CD62E- and ROS-Responsive ETS Improves Cartilage Repair by Inhibiting Endothelial Cell Activation through OPA1-Mediated Mitochondrial Homeostasis.

Background: In the environment of cartilage injury, the activation of vascular endothelial cell (VEC), marked with excessive CD62E and reactive oxygen species (ROS), can affect the formation of hyaluronic cartilage. Therefore, we developed a CD62E- and ROS-responsive drug delivery system using E-selectin binding peptide, Thioketal, and silk fibroin (ETS) to achieve targeted delivery and controlled release of Clematis triterpenoid saponins (CS) against activated VEC, and thus promote cartilage regeneration. Methods: We prepared and characterized ETS/CS and verified their CD62E- and ROS-responsive properties in vitro. We investigated the effect and underlying mechanism of ETS/CS on inhibiting VEC activation and promoting chondrogenic differentiation of bone marrow stromal cells (BMSCs). We also analyzed the effect of ETS/CS on suppressing the activated VEC-macrophage inflammatory cascade in vitro. Additionally, we constructed a rat knee cartilage defect model and administered ETS/CS combined with BMSC-containing hydrogels. We detected the cartilage differentiation, the level of VEC activation and macrophage in the new tissue, and synovial tissue. Results: ETS/CS was able to interact with VEC and inhibit VEC activation through the carried CS. Coculture experiments verified ETS/CS promoted chondrogenic differentiation of BMSCs by inhibiting the activated VEC-induced inflammatory cascade of macrophages via OPA1-mediated mitochondrial homeostasis. In the rat knee cartilage defect model, ETS/CS reduced VEC activation, migration, angiogenesis in new tissues, inhibited macrophage infiltration and inflammation, promoted chondrogenic differentiation of BMSCs in the defective areas. Conclusions: CD62E- and ROS-responsive ETS/CS promoted cartilage repair by inhibiting VEC activation and macrophage inflammation and promoting BMSC chondrogenesis. Therefore, it is a promising therapeutic strategy to promote articular cartilage repair.

Inhibition of HDAC6 promotes microvascular endothelial cells to phagocytize myelin debris and reduces inflammatory response to accelerate the repair of spinal cord injury.

AIMS: To identify an effective strategy for promoting microvascular endothelial cells (MECs) to phagocytize myelin debris and reduce secretion of inflammatory factors following spinal cord injury (SCI). METHODS: We established a coculture model of myelin debris and vascular-like structures. The efficiency with which MECs phagocytize myelin debris under different conditions was examined via ELISA, flow cytometry, and immunofluorescence. Tubastatin-A was used to interfere with the coculture model. The anti-inflammatory effects of Tubastatin-A were observed by HE staining, flow cytometry, immunofluorescence, and ELISA. RESULTS: MECs phagocytized myelin debris via IgM opsonization, and phagocytosis promoted the secretion of inflammatory factors, whereas IgG-opsonized myelin debris had no effect on inflammatory factors. Application of the HDAC6 inhibitor Tubastatin-A increased the IgG levels and decreased the IgM levels by regulating the proliferation and differentiation of B cells. Tubastatin-A exerted a regulatory effect on the HDAC6-mediated autophagy-lysosome pathway, promoting MECs to phagocytize myelin debris, reducing the secretion of inflammatory factors, and accelerating the repair of SCI. CONCLUSIONS: Inhibition of HDAC6 to regulate the immune-inflammatory response and promote MECs to phagocytize myelin debris may represent a novel strategy in the treatment of SCI.

Celebrating Diversity With Subtask Specialization in Shared Multiagent Reinforcement Learning.

Subtask decomposition offers a promising approach for achieving and comprehending complex cooperative behaviors in multiagent systems. Nonetheless, existing methods often depend on intricate high-level strategies, which can hinder interpretability and learning efficiency. To tackle these challenges, we propose a novel approach that specializes subtasks for subgroups by employing diverse observation representation encoders within information bottlenecks. Moreover, to enhance the efficiency of subtask specialization while promoting sophisticated cooperation, we introduce diversity in both optimization and neural network architectures. These advancements enable our method to achieve state-of-the-art performance and offer interpretable subtask factorization across various scenarios in Google Research Football (GRF).

Iron induces B cell pyroptosis through Tom20-Bax-caspase-gasdermin E signaling to promote inflammation post-spinal cord injury.

BACKGROUND: Immune inflammatory responses play an important role in spinal cord injury (SCI); however, the beneficial and detrimental effects remain controversial. Many studies have described the role of neutrophils, macrophages, and T lymphocytes in immune inflammatory responses after SCI, although little is known about the role of B lymphocytes, and immunosuppression can easily occur after SCI. METHODS: A mouse model of SCI was established, and HE staining and Nissl staining were performed to observe the pathological changes. The size and morphology of the spleen were examined, and the effects of SCI on spleen function and B cell levels were detected by flow cytometry and ELISA. To explore the specific mechanism of immunosuppression after SCI, B cells from the spleens of SCI model mice were isolated using magnetic beads and analyzed by 4D label-free quantitative proteomics. The level of inflammatory cytokines and iron ions were measured, and the expression of proteins related to the Tom20 pathway was quantified by western blotting. To clarify the relationship between iron ions and B cell pyroptosis after SCI, we used FeSO4 and CCCP, which induce oxidative stress to stimulate SCI, to interfere with B cell processes. siRNA transfection to knock down Tom20 (Tom20-KD) in B cells and human B lymphocytoma cell was used to verify the key role of Tom20. To further explore the effect of iron ions on SCI, we used deferoxamine (DFO) and iron dextran (ID) to interfere with SCI processes in mice. The level of iron ions in splenic B cells and the expression of proteins related to the Tom20-Bax-caspase-gasdermin E (GSDME) pathway were analyzed. RESULTS: SCI could damage spleen function and lead to a decrease in B cell levels; SCI upregulated the expression of Tom20 protein in the mitochondria of B cells; SCI could regulate the concentration of iron ions and activate the Tom20-Bax-caspase-GSDME pathway to induce B cell pyroptosis. Iron ions aggravated CCCP-induced B cell pyroptosis and human B lymphocytoma pyroptosis by activating the Tom20-Bax-caspase-GSDME pathway. DFO could reduce inflammation and promote repair after SCI by inhibiting Tom20-Bax-caspase-GSDME-induced B cell pyroptosis. CONCLUSIONS: Iron overload activates the Tom20-Bax-caspase-GSDME pathway after SCI, induces B cell pyroptosis, promotes inflammation, and aggravates the changes caused by SCI. This may represent a novel mechanism through which the immune inflammatory response is induced after SCI and may provide a new key target for the treatment of SCI.

Construction and optimization of a coculture system of mouse brain microvascular endothelial cells and myelin debris.

Microvascular endothelial cells are a newly discovered cell type involved in the phagocytosis of myelin debris, which play a key role in the repair of spinal cord injuries. Several methods for the preparation of myelin debris and parameters for constructing a coculture system of microvascular endothelial cells and myelin debris are available, but no systematic studies have yet been conducted, which hinders further exploration of the mechanisms of demyelinating disease repair. Herein, we aimed to develop a standardized method for this process. Myelin debris of different sizes was obtained from the brains of C57BL/6 mice by stripping the brains under aseptic conditions, multiple grinding, gradient centrifugation, etc. Transmission electron microscopy and nanoparticle size analysis were used to characterize myelin debris. Microvascular endothelial cells were cultured on a matrix gel, and myelin debris of different sizes (fluorescently labeled using CFSE) was placed in coculture after forming a vascular-like structure. Subsequently, myelin debris of different concentrations was cocultured in the vascular-like structure, and phagocytosis of myelin debris by microvascular endothelial cells was detected using immunofluorescence staining and flow cytometry. We found that myelin debris could be successfuly obtained from the mouse brain with secondary grinding and other steps and cocultured with microvascular endothelial cells at a concentration of 2 mg/mL, which promoted the phagocytosis of microvascular endothelial cells. In conclusion, we provide a reference for the protocol of a coculture system of microvascular endothelial cells and myelin debris.

Acetate attenuates hyperoxaluria-induced kidney injury by inhibiting macrophage infiltration via the miR-493-3p/MIF axis.

Hyperoxaluria is well known to cause renal injury and end-stage kidney disease. Previous studies suggested that acetate treatment may improve the renal function in hyperoxaluria rat model. However, its underlying mechanisms remain largely unknown. Using an ethylene glycol (EG)-induced hyperoxaluria rat model, we find the oral administration of 5% acetate reduced the elevated serum creatinine, urea, and protected against hyperoxaluria-induced renal injury and fibrosis with less infiltrated macrophages in the kidney. Treatment of acetate in renal tubular epithelial cells in vitro decrease the macrophages recruitment which might have reduced the oxalate-induced renal tubular cells injury. Mechanism dissection suggests that acetate enhanced acetylation of Histone H3 in renal tubular cells and promoted expression of miR-493-3p by increasing H3K9 and H3K27 acetylation at its promoter region. The miR-493-3p can suppress the expression of macrophage migration inhibitory factor (MIF), thus inhibiting the macrophages recruitment and reduced oxalate-induced renal tubular cells injury. Importantly, results from the in vivo rat model also demonstrate that the effects of acetate against renal injury were weakened after blocking the miR-493-3p by antagomir treatment. Together, these results suggest that acetate treatment ameliorates the hyperoxaluria-induced renal injury via inhibiting macrophages infiltration with change of the miR-493-3p/MIF signals. Acetate could be a new therapeutic approach for the treatment of oxalate nephropathy.

Wen-Shen-Tong-Luo-Zhi-Tong Decoction regulates bone-fat balance in osteoporosis by adipocyte-derived exosomes.

CONTEXT: Wen-Shen-Tong-Luo-Zhi-Tong (WSTLZT) Decoction is a Chinese prescription with antiosteoporosis effects, especially in patients with abnormal lipid metabolism. OBJECTIVE: To explore the effect and mechanism of WSTLZT on osteoporosis (OP) through adipocyte-derived exosomes. MATERIALS AND METHODS: Adipocyte-derived exosomes with or without WSTLZT treated were identified by transmission electron microscopy, nanoparticle tracking analysis (NTA) and western blotting (WB). Co-culture experiments for bone marrow mesenchymal stem cells (BMSCs) and exosomes were performed to examine the uptake and effect of exosome in osteogenesis and adipogenic differentiation of BMSC. MicroRNA profiles, luciferase and IP were used for exploring specific mechanisms of exosome on BMSC. In vivo, 80 Balb/c mice were randomly divided into four groups: Sham, Ovx, Exo (30 µg exosomes), Exo-WSTLZT (30 µg WSTLZT-exosomes), tail vein injection every week. After 12 weeks, the bone microstructure and marrow fat distribution were analysed by micro-CT. RESULTS: ALP, Alizarin red and Oil red staining showed that WSTLZT-induced exosomes from adipocyte can regulate osteoblastic and adipogenic differentiation of BMSC. MicroRNA profiles observed that WSTLZT treatment resulted in 87 differentially expressed miRNAs (p < 0.05). MiR-122-5p with the greatest difference was screened by q-PCR (p < 0.01). The target relationship between miR-122-5p and SPRY2 was tested by luciferase and IP. MiR-122-5p negatively regulated SPRY2 and elevated the activity of MAPK signalling pathway, thereby regulating the osteoblastic and adipogenic differentiation of BMSC. In vivo, exosomes can not only improve bone microarchitecture but also significantly reduce accumulation of bone marrow adipose. CONCLUSIONS: WSTLZT can exert anti-OP effect through SPRY2 via the MAKP signalling by miR-122-5p carried by adipocyte-derived exosomes.

circHIPK3 prevents cardiac senescence by acting as a scaffold to recruit ubiquitin ligase to degrade HuR.

Rational: Senescence is a major aging process that contributes to the development of cardiovascular diseases, but the underlying molecular mechanisms remain largely unknown. One reason is due to the lack of suitable animal models. We aimed to generate a cardiomyocyte (CM)-specific senescent animal model, uncover the underlying mechanisms, and develop new therapies for aging associated cardiac dysfunction. Methods: The gain/loss of circHIPK3 approach was used to explore the role of circHIPK3 in cardiomyocyte (CM) senescence. To investigate the mechanisms of circHIPK3 function in cardiac senescence, we generated CM-specific tamoxifen-induced circHIPK3 knockout (CKO) mice. We also applied various analyses including PCR, Western blot, nuclear and cytoplasmic protein extraction, immunofluorescence, echocardiography, RNA immunoprecipitation assay, RNA-pulldown assay, and co-immunoprecipitation. Results: Our novel CKO mice exhibited worse cardiac function, decreased circHIPK3 expression and telomere length shortening in the heart. The level of the senescence-inducer p21 in the hearts of CKO mice was significantly increased and survival was poor compared with control mice. In vitro, the level of p21 in CMs was significantly decreased by circHIPK3 overexpression, but increased by circHIPK3 silencing. We showed that circHIPK3 was a scaffold for p21 mRNA-binding protein HuR and E3 ubiquitin ligase ß-TrCP. circHIPK3 silencing weakened the interaction between HuR and ß-TrCP, reduced HuR ubiquitination, and enhanced the interaction between HuR and p21 mRNA. Moreover, we found that mice injected with human umbilical cord mesenchymal stem cell-derived exosomes (UMSC-Exos) showed increased circHIPK3 levels, decreased levels of p21, longer telomere length, and good cardiac function. However, these beneficial effects exerted by UMSC-Exos were inhibited by silencing circHIPK3. Conclusions: We successfully generated CM-specific CKO mice for aging research. Our results showed that deletion of circHIPK3 led to exaggerated CM senescence and decreased cardiac function. As a scaffold, circHIPK3 enhanced the binding of E3 ubiquitin ligase ß-TrCP and HuR in the cytoplasm, leading to the ubiquitination and degradation of HuR and reduced p21 activity. In addition, UMSC-Exos exerted an anti-senescence and cardio-protective effect by delivering circHIPK3. These findings pave the way to the development of new therapies for aging associated cardiac dysfunction.

Dearomative Aminocarbonylation of Arenes via Bifunctional Coordination to Chromium.

Amides are ubiquitous in physical and life sciences. Given the significant abundance of arenes, dearomative aminocarbonylation of arenes would lead to a large and underexplored chemical space for amide discovery. However, such reactions are challenging due to the high degree of resonance stabilization and selectivity issues. Herein, we disclose an unprecedented dearomative trifluoromethylative aminocarbonylation of arenes via bifunctional coordination to chromium, providing a modular platform for the construction of amides possessing trifluoromethyl (CF3 ) groups and three-dimensional rings. Its versatility further enabled a switchable difluoromethylation or trifluoromethylation aminocarbonylation of arene C-H bonds. A possible mechanism was proposed based on control experiments. Finally, the synthetic utility was well demonstrated by diverse applications in the total synthesis of CF3 -functionalized amide-type drugs, including praziquantel, nateglinide, maraviroc and alloyohimbane.

Mechanical Stretch Promotes Macrophage Polarization and Inflammation via the RhoA-ROCK/NF-κB Pathway.

Macrophages play an essential role in the pathogenesis of most inflammatory diseases. Recent studies have shown that mechanical load can influence macrophage function, leading to excessive and uncontrolled inflammation and even systemic damage, including cardiovascular disease and knee osteoarthritis. However, the molecular mechanism remains unclear. In this study, murine RAW264.7 cells were treated with mechanical stretch (MS) using the Flexcell-5000T Tension System. The expression of inflammatory factors and cytokine release were measured by RT-qPCR, ELISA, and Western blotting. The protein expression of NF-κB p65, Iκb-α, p-Iκb-α, RhoA, ROCK1, and ROCK2 was also detected by Western blotting. Then, Flow cytometry was used to detect the proportion of macrophage subsets. Meanwhile, Y-27632 dihydrochloride, a ROCK inhibitor, was added to knockdown ROCK signal transduction in cells. Our results demonstrated that MS upregulated mRNA expression and increased the secretion levels of proinflammatory factors iNOS, IL-1ß, TNF-α, and IL-6. Additionally, MS significantly increased the proportion of CD11b+CD86+ and CD11b+CD206+ subsets in RAW264.7 macrophages. Furthermore, the protein expression of RhoA, ROCK1, ROCK2, NF-κB p65, and IκB-α increased in MS-treated RAW264.7 cells, as well as the IL-6 and iNOS. In contrast, ROCK inhibitor significantly blocked the activation of RhoA-ROCK and NF-κB pathway, decreased the protein expression of IL-6 and iNOS, reduced the proportion of CD11b+CD86+ cells subpopulation, and increased the proportion of CD11b+CD206+ cell subpopulation after MS. These data indicate that mechanical stretch can regulate the RAW264.7 macrophage polarization and enhance inflammatory responses in vitro, which may contribute to activation the RhoA-ROCK/NF-κB pathway.

Quercetin Attenuates Osteoporosis in Orchiectomy Mice by Regulating Glucose and Lipid Metabolism via the GPRC6A/AMPK/mTOR Signaling Pathway.

Quercetin, a flavonoid found in natural medicines, has shown a role in disease prevention and health promotion. Moreover, because of its recently identified contribution in regulating bone homeostasis, quercetin may be considered a promising agent for improving bone health. This study aimed to elucidate the role of quercetin in androgen deprivation therapy-induced osteoporosis in mice. C57BL/6 mice were subjected to orchiectomy, followed by quercetin treatment (75 and 150 mg/kg/d) for 8 weeks. Bone microstructure was then assessed by micro-computed tomography, and a three-point bending test was used to evaluate the biomechanical parameters. Hematoxylin and eosin (H&E) staining was used to examine the shape of the distal femur, gastrocnemius muscle, and liver. The balance motion ability in mice was evaluated by gait analysis, and changes in the gastrocnemius muscle were observed via Oil red O and Masson's staining. ELISA and biochemical analyses were used to assess markers of the bone, glucose, and lipid metabolism. Western blotting analyses of glucose and lipid metabolism-related protein expression was performed, and expression of the GPCR6A/AMPK/mTOR signaling pathway-related proteins was also assessed. After 8 weeks of quercetin intervention, quercetin-treated mice showed increased bone mass, bone strength, and improved bone microstructure. Additionally, gait analysis, including stride length and frequency, were significantly increased, whereas a reduction of the stride length and gait symmetry was observed. H&E staining of the gastrocnemius muscle showed that the cross-sectional area of the myofibers had increased significantly, suggesting that quercetin improves balance, motion ability, and muscle mass. Bone metabolism improvement was defined by a reduction of serum levels of insulin, triglycerides, total cholesterol, and low-density lipoprotein, whereas levels of insulin-like growth factor-1 and high-density lipoprotein were increased after quercetin treatment. Expression of proteins involved in glucose uptake was increased, whereas that of proteins involved in lipid production was decreased. Moreover, the GPRC6A and the phospho-AMPK/AMPK expression ratio was elevated in the liver and tibia tissues. In contrast, the phospho-mTOR/mTOR ratio was reduced in the quercetin group. Our findings indicate that quercetin can reduce the osteoporosis induced by testosterone deficiency, and its beneficial effects might be associated with the regulation of glucose metabolism and inhibition of lipid metabolism via the GPCR6A/AMPK/mTOR signaling pathway.

The Mechanisms Underlying the Beneficial Effects of Stem Cell-Derived Exosomes in Repairing Ischemic Tissue Injury.

Ischemic diseases are life-threatening, and the incidence increases as people's lifestyles change. Medications and surgical intervention offer limited benefit, and stem cell therapy has emerged as a potential approach for treating ischemic diseases. The exosomes secreted by stem cells have attracted more attention because they do not trigger the immune response and can be used as drug carriers. The non-coding RNA (ncRNA) carried by exosomes plays a key role in mediating exosome's beneficial effect, which can be further enhanced when combined with nanomaterials to improve its retention time. Here, we review the downstream target molecules and signal pathways of ncRNA and summarize recent advances of some nanomaterials used to encapsulate exosomes and promote ischemic tissue repair. We highlight the imprinting of exosomes from parent cells and discuss how the inflammasome pathway may be targeted for the development of novel therapy for ischemic diseases.

A new method for primary culture of microglia in rats with spinal cord injury.

At present, the primary culture method of microglia is complicated, and the culture of spinal cord microglia is rare, so we will explore to establish a new and efficient primary culture method of microglia in rats with spinal cord injury (SCI). The SCI model of SD rats was established by modified A11en's method, and the model of SCI was performed on 1 d, 3 d, 7 d and 14 d respectively. Then the injured spinal cord was removed, mechanically separated and filtered. The morphology of microglia was observed the next day and its purity was identified by CD11b and Iba1 immunofluorescence labeling. According to the above results, the morphological changes of microglia after 3 d of SCI were observed at 1 d, 2 d and 4 d. The results showed that the purity of microglia was 98%. The number of microglia after 3 d of SCI was the most. After SCI, the migration ability of microglia was enhanced, the number of microglia in the injured area increased, and the number was the highest at 3 d, then gradually decreased. In addition, the microglia after SCI would gradually change from active state to resting state with the passage of time. Therefore, we can use a simple and efficient mechanical separation method to extract primary microglia, which provides the basis for the study of microglia.

Cartilage Repair Using Clematis Triterpenoid Saponin Delivery Microcarrier, Cultured in a Microgravity Bioreactor Prior to Application in Rabbit Model.

Cartilage tissue engineering provides a promising method for the repair of articular cartilage defects, requiring appropriate biological scaffolds and necessary growth factors to enhance the efficiency of cartilage regeneration. Here, a silk fibroin (SF) microcarrier and a clematis triterpenoid saponin delivery SF (CTS-SF) microcarrier were prepared by the high-voltage electrostatic differentiation and lyophilization method, and chondrocytes were carried under the simulated microgravity condition by a rotating cell culture system. SF and CTS-SF microspheres were relatively uniform in size and had a porous structure with good swelling and cytocompatibility. Further, CTS-SF microcarriers could sustainably release CTSs in the monitored 10 days. Compared with the monolayer culture, chondrocytes under the microgravity condition maintained a better chondrogenic phenotype and showed better proliferation ability after culture on microcarriers. Moreover, the sustained release of CTS from CTS-SF microcarriers upregulated transforming growth factor-ß, Smad2, and Smad3 signals, contributing to promote chondrogenesis. Hence, the biophysical effects of microgravity and bioactivities of CTS-ST were used for chondrocyte expansion and phenotype maintenance in vitro. With prolonged expansion, SF- and CTS-SF-based microcarrier-cell composites were directly implanted in vivo to repair rabbit articular defects. Gross evaluations, histopathological examinations, and biochemical analysis indicated that SF- and CTS-SF-based composites exhibited cartilage-like tissue repair compared with the nontreated group. Further, CTS-SF-based composites displayed superior hyaline cartilage-like repair that integrated with the surrounding cartilage better and higher cartilage extracellular matrix content. In conclusion, these results provide an alternative preparation method for drug-delivered SF microcarrier and a culture method for maintaining the chondrogenic phenotype of seed cells based on the microgravity environment. CTS showed its bioactive function, and the application of CTS-SF microcarriers can help repair and regenerate cartilage defects.

Human Umbilical Cord Mesenchymal Stem Cell Derived Exosomes Delivered Using Silk Fibroin and Sericin Composite Hydrogel Promote Wound Healing.

Recent studies have shown that the hydrogels formed by composite biomaterials are better choice than hydrogels formed by single biomaterial for tissue repair. We explored the feasibility of the composite hydrogel formed by silk fibroin (SF) and silk sericin (SS) in tissue repair for the excellent mechanical properties of SF, and cell adhesion and biocompatible properties of SS. In our study, the SF SS hydrogel was formed by SF and SS protein with separate extraction method (LiBr dissolution for SF and hot alkaline water dissolution for SS), while SF-SS hydrogel was formed by SF and SS protein using simultaneous extraction method (LiBr dissolution for SF and SS protein). The effects of the two composite hydrogels on the release of inflammatory cytokines from macrophages and the wound were analyzed. Moreover, two hydrogels were used to encapsulate and deliver human umbilical cord mesenchymal stem cell derived exosomes (UMSC-Exo). Both SF SS and SF-SS hydrogels promoted wound healing, angiogenesis, and reduced inflammation and TNF-α secretion by macrophages. These beneficial effects were more significant in the experimental group treated by UMSC-Exo encapsulated in SF-SS hydrogel. Our study found that SF-SS hydrogel could be used as an excellent alternative to deliver exosomes for tissue repair.

Inhibition of LIM kinase reduces contraction and proliferation in bladder smooth muscle.

Overactive bladder (OAB) is the most bothersome symptom in lower urinary tract symptoms (LUTS). Current pharmacologic treatment aims to inhibit detrusor contraction; however, shows unsatisfied efficacy and high discontinuation rate. LIM kinases (LIMKs) promote smooth muscle contraction in the prostate; however, their function in the bladder smooth muscle remains unclear. Here, we studied effects of the LIMK inhibitors on bladder smooth muscle contraction and proliferation both in vitro and in vivo experiments. Bladder expressions of LIMKs are elevated in OAB rat detrusor tissues. Two LIMK inhibitors, SR7826 and LIMKi3, inhibit contraction of human detrusor strip, and cause actin filament breakdown, as well as cell proliferation reduction in cultured human bladder smooth muscle cells (HBSMCs), paralleled by reduced cofilin phosphorylation. Silencing of LIMK1 and LIMK2 in HBSMCs resulted in breakdown of actin filaments and decreased cell proliferation. Treatment with SR7826 or LIMKi3 decreased micturition frequency and bladder detrusor hypertrophy in rats with bladder outlet obstruction. Our study suggests that LIMKs may promote contraction and proliferation in the bladder smooth muscle, which could be inhibited by small molecule LIMK inhibitors. LIMK inhibitors could be a potential therapeutic strategy for OAB- related LUTS.

Icariin promotes the repair of PC12 cells by inhibiting endoplasmic reticulum stress.

BACKGROUND: Endoplasmic reticulum stress (ERS) is one of the main mechanisms of spinal cord injury (SCI) pathology and can affect the physiological state of neurons. Icariin (ICA), the main pharmacological component of Epimedium, can relieve the symptoms of patients with SCI and has obvious protective effects on neurons through ERS. METHODS: PC12 cells were induced to differentiate into neurons by nerve growth factor and identified by flow cytometry. Cell proliferation was detected by CCK8 method, cell viability was detected by SRB assay, apoptosis was detected by flow cytometry and microstructure of ER was observed by transmission electron microscope. Western blot was used to detect the protein expression of CHOP and Grp78, and qPCR was used to detect the mRNA expression of CHOP and Grp78. RESULTS: The results of CCK8, SRB and flow cytometry showed that ICA could relieve ERS and reduce apoptosis of PC12 cells. The results of transmission microscope showed that ICA could reduce apoptosis of PC12 cells caused by ERS. The results of Western blot and q-PCR showed that ICA could inhibit ERS by down-regulating the expression of CHOP and Grp78. CONCLUSIONS: ICA can inhibit ERS and promote the repair of PC12 cells by down-regulating the expression of CHOP and Grp78. ICA has the potential to promote the recovery of spinal cord injury.

Jisuikang Promotes the Repair of Spinal Cord Injury in Rats by Regulating NgR/RhoA/ROCK Signal Pathway.

Jisuikang (JSK) is an herbal formula composed of many kinds of traditional Chinese medicine, which has been proved to be effective in promoting the rehabilitation of patients with spinal cord injury (SCI) after more than ten years of clinical application. However, the mechanisms of JSK promoting nerve regeneration are yet to be clarified. The aim of this study was to investigate the effects of JSK protecting neurons, specifically the regulation of NgR/RhoA/ROCK signal pathway. The motor function of rats was evaluated by the BBB score and inclined plate test, Golgi staining and transmission electron microscope were used to observe the microstructure of nerve tissue, and fluorescence double-labeling method was used to detect neuronal apoptosis. In this study, we found that JSK could improve the motor function of rats with SCI, protect the microstructure (mitochondria, endoplasmic reticulum, and dendritic spine) of neurons, and reduce the apoptosis rate of neurons in rats with SCI. In addition, JSK could inhibit the expression of Nogo receptor (NgR) in neurons and the NgR/RhoA/ROCK signal pathway in rats with SCI. These results indicated JSK could improve the motor function of rats with SCI by inhibiting the NgR/RhoA/ROCK signal pathway, which suggests the potential applicability of JSK as a nerve regeneration agent.

[Research progress of different mechanical stimulation regulating chondrocytes metabolism].

As a kind of mechanical effector cells, chondrocytes can produce a variety of physical and chemical signals under the stimulation of multiaxial load in vivo, which affect their own growth, development and apoptosis. Therefore, simulating the mechanical environment in vivo has become a research hotspot in the culture of chondrocytes in vitro. Although a large number of reports have fully proved that different mechanical stimulation can regulate the metabolism of chondrocytes, the loading scheme has not been agreed. Starting from different mechanical forms, this review will explore the differences in the regulation of chondrocyte metabolism by different mechanical stimuli, so as to find an advantage scheme to promote the growth and proliferation of chondrocytes and to develop a more stable, effective and reliable experimental strategy.

Alteration of the gut microbiota by vinegar is associated with amelioration of hyperoxaluria-induced kidney injury.

Hyperoxaluria is well known to cause renal injury and end-stage kidney disease. Previous studies suggested that the renal function of rats with hyperoxaluria was improved after dietary vinegar intake. However, its underlying mechanisms remain largely unknown. The aim of the present study was to examine changes of gut microbiota and blood and urinary metabolites that associate with changes in kidney function to identify mechanisms involved with vinegar induced amelioration of hyperoxaluria-induced kidney injury. Using an ethylene glycol (EG)-induced hyperoxaluria rat model, we evaluated the effects of the vinegar on renal injury. Oral administration of vinegar (2 ml kg-1 day-1) reduced the elevated serum creatinine, BUN, and protected against hyperoxaluria-induced renal injury, renal fibrosis, and inflammation. Gut microbiota analysis of 16S rRNA gene in the hyperoxaluria-induced renal injury rats showed that vinegar treatment altered their microbial composition, especially the recovery of the levels of the Prevotella, Ruminiclostridium, Alistipes and Paenalcaligenes genus, which were significantly increased in the hyperoxaluria-induced renal injury rats. Additionally, liquid chromatography-mass spectrometry (LC-MS)-based metabolome analysis showed that total of 35 serum and 42 urine metabolites were identified to be associated with protective effects of vinegar on hyperoxaluria-induced renal injury rats. Most of these metabolites were involved in thiamine metabolism, glycerol phosphate shuttle, biotin metabolism, phosphatidylcholine biosynthesis and membrane lipid metabolism. Importantly, the effects of vinegar against renal injury were weakened after depletion of gut microbiota by antibiotic treatment. These results suggest that vinegar treatment ameliorates the hyperoxaluria-induced renal injury by improving the gut microbiota and metabolomic profiles.