ABCB1 and COMT (P > .05). Among patients with CC genotype in ABCB1, the pain scores and total consumption of sufentanil at awakening, 2 and 6 hours after surgery were higher compared to TT and CT genotypes (P < .05), while there were no statistically significant differences between TT and CT genotypes (P > .05). Among patients with AA genotype in COMT, the pain scores and total consumption of sufentanil at awakening, 2, 6, 12, and 24 hours after surgery were higher compared to AG and GG genotypes (P < .05), while there were no statistically significant differences between AG and GG genotypes (P > .05). There were no statistically significant differences in adverse reactions between different genotypes of ABCB1 and COMT (P > .05). The polymorphisms of COMT gene rs4680 and ABCB1 gene rs1045642 are associated with the analgesic effect and consumption of sufentanil in pediatric patients after fracture surgery.

Mineralocorticoid promotes intestinal inflammation through receptor dependent IL17 production in ILC3s.

Aldosterone is a key mineralocorticoid involved in regulating the concentration of blood electrolytes and physiological volume balance. Activation of mineralocorticoid receptor (MR) has been recently reported to participate in adaptive and innate immune responses under inflammation. Here, we evaluated the role of aldosterone and MR in inflammation bowel diseases (IBD). Aldosterone elevated in the colon of DSS-induced colitis mice. Aldosterone addition induced IL17 production and ROS/RNS level in group 3 innate lymphoid cells (ILC3s) and exacerbated intestinal injury. A selective mineralocorticoid receptor antagonism, eplerenone, inhibited IL17-producing ILC3s and its ROS/RNS production, protected mice from DSS-induced colitis. Mice lacking Nr3c2 (MR coding gene) in ILC3s exhibited decreased IL17 and ROS/RNS production, which alleviated colitis and colitis-associated colorectal cancer (CAC). Further experiments revealed that MR could directly bind to IL17A promoter and facilitate its transcription, which could be enhanced by aldosterone. Thus, our findings demonstrated the critical role of aldosterone-MR-IL17 signaling in ILC3s and gut homeostasis, indicating the therapeutic strategy of eplerenone in IBD clinical trial.

Platelet ITGA2B inhibits caspase-8 and Rip3/Mlkl-dependent platelet death though PTPN6 during sepsis.

Platelets play an important role in the pathogenesis of sepsis and platelet transfusion is a therapeutic option for sepsis patients, although the exact mechanisms have not been elucidated so far. ITGA2B encodes the αIIb protein in platelets, and its upregulation in sepsis is associated with increased mortality rate. Here, we generated a Itga2b (Q887X) knockin mouse, which significantly reduced ITGA2B expression of platelet and megakaryocyte. The decrease of ITGA2B level aggravated the death of septic mice. We analyzed the transcriptomic profiles of the platelets using RNA sequencing. Our findings suggest that ITGA2B upregulates PTPN6 in megakaryocytes via the transcription factors Nfkb1 and Rel. Furthermore, PTPN6 inhibits platelet apoptosis and necroptosis during sepsis by targeting the Ripk1/Ripk3/Mlkl and caspase-8 pathways. This prevents Kupffer cells from rapidly clearing activated platelets, and eventually maintains vascular integrity during sepsis. Our findings indicate a new function of ITGA2B in the regulation of platelet death during sepsis.

Analysis of intercellular communication in the osteosarcoma microenvironment based on single cell sequencing data.

Osteosarcoma (OS) is the most common primary bone cancer in children and young adults, patient survival rates have not improved in recent decades. To further understand the interrelationship between different cell types in the tumor microenvironment of osteosarcoma, we comprehensively analyzed single-cell sequencing data from six patients with untreated osteosarcoma. Nine major cell types were identified from a total of 46,046 cells based on unbiased clustering of gene expression profiles and canonical markers. Osteosarcoma from different patients display heterogeneity in cellular composition. Myeloid cells were the most commonly represented cell type, followed by osteoblastic and TILs. Copy number variation (CNV) results identified amplifications and deletions in malignant osteoblastic cells and fibroblasts. Trajectory analysis based on RNA velocity showed that osteoclasts in the OS microenvironment could be differentiated from myeloid cells. Furthermore, we explored the intercellular communications in OS microenvironment and identified multiple ligand-receptor pairs between myeloid cells, osteoblastic cells and their cells, including 21 ligand-receptor pair genes that significantly associated with survival outcomes. Importantly, we found chemotherapy may have an effect on cellular communication in the OS microenvironment by analyzing single-cell sequencing data from seven primary osteosarcoma patients who received chemotherapy. We believe these observations will improve our understanding of potential mechanisms of microenvironment contributions to OS progression and help identify potential targets for new treatment development in the future.

Single-cell RNA-seq of heart reveals intercellular communication drivers of myocardial fibrosis in diabetic cardiomyopathy.

Myocardial fibrosis is the characteristic pathology of diabetes-induced cardiomyopathy. Therefore, an in-depth study of cardiac heterogeneity and cell-to-cell interactions can help elucidate the pathogenesis of diabetic myocardial fibrosis and identify treatment targets for the treatment of this disease. In this study, we investigated intercellular communication drivers of myocardial fibrosis in mouse heart with high-fat-diet/streptozotocin-induced diabetes at single-cell resolution. Intercellular and protein-protein interaction networks of fibroblasts and macrophages, endothelial cells, as well as fibroblasts and epicardial cells revealed critical changes in ligand-receptor interactions such as Pdgf(s)-Pdgfra and Efemp1-Egfr, which promote the development of a profibrotic microenvironment during the progression of and confirmed that the specific inhibition of the Pdgfra axis could significantly improve diabetic myocardial fibrosis. We also identified phenotypically distinct Hrchi and Postnhi fibroblast subpopulations associated with pathological extracellular matrix remodeling, of which the Hrchi fibroblasts were found to be the most profibrogenic under diabetic conditions. Finally, we validated the role of the Itgb1 hub gene-mediated intercellular communication drivers of diabetic myocardial fibrosis in Hrchi fibroblasts, and confirmed the results through AAV9-mediated Itgb1 knockdown in the heart of diabetic mice. In summary, cardiac cell mapping provides novel insights into intercellular communication drivers involved in pathological extracellular matrix remodeling during diabetic myocardial fibrosis.

Astrocyte-Derived TNF-α-Activated Platelets Promote Cerebral Ischemia/Reperfusion Injury by Regulating the RIP1/RIP3/AKT Signaling Pathway.

Ischemic stroke is a clinical syndrome caused by the disruption of blood flow into cerebral tissues and is associated with high disability and mortality rates. Studies have established the pathological role of platelets in cerebral ischemia/reperfusion (I/R) injury, although the underlying mechanism of action remains largely unclear. In this study, we created an I/R mouse model via middle cerebral artery occlusion and reperfusion (MCAO/R) and analyzed the transcriptomic profiles of the ipsilateral and contralateral cortices using RNA-seq. We found that cerebral I/R injury induced platelet invasion and accumulation in the cerebral cortex by stimulating TNF-α secretion from activated astrocytes in the ischemic region, while TNF-α expression enhanced platelet reactivity through the RIP1/RIP3/AKT pathway. Furthermore, the inoculation of TNF-α-stimulated platelets aggravated I/R injury in mice, whereas the administration of anti-TNF-α antibodies at the onset of reperfusion alleviated ischemic damage. The RNA-seq results further showed that AP-1 transcriptionally activated TNF-α in the I/R-injured cortex by directly binding to the promoter region. These findings provide novel insights into the pathological role of platelets activated by reactive astrocyte-derived TNF-α in cerebral I/R injury.

Glaucocalyxin a protect liver function via inhibiting platelet over-activation during sepsis.

BACKGROUND: Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) is a perennial herb, and is traditionally used as folk medicine for treating inflammatory diseases and cancer. Gaucocalyxin A (GLA) is an ent­kaurane diterpenoid that is isolated from the aerial parts of R. japonica (Burm. f.) var. glaucocalyx (Maxim.). In a recent study, we found that GLA protects against acute liver dysfunction induced by Escherichia coli, which is likely related to its anti-inflammatory effects. However, the mechanism by which GLA protects liver injury during sepsis is unknown. AIM: To evaluate the anti-inflammatory function of GLA and its regulatory effect on platelet function. METHOD: An in vivo model of sepsis was established by inoculating mice with E. coli. Live function and platelet activation were evaluated through standard assays. The levels of pro-inflammatory factors were measured through ELISA and qRT-PCR. RESULTS: GLA alleviated liver dysfunction in the mouse model of sepsis. GLA-treated mice displayed lower complement activation and liver dysfunction after E. coli infection. GLA alleviated the decrease in peripheral platelet counts by inhibiting their clearance by Kupffer cells in liver. Furthermore, GLA inhibited platelet activation through the RIP1/RIP3/AKT pathway and downregulated C3aR expression on the platelets, thereby inhibiting liver injury and dysfunction due to excessive complement activation. CONCLUSION: GLA can inhibit platelet activation by reducing surface expression of C3aR, which protect the liver from injury induced by excessive complement activation. GLA is a novel therapeutic agent for controlling sepsis-related liver dysfunction.

Madecassic Acid Ameliorates the Progression of Osteoarthritis: An in vitro and in vivo Study.

Purpose: Osteoarthritis (OA) places a significant burden on society and finance, and there is presently no effective treatment besides late replacement surgery and symptomatic relief. The therapy of OA requires additional research. Madecassic acid (MA) is the first native triterpenoid compound extracted from Centella asiatica, which has a variety of anti-inflammatory effects. However, the role of MA in OA therapy has not been reported. This study aimed to explore whether MA could suppress the inflammatory response, preserve and restore chondrocyte functions, and ameliorate the progression of OA in vitro and in vivo. Methods: Rat primary chondrocytes were treated with IL-1ß to simulate inflammatory environmental conditions and OA in vitro. We examined the effects of MA at concentrations ranging from 0 to 200 µM on the viability of rat chondrocytes and selected 10 µM for further study. Using qRT-PCR, immunofluorescent, immunocytochemistry, and Western blotting techniques, we identified the potential molecular mechanisms and signaling pathways that are responsible for these effects. We established an OA rat model by anterior cruciate ligament transection (ACLT). The animals were then periodically injected with MA into the knee articular cavity. Results: We found that MA could down-regulate the IL-1ß-induced up-regulation of COX-2, iNOS and IL-6 and restore the cytoskeletal integrity of chondrocytes treated with IL-1ß. Moreover, MA protects chondrocytes from IL-1ß-induced ECM degradation by upregulating ECM synthesis related protein expression, including collagen-II and ACAN, and further down-regulating ECM catabolic related protein expression, including MMP-3 and MMP-13. Furthermore, we found that NF-κB/IκBα and PI3K/AKT signaling pathways were involved in the regulatory effects of MA on the inflammation inhibition and promotion of ECM anabolism on IL-1ß-induced chondrocytes. Conclusion: These findings suggest that MA appears to be a potentially small molecular drug for rat OA.

Hypothermia Prevents Cardiac Dysfunction during Acute Ischemia Reperfusion by Maintaining Mitochondrial Bioenergetics and by Promoting Hexokinase II Binding to Mitochondria.

Background: Hypothermia (H), cardioplegia (CP), and both combined (HCP) are known to be protective against myocardial ischemia reperfusion (IR) injury. Mitochondria have molecular signaling mechanisms that are associated with both cell survival and cell death. In this study, we investigated the dynamic changes in proapoptotic and prosurvival signaling pathways mediating H, CP, or HCP-induced protection of mitochondrial function after acute myocardial IR injury. Methods: Rats were divided into five groups. Each group consists of 3 subgroups based on a specific reperfusion time (5, 20, or 60 min) after a 25-min global ischemia. The time control (TC) groups were not subjected to IR but were perfused with 37 °C Krebs-Ringer's (KR) buffer, containing 4.5 mM K+, in a specific perfusion protocol that corresponded with the duration of each IR protocol. The IR group (control) was perfused for 20 min with KR, followed by 25-min global ischemia, and then KR reperfusion for 5, 20, or 60 min. The treatment groups were exposed to 17 °C H, 37 °C CP (16 mM K+), or HCP (17 °C + CP) for 5 min before ischemia and for 2 min on reperfusion before switching to 37 °C KR perfusion for the remainder of each of the reperfusion times. Cardiac function and mitochondrial redox state (NADH/FAD) were monitored online in the ex vivo hearts before, during, and after ischemia. Mitochondria were isolated at the end of each specified reperfusion time, and changes in O2 consumption, membrane potential (ΔΨ m), and Ca2+ retention capacity (CRC) were assessed using complex I and complex II substrates. In another set of hearts, mitochondrial and cytosolic fractions were isolated after a specified reperfusion time to conduct western blot assays to determine hexokinase II (HKII) and Bax binding/translocation to mitochondria, cytosolic pAkt levels, and cytochrome c (Cyto-c) release into the cytosol. Results: H and HCP were more protective of mitochondrial integrity and, concomitantly, cardiac function than CP alone; H and HCP improved post-ischemic cardiac function by (1) maintaining mitochondrial bioenergetics, (2) maintaining HKII binding to mitochondria with an increase in pAkt levels, (3) increasing CRC, and (4) decreasing Cyto-c release during reperfusion. Bax translocation/binding to mitochondria was unaffected by any treatment, regardless of cardiac functional recovery. Conclusions: Hypothermia preserved mitochondrial function and cardiac function, in part, by maintaining mitochondrial bioenergetics, by retaining HKII binding to mitochondria via upstream pAkt, and by reducing Cyto-c release independently of Bax binding to mitochondria.

RIPK1-RIPK3 mediates myocardial fibrosis in type 2 diabetes mellitus by impairing autophagic flux of cardiac fibroblasts.

Receptor-interacting protein kinase 1 (RIPK1) and 3 (RIPK3) are critical regulators of programmed necrosis or necroptosis. However, the role of the RIPK1/RIPK3 signaling pathway in myocardial fibrosis and related diabetic cardiomyopathy is still unclear. We hypothesized that RIPK1/RIPK3 activation mediated myocardial fibrosis by impairing the autophagic flux. To this end, we established in vitro and in vivo models of type 2 diabetes mellitus with high glucose fat (HGF) medium and diet respectively. HGF induced myocardial fibrosis, and impaired cardiac diastolic and systolic function by activating the RIPK1/RIPK3 pathway, which increased the expression of autophagic related proteins such as LC3-II, P62 and active-cathepsin D. Inhibition of RIPK1 or RIPK3 alleviated HGF-induced death and fibrosis of cardiac fibroblasts by restoring the impaired autophagic flux. The autophagy blocker neutralized the effects of the RIPK1 inhibitor necrostatin-1 (Nec-1) and RIPK3 inhibitor GSK872 (GSK). RIPK1/RIPK3 inhibition respectively decreased the levels of RIPK3/p-RIPK3 and RIPK1/p-RIPK1. P62 forms a complex with RIPK1-RIPK3 and promotes the binding of RIPK1 and RIPK3, silencing of RIPK1 decreased the association of RIPK1 with P62 and the binding of P62 to LC3. Furthermore, inhibition of both kinases in combination with a low dose of Nec-1 and GSK in the HGF-treated fibroblasts significantly decreased cell death and fibrosis, and restored the autophagic flux. In the diabetic rat model, Nec-1 (1.65 mg/kg) treatment for 4 months markedly alleviated myocardial fibrosis, downregulated autophagic related proteins, and improved cardiac systolic and diastolic function. In conclusion, HGF induces myocardial fibrosis and cardiac dysfunction by activating the RIPK1-RIPK3 pathway and by impairing the autophagic flux, which is obviated by the pharmacological and genetic inhibition of RIPK1/RIPK3.

Tumor Necrosis Factor-Alpha Disrupts Cx43-Mediated Corneal Endothelial Gap Junction Intercellular Communication.

Connexin43 (Cx43)-mediated gap junctions are vital in maintaining corneal endothelium homeostasis. Tumor necrosis factor-alpha (TNF-α) is among the most important inflammatory factors which cause corneal endothelial dysfunction in various eye diseases. However, the effect of TNF-α on Cx43-mediated gap junctions of the corneal endothelium remains undefined. In the current research, we determined the effect of TNF-α on gap junction intercellular communication (GJIC) in rabbit corneal endothelium. To evaluate alterations of GJIC, if any, we treated ex vivo cultured rabbit corneal endothelium with different concentrations of TNF-α (2-20 ng/ml). The localization of Cx43 was analyzed by immunostaining, while RT-qPCR and western blot were used to profile the expression of Cx43 and zonula occludens-1 (ZO-1). The association between ZO-1 and Cx43 was evaluated using immunoprecipitation and double staining. GJIC activity was determined by the scrap loading and dye transfer assay (SLDT). Our data demonstrated that a high concentration of TNF-α (10 ng/ml and 20 ng/ml) disrupts the Cx43 mediated gap junction distribution in rabbit corneal endothelium and suppresses the expression of Cx43 protein. Furthermore, rabbit corneal endothelial GJIC was inhibited due to the decreased association between the ZO-1 and Cx43 proteins. Current results demonstrate that TNF-α inhibits corneal endothelial GJIC via decreasing the association between ZO-1 and Cx43, disrupting the distribution of Cx43, and downregulating the expression of Cx43 protein. This study offers a new theoretical foundation for diagnosing and treating corneal endothelial cell decompensation induced by elevated TNF-α in various eye diseases.