How to Set Working Cannula in Endoscopic-Assisted Transforaminal Lumbar Interbody Fusion: A Morphometric Analysis Based on Computed Tomography.

OBJECTIVES: There is a high risk of nerve root injury during endoscopic-assisted transforaminal lumbar interbody fusion (Endo-TLIF). This study used computed tomography (CT) imaging to assess the relationship between the exiting nerve root and its surroundings, and the corresponding intervertebral disc. We also measured the approximate position and angle for the placement of the working cannula to reduce the risk of nerve root injury during Endo-TLIF procedures in the Chinese population. METHODS: This retrospective study was conducted at our institution between December 2021 and December 2022. A total of 115 patients suffering from low back pain were recruited for the study. For each participant, three-dimensional (3D) vertebral models of the lumbar segments from L3 to S1 were constructed based on their CT images. The nerve root-disc distance, cannula insertion bypass distance and angle, foraminal height and width, exiting nerve root height, and nerve root-pedicle distance were measured. A paired t-test was used to compare measurements between the left and right sides, while inter- and intraobserver reproducibility was assessed using the intraclass correlation coefficient (ICC). RESULTS: From L3/4 to L5/S1 segments, the ideal cannula insertion distance range was 37.51 ± 4.91-120.38 ± 37.71 mm at L3/4; 42.38 ± 5.29-116.25 ± 27.22 mm at L4/5; and 37.78 ± 4.86-69.26 ± 12.64 mm at L5/S1. The appropriate cannula insertion angle range was 30.86° ± 5.05°-62.59° ± 6.66° at L3/4; 34.30° ± 4.73°-60.88° ± 7.34° at L4/5; and 35.89° ± 4.18°-47.65° ± 7.38° at L5/S1. The height of the intervertebral foramen (IVF) gradually decreased, and the width steadily increased. The exiting nerve root height and the nerve root-pedicle distance slightly decreased caudally. CONCLUSION: From L3/4 to L5/S1, the range of working cannula insertion distance and angle gradually decreased, and the exiting nerve root height occupying the IVF gradually increased. Our measurement can reduce the risk of nerve root injury caused by inserting the working cannula during Endo-TLIF.

Exploring the tumor microenvironment: Chemokine-related genes and immunotherapy/chemotherapy response in clear-cell renal cell carcinoma.

BACKGROUND: The treatment of clear-cell renal cell carcinoma (ccRCC) remains challenge. Chemokines laid impact on the proliferation and metastasis of cancer cells. The objective was to identify the chemokine-related genes and construct a prognostic model for ccRCC. METHODS: Bulk transcriptomic data (n = 531), single-cell RNA sequencing (scRNA-seq) dataset GSE159115, and other validation cohorts were acquired from the Cancer Genome Atlas Program (TCGA) and GEO databases. All clustering analysis was conducted by Seurat R package. Gene set enrichment analysis (GSEA), immune infiltration analysis, single nucleotide variations (SNV) analysis, and predictive response analysis of immunotherapy/chemotherapy were conducted. 786-O and A498 cell lines were cultured and applied into CCK-8, Western blot, and RT-qPCR kits. RESULTS: Univariate Cox analysis was used to screen out chemokine-related genes related to survival. ZIC2, SMIM24, COL7A1, IGF2BP3, ITPKA, ADAMTS14, CYP3A7, and AURKB were identified and applied for the construction of the prognostic model. High-risk group had a poorer prognosis than the low-risk group in each dataset. Memory CD8+ T cells, macrophages, and memory B cells were higher in the high-risk group, while the content of basophils was higher in the low-risk group. Bortezomib_1191, Dactinomycin_1911, Docetaxel_1007, and Daporinad_1248 were more sensitive to high-risk groups than low-risk groups. Moreover, we found that IGF2BP3 significantly elevated in both 786-O and A498 cell lines resistance to sunitinib. Knockdown of IGF2BP3 markedly reduced ccRCC cell migration and viability. CONCLUSION: Our study has yielded a novel prognostic model of chemokine-related genes based on comprehensive transcriptional atlas of ccRCC patients, shedding light on the significant impact of the tumor microenvironment on biology and immunotherapy response of ccRCC. We identified IGF2BP3 as a pivotal regulator in regulating ccRCC resistance to sunitinib.

Naringenin reduces oxidative stress and necroptosis, apoptosis, and pyroptosis in random-pattern skin flaps by enhancing autophagy.

BACKGROUND: Random skin flap grafting is one of the most commonly used techniques in plastic and orthopedic surgery. However, necrosis resulting from ischemia and ischemia-reperfusion injury in the distal part of the flap can severely limit the clinical application of the flap. Studies have revealed that naringenin reduces pyroptosis, apoptosis, and necroptosis, inhibits oxidative stress, and promotes autophagy. In this study, the effects of Naringenin on flap viability and its underlying mechanism were evaluated. METHODS: Mice with random skin flaps were randomly allocated to control, Naringenin, and Naringenin + 3-methyladenine groups. On postoperative day 7, flap tissues were collected to estimate angiogenesis, necroptosis, apoptosis, pyroptosis, oxidative stress, and autophagy via hematoxylin and eosin staining, immunofluorescence, and immunohistochemistry. RESULTS: The results revealed that naringenin promoted the viability of the random flaps as well as angiogenesis, while inhibiting oxidative stress and decreasing pyroptosis, apoptosis, and necroptosis. These effects were reversed by the autophagy inhibitor 3-methyladenine. CONCLUSIONS: The findings indicated that naringenin treatment could promote flap survival by inhibiting pyroptosis, apoptosis, necroptosis, and alleviating oxidative stress, caused by the activation of autophagy.

UBC9-mediated SUMOylation of Lamin B1 enhances DNA-damage-induced nuclear DNA leakage and autophagy after spinal cord injury.

Recent studies have shown that nucleophagy can mitigate DNA damage by selectively degrading nuclear components protruding from the nucleus. However, little is known about the role of nucleophagy in neurons after spinal cord injury (SCI). Western blot analysis and immunofluorescence were performed to evaluate the nucleophagy after nuclear DNA damage and leakage in SCI neurons in vivo and NSC34 expression in primary neurons cultured with oxygen-glucose deprivation (OGD) in vitro, as well as the interaction and colocalization of autophagy protein LC3 with nuclear lamina protein Lamin B1. The effect of UBC9, a Small ubiquitin-related modifier (SUMO) E2 ligase, on Lamin B1 SUMOylation and nucleophagy was examined by siRNA transfection or 2-D08 (a small-molecule inhibitor of UBC9), immunoprecipitation, and immunofluorescence. In SCI and OGD injured NSC34 or primary cultured neurons, neuronal nuclear DNA damage induced the SUMOylation of Lamin B1, which was required by the nuclear Lamina accumulation of UBC9. Furthermore, LC3/Atg8, an autophagy-related protein, directly bound to SUMOylated Lamin B1, and delivered Lamin B1 to the lysosome. Knockdown or suppression of UBC9 with siRNA or 2-D08 inhibited SUMOylation of Lamin B1 and subsequent nucleophagy and protected against neuronal death. Upon neuronal DNA damage and leakage after SCI, SUMOylation of Lamin B1 is induced by nuclear Lamina accumulation of UBC9. Furthermore, it promotes LC3-Lamin B1 interaction to trigger nucleophagy that protects against neuronal DNA damage.

Pathogenomics model for personalized medicine in cervical cancer: Cross-talk of gene expressions and pathological images related to oxidative stress.

An increasing number of studies have shown that oxidative stress plays an important role in the development and progression of cancer. Cervical cancer (CC) is a disease of unique complexity that tends to exhibit high heterogeneity in molecular phenotypes. We aim here to characterize molecular features of cervical cancer by developing a classification system based on oxidative stress-related gene expression profiles. In this study, we obtained gene expression profiling data for cervical cancer from the TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus) (GSE44001) databases. Oxidative stress-related genes used for clustering were obtained from GeneCards. Patients with cervical cancer were divided into two subtypes (C1 and C2) by non-negative matrix factorization (NMF) classification. By performing Kaplan-Meier survival analysis, differential expression analysis, and gene set enrichment analysis (GSEA) between the two subtypes, we found that subtype C2 had a worse prognosis and was highly enriched for immune-related pathways as well as epithelial-mesenchymal transition (EMT) pathways. Subsequently, we performed metabolic pathway analysis, gene mutation landscape analysis, immune microenvironment analysis, immunotherapy response analysis, and drug sensitivity analysis of the two isoforms. The results showed that the isoforms were significantly different between metabolic pathway enrichment and the immune microenvironment, and the chromosomes of subtype C1 were more unstable. In addition, we found that subtype C2 tends to respond to treatment with anti-CTLA4 agents, a conclusion that coincides with high chromosomal variation in C1, as well as C2 enrichment of immune-related pathways. Then, we screened 10 agents that were significantly susceptible to C2 subtype. Finally, we constructed pathogenomics models based on pathological features and linked them to molecular subtypes. This study establishes a novel CC classification based on gene expression profiles of oxidative stress-related genes and elucidates differences between immune microenvironments between CC subtypes, contributing to subtype-specific immunotherapy and drug therapy.

SUMOylation of Rho-associated protein kinase 2 induces goblet cell metaplasia in allergic airways.

Allergic asthma is characterized by goblet cell metaplasia and subsequent mucus hypersecretion that contribute to the morbidity and mortality of this disease. Here, we explore the potential role and underlying mechanism of protein SUMOylation-mediated goblet cell metaplasia. The components of SUMOylaion machinery are specifically expressed in healthy human bronchial epithelia and robustly upregulated in bronchial epithelia of patients or mouse models with allergic asthma. Intratracheal suppression of SUMOylation by 2-D08 robustly attenuates not only allergen-induced airway inflammation, goblet cell metaplasia, and hyperreactivity, but IL-13-induced goblet cell metaplasia. Phosphoproteomics and biochemical analyses reveal SUMOylation on K1007 activates ROCK2, a master regulator of goblet cell metaplasia, by facilitating its binding to and activation by RhoA, and an E3 ligase PIAS1 is responsible for SUMOylation on K1007. As a result, knockdown of PIAS1 in bronchial epithelia inactivates ROCK2 to attenuate IL-13-induced goblet cell metaplasia, and bronchial epithelial knock-in of ROCK2(K1007R) consistently inactivates ROCK2 to alleviate not only allergen-induced airway inflammation, goblet cell metaplasia, and hyperreactivity, but IL-13-induced goblet cell metaplasia. Together, SUMOylation-mediated ROCK2 activation is an integral component of Rho/ROCK signaling in regulating the pathological conditions of asthma and thus SUMOylation is an additional target for the therapeutic intervention of this disease.

Thymoquinone has a synergistic effect with PHD inhibitors to ameliorate ischemic brain damage in mice.

BACKGROUND: A blockage in a blood vessel can cause reduced blood flow to the brain, which eventually leads to the death of surrounding tissue. Several studies have attempted to develop an effective intervention to reverse this process and improve the health status of affected individuals. Due to its indirect effect on cellular functions and metabolism, the hypoxia-inducible factor (HIF-1α) protein has been proposed as a promising transcription factor in the treatment of stroke. PURPOSE: The current study aims to explore the relation between HIF-1 α and thymoquinone (TQ) in the attenuation of ischemic brain damage and the possible mechanism of this relation to reduce cell death. METHODS: For this purpose, dimethyloxallyl glycine (DMOG), 8 mg/kg, Acriflavine (ACF), 1.5 mg/kg, and both combined with TQ (5 mg/kg) were assessed. Male C57 mice were used to establish an ischemic stroke model by using endothelin-1 (ET-1) (400 pmole/µl) intra- cranial injection. The ultrastructure alterations of neuronal soma, axons, and mitochondria after stroke and treatment were well addressed. Besides, the expression levels of VEGF, HIF-1α, Nrf2, and HO-1 were evaluated. Meanwhile, apoptosis and autophagy-related proteins were also investigated. RESULTS: Treatment of ischemic stroke by TQ can activate the HIF-1α pathway and its downstream genes such as VEGF, TrkB, and PI3K, which in turn enhance angiogenesis and neurogenesis. Our study revealed that TQ has the same effect as DMOG to activate HIF-1 α and can improve motor dysfunction after ischemic stroke. Further, we demonstrated that both TQ and DMOG effectively attenuate the organelle's damage following ischemic stroke, which was confirmed by the cryogenic transmission electron microscope. The synergistic effect of TQ and DMOG may lead to a chemo-modulation action in the autophagy process after stroke onset and this result is validated by the western blot and rt-qPCR techniques. CONCLUSION: Our finding revealed the potential role of TQ as a HIF-1 α activator to reduce cell death, modulate autophagy and decrease the infarct volume after ischemic stroke onset. The neuroprotective effect of TQ is achieved by decreasing the inflammation and increasing angiogenesis as well as neurogenesis via induction of the HIF-1α-VEGF/Nrf2-HO-1-TrkB-PI3K pathway.

Probiotic-fermented Pueraria lobata (Willd.) Ohwi alleviates alcoholic liver injury by enhancing antioxidant defense and modulating gut microbiota.

BACKGROUND: Pueraria lobata (Willd.) Ohwi (PL) has been used in China to detoxify alcohol and protect the liver for millennia, though its mechanism of liver protection has not been elucidated. However, fermentation is considered to be one of the effective ways to enhance the efficacy of traditional Chinese medicine. The aim of this study was to investigate the hepatoprotective mechanism of probiotic-fermented PL (FPL). Sprague Dawley rats were administered with FPL followed by gavage of alcohol for seven consecutive days; following that, liver injury levels were evaluated in rats. RESULTS: FPL ameliorated lipid accumulation and inflammation levels in rats. Meanwhile, the levels of ethanol dehydrogenase, acetaldehyde dehydrogenase, and cytochrome P4502E1 were elevated by FPL treatment. It was observed that the levels of catalase, superoxide dismutase, and glutathione peroxidase were elevated, and the expression of nuclear transcriptional factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 genes and proteins were increased by FPL treatment, demonstrating that the Nrf2-mediated signal pathway was activated. Furthermore, FPL restored the composition of the gut microbiota with an increase in the abundances of Firmicutes and Lactobacillus and a decrease in the abundances of Bacteroidota and Akkermansia. Additionally, a strong correlation was found between the gut microbiota and the antioxidant parameters. CONCLUSION: The results indicate that FPL possesses an excellent protective effect in alcoholic liver injury. Our findings are beneficial to the development of hepatoprotective nutraceuticals for alcoholics. © 2022 Society of Chemical Industry.

T851I mutation of human large tumor suppressor 1 disrupts its kinase activity and tumor-suppressor functions.

AIM: Large tumor suppressor 1 (LATS1) is a Ser/Thr kinase to mediate Hippo signaling pathway and plays a pivotal role in tumor suppression. By searching the COSMIC database, we found a somatic missense mutation (NM_004690.4:c.2552C>T) of human LATS1 (NP_004681.1:p.851T>I) in two colorectal cancer cell lines, and investigated the role and underlying mechanism of this mutation in the colorectal tumorigenesis. MAIN METHODS: We performed structural and biochemistry analyses to investigate the role of LATS1 T851I mutation in Hippo signaling activation and used the mouse xenograft model to assess the role of this mutation in the colorectal tumorigenesis. KEY FINDINGS: By structural and biochemistry approaches, we propose that T851 is an active residue other than Ser909 on the activation loop and is essential for LATS1 phosphorylation and kinase activity. We then reveal that T851I mutation in LATS1 not only destabilizes the phospho-Thr1079-LATS1, a prerequisite of LATS1 kinase activity, but also reduces its binding to the downstream effectors, YAP and TAZ. As a result, T851I mutation in LATS1 attenuates Hippo signaling and decreases its tumor-suppressor functions in the colorectal cancer. SIGNIFICANCE: The present study identifies the T851 as an essential residue for LATS1 kinase activity and uncovers the T851I mutation of LATS1 and consequent Hippo signaling suppression as a hitherto uncharacterized mechanism controlling colorectal tumorigenesis.

Does anxiety influence the prognosis of percutaneous transforaminal endoscopic discectomy in the treatment of lumbar disc herniation? A preliminary propensity score matching analysis.

PURPOSE: Previous reports revealed a correlation between psychological problems and spinal surgery. There is a lack of knowledge on the effect of anxiety on the percutaneous transforaminal endoscopic discectomy (PTED) outcome at the two year follow-up. The purpose of this study is to investigate changes in anxiety after PTED among patients with lumbar disc herniation (LDH), to compare the effect of anxiety on the prognosis using propensity score matching analysis, and to identify the related parameters of anxiety. METHODS: A total of 145 patients with LDH requiring PTED surgery were included. Twenty-six LDH patients with anxiety were matched with 26 control patients utilizing propensity score matching analysis. The demographic and peri-operative data were collected and analyzed. A correlation analysis was utilized. RESULTS: Both groups achieved significant improvements in visual analogue scale (VAS) scores for pain, Japanese Orthopedic Association (JOA) scores for neurological deficit, and 36-item Short-Form Health Survey (SF-36) scores and Oswestry Disability Index (ODI) scores for quality of life. A statistical difference was detected between the pre-operative and the post-operative Zung Self-Rating Anxiety Scale scores in the anxiety cohort. However, the difference between the anxiety group and the control group was statistically significant in the aforementioned parameters. The VAS, JOA, ODI and the SF-36 scores, and the disease duration were associated with pre-operative anxiety. CONCLUSION: PTED may provide significant improvements in clinical outcomes and symptoms of anxiety. A negative impact on the patient's prognosis may be caused by the presence of anxiety. Pain severity, neurological deficit, disease duration, and quality of life were associated with anxiety.

Hypoxia inducible factor-1 (HIF-1α) reduced inflammation in spinal cord injury via miR-380-3p/ NLRP3 by Circ 0001723

BACKGROUND: Spinal cord injury (SCI) is a severe central nervous system trauma. The present study aimed to evaluate the effect of HIF-1α on inflammation in spinal cord injury (SCI) to uncover the molecular mechanisms of anti-inflammation. RESULTS: HIF-1α was reduced in SCI model rats and HIF-1α activation reduced TNF-α, IL-1ß, IL-6 and IL-18 levels in SCI model rats. Meanwhile, Circ 0001723 expression was down-regulated and miR-380-3p expression was up-regulated in SCI model rats. In vitro model, down-regulation of Circ 0001723 promoted TNF-α, IL-1ß, IL-6 and IL-18 levels, compared with control negative group. However, over-expression of Circ 0001723 reduced TNF-α, IL-1ß, IL-6 and IL-18 levels in vitro model. Down-regulation of Circ 0001723 suppressed HIF-1α protein expressions and induced NLRP3 and Caspase-1 protein expressions in vitro model by up-regulation of miR-380-3p. Next, inactivation of HIF-1α reduced the pro-inflammation effects of Circ 0001723 in vitro model. Then, si-NLRP3 also inhibited the pro-inflammation effects of Circ 0001723 in vitro model via promotion of autophagy. CONCLUSIONS: We concluded that HIF-1α reduced inflammation in spinal cord injury via miR-380-3p/ NLRP3 by Circ 0001723.

CRTH2 antagonist, CT­133, effectively alleviates cigarette smoke-induced acute lung injury.

AIMS: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), characterized by overwhelming lung inflammation, are associated with high mortality. Cigarette smoke (CS) is one of the major causes of ALI/ARDS. Since high expression of prostaglandin (PG) D2 has been observed in CS-induced lung injury. Currently, no effective pharmacological therapies are available to treat ALI, and supportive therapies remain the mainstay of treatment. Therefore, we investigated the protective effect of CT­133, a newly discovered selective CRTH2 antagonist, on CS-induced ALI in vivo and in vitro. MAIN METHODS: CT­133 (10 and 30 mg/kg), dexamethasone (1 mg/kg) and normal saline were intratracheally administrated 1 hr prior to whole-body CS-exposure for seven consecutive days to study the key characteristics of ALI. Subsequently, CSE (4%)- and PGD2-stimulated RAW 264.7 macrophages were used to evaluate the protective effect of CT­133. KEY FINDINGS: CT­133 remarkably attenuated infiltration of inflammatory cells, neutrophils, and macrophages in the BALF, albumin contents, expression of IL­1ß, IL­6, TNF­α and KC, lung myeloperoxidase (MPO) activity and lung histopathological alterations caused by CS exposure in mice. Moreover, CT­133 not only reversed the uncontrolled secretion of IL­1ß, IL-6, TNF­α and KC from CSE- and PGD2-stimulated RAW 264.7 macrophages but also augmented IL-10 production in both in vivo and in vitro studies. Additionally, CT­133 alleviated in vitro neutrophil migration chemoattracted by PGD2. SIGNIFICANCE: Our results provide the first evidence that targeting CRTH2 could be a new potential therapeutic option to treat CS-induced ALI.

High expression of Sonic hedgehog in allergic airway epithelia contributes to goblet cell metaplasia.

Sonic hedgehog (SHH) is abundantly expressed and critical for morphogenesis in embryonic lungs; however, SHH expression drops to a much lower level in mice from E17.5 and in humans from the 21st gestational week. We find that SHH expression is robustly upregulated in the airway epithelia of children with asthma or mouse models with allergic airway disease. Specifically, airway-specific SMO loss of function significantly suppresses allergen-induced goblet cell phenotypes, whereas an airway-specific SMO gain of function markedly enhances the goblet cell phenotypes in mouse models with allergic airway disease. Notably, intratracheal administration with SHH-neutralizing antibody or cyclopamine robustly attenuates goblet cell phenotypes in mouse models with allergic airway disease. Finally, we identify that Muc5AC gene encoding MUC5AC mucin serves as a direct target of GLI transcriptional factors in response to SHH, whereas the SAM-pointed domain-containing ETS transcription factor and Forkhead box A2, critical transcriptional factors for goblet cell phenotypes, both function as the effectors of GLIs in response to SHH stimulation. Together, the upregulation of SHH expression in allergic bronchial epithelia contributes to goblet cell metaplasia; thus, blockage of SHH signaling is a rational approach in a therapeutic intervention of epithelial remodeling in chronic airway diseases.

Cdc42 Is Essential for Both Articular Cartilage Degeneration and Subchondral Bone Deterioration in Experimental Osteoarthritis.

Cdc42, a member of Rho family small guanosine triphosphatases (GTPases), is critical for cartilage development. We investigated the roles of Cdc42 in osteoarthritis and explored the potential mechanism underlying Cdc42-mediated articular cartilage degeneration and subchondral bone deterioration. Cdc42 is highly expressed in both articular cartilage and subchondral bone in a mouse osteoarthritis model with surgical destabilization of the medial meniscus (DMM) in the knee joints. Specifically, genetic disruption of Cdc42, knockdown of Cdc42 expression, or inhibition of Cdc42 activity robustly attenuates the DMM-induced destruction, hypertrophy, high expression of matrix metallopeptidase-13 and collagen X, and activation of Stat3 in articular cartilages. Notably, genetic disruption of Cdc42, knockdown of Cdc42 expression or inhibition of Cdc42 activity significantly restored the increased numbers of mesenchymal stem cells, osteoprogenitors, osteoblasts, osteoclasts, and neovascularized vessels, the increased bone mass, and the activated Erk1/2, Smad1/5 and Smad2 in subchondral bone of DMM-operated mice. Mechanistically, Cdc42 mediates interleukin-1ß-induced interleukin-6 production and subsequent Jak/Stat3 activation to regulate chondrocytic inflammation, and also lies upstream of Erk/Smads to regulate subchondral bone remodeling during transform growth factor-ß1 signaling. Cdc42 is apparently required for both articular cartilage degeneration and subchondral bone deterioration of osteoarthritis, thus, interventions targeting Cdc42 have potential in osteoarthritic therapy. © 2018 American Society for Bone and Mineral Research.

Inhibition of Myosin Light-Chain Kinase Enhances the Clearance of Lipopolysaccharide-Induced Lung Inflammation Possibly by Accelerating Neutrophil Apoptosis.

Neutrophils are a population of inflammatory cells involved in acute lung injury (ALI), and lipopolysaccharide (LPS)-induced prolonged neutrophil survival and delayed neutrophil apoptosis hinder the alleviation of lung inflammation. Myosin light-chain kinase (MLCK) involved the RhoA/Rho kinase signaling pathway responsible for the cytoskeletal arrangement, and previous studies have revealed that inhibition of MLCK induces apoptosis in vitro and in vivo. In this study, glycogen-induced neutrophils isolated from rats or mice were incubated with ML-7, a MLCK-specific inhibitor, and LPS-induced ALI mice administrated with ML-7 were investigated, to demonstrate the roles of MLCK in neutrophil apoptosis as well as its possibility of contributing to the clearance of inflammation. We found that ML-7 dramatically promoted neutrophil apoptosis that possibly signal through the p38 to upregulate the expression of the apoptotic proteins caspase-9 and B-cell lymphoma 2 and to downregulate the expression of the antiapoptotic protein Bcl-2-associated X protein and myeloid cell leukemia-1. In mice, ML-7 accelerated the clearance of inflammation in LPS-induced ALI through attenuating neutrophil accumulation, histopathological changes, and pulmonary edema. ML-7 promoted elimination of inflammation possibly by accelerating neutrophil apoptosis and macrophage-mediated clearance. Moreover, ML-7 also reduced the LPS-induced production of proinflammatory cytokines interleukin-1ß and tumor necrosis factor-α, and the activity of myeloperoxidase. Taken together, the present study uncovers a hitherto uncharacterized role of MLCK in neutrophil apoptosis that contributes to the alleviation of inflammation in response to LPS.

Ginkgolide B functions as a determinant constituent of Ginkgolides in alleviating lipopolysaccharide-induced lung injury.

Ginkgolides are the major bioactive components of Ginkgo biloba extracts, however, the exact constituents of Ginkgolides contributing to their pharmacological effects remain unknown. Herein, we have determined the anti-inflammatory effects of Ginkgolide B (GB) and Ginkgolides mixture (GM) at equivalent dosages against lipopolysaccharide (LPS)-induced inflammation. RAW 264.7 cell culture model and mouse model of LPS-induced lung injury were used to evaluate in vitro and in vivo effects of GB and GM, respectively. In RAW 264.7 cells, GB and GM at equivalent dosages exhibit an identical capacity to attenuate LPS-induced inducible nitric oxide synthase mRNA and protein expression and subsequent NO production. Likewise, GB and GM possess almost the same potency in attenuating LPS-induced expression and activation of nuclear factor kappa B (p65) and subsequent increases in tumor necrosis factor-α mRNA levels. In LPS-induced pulmonary injury, GB and GM at the equivalent dosages have equal efficiency in attenuating the accumulation of inflammatory cells, including neutrophils, lymphocytes, and macrophages, and in improving the histological damage of lungs. Moreover, GB and GM at equivalent dosages decrease the exudation of plasma protein to the same degree, whereas GM is superior to GB in alleviating myeloperoxidase activities. Finally, though GB and GM at equivalent dosages appear to reduce LPS-induced IL-1ß mRNA and protein levels and IL-10 protein levels to the same degree, GM is more potent than GB to attenuate the IL-10 mRNA levels. Taken together, this study demonstrates that GB functions as the determinant constituent of Ginkgolides in alleviating LPS-induced lung injury.

Smoothened-independent activation of hedgehog signaling by rearranged during transfection promotes neuroblastoma cell proliferation and tumor growth.

BACKGROUND: Rearranged during transfection (RET) proto-oncogene encodes a receptor tyrosine kinase for glial cell line-derived neurotrophic factor (GDNF) signaling, and high RET expression is closely related to the tumorigenesis and malignancy of neuroblastoma(NB). METHODS: We have investigated whether RET signals through hedgehog (HH) pathway in NB cell proliferation and tumor growth by in vitro cell culture and in vivo xenograft approaches. RESULTS: The key members of both GDNF/RET and HH/GLI pathways are expressed in NB cell lines to different extents. Knockdown of RET in NB cells significantly attenuates the activity of HH signaling, whereas overexpression of RET robustly enhances the output of transcriptional activation by HH. Likewise, activation of RET by GDNF induces HH signaling, whereas knockdown of RET attenuates both basal and GDNF-induced activities of HH signaling. Moreover, protein kinase B lies on the downstream of GDNF/RET signaling module to inhibit the GSK3ß, resulting in activation of HH signaling. Furthermore, either knockdown of RET by shRNA or inhibition of HH pathway by cyclopamine attenuates not only basal but also GDNF-induced proliferation of SH-SY5Y cells, and knockdown of either RET or smoothened in SH-SY5Y cell xenografts significantly attenuated the tumor growth. Finally, inhibition of HH signaling by GLI1 and GLI2 inhibitor, Gant61, reduces not only basal but also RET-induced proliferation of SH-SY5Y cells and outgrowth of xenografts. CONCLUSION: GDNF/RET/AKT/GSK3ß signaling module activates HH pathway to stimulate NB cells proliferation and tumor outgrowth. GENERAL SIGNIFICANCE: Targeting HH pathway is a rational approach for therapeutic intervention of NB with high RET expression.

Phosphodiesterase 5/protein kinase G signal governs stemness of prostate cancer stem cells through Hippo pathway.

Cancer stem cells (CSC) are critical for initiation, metastasis, and relapse of cancers, however, the underlying mechanism governing stemness of CSC remains unknown. Herein, we have investigated the roles of phosphodiesterase 5 (PDE5) in stemness of prostate cancer cells. Both PDE5 and WW domain-containing transcription regulator protein-1 (TAZ), a core effector of Hippo pathway, are highly expressed in the PC3-derived cancer stem cells (PCSC). Either TAZ knockdown or inhibition of PDE5 activity attenuated colony formation, altered expression patterns of stem cell markers, and enhanced cisplatin cytotoxicity, resulting in attenuation of stemness in PCSC. In addition, inhibition of PDE5 activity by its specific inhibitors activates cGMP-dependent protein kinase G (PKG), which in turn induces MST/LATS kinases, resulting in cytosolic degradation of TAZ and activation of Hippo pathway. Accordingly, knockdown of TAZ almost completely abolished PDE5 inhibitor-induced attenuation in stemness in cultured PCSC, whereas knockdown of TAZ not only abolished PDE5 inhibitor-induced attenuation in stemness but also facilitated PDE5 inhibitor-induced trans-differentiation in PCSC xenografts. Together, the present study has uncovered that PDE/cGMP/PKG signal targets to Hippo/TAZ pathway in maintaining stemness of PCSC, and suggested that PDE5 inhibitors in combination with chemotherapeutic agents could effectively prevent initiation, metastasis, and relapse of prostate cancer.

Glioma-associated Oncogene 2 Is Essential for Trophoblastic Fusion by Forming a Transcriptional Complex with Glial Cell Missing-a.

Cell-cell fusion of human villous trophoblasts, referred to as a process of syncytialization, acts as a prerequisite for the proper development and functional maintenance of the human placenta. Given the fact that the main components of the Hedgehog signaling pathway are expressed predominantly in the syncytial layer of human placental villi, in this study, we investigated the potential roles and underlying mechanisms of Hedgehog signaling in trophoblastic fusion. Activation of Hedgehog signaling by a variety of approaches robustly induced cell fusion and the expression of syncytial markers, whereas suppression of Hedgehog signaling significantly attenuated cell fusion and the expression of syncytial markers in both human primary cytotrophoblasts and trophoblast-like BeWo cells. Moreover, among glioma-associated oncogene (GLI) family transcriptional factors in Hedgehog signaling, knockdown of GLI2 but not GLI1 and GLI3 significantly attenuated Hedgehog-induced cell fusion, whereas overexpression of the GLI2 activator alone was sufficient to induce cell fusion. Finally, GLI2 not only stabilized glial cell missing-a, a pivotal transcriptional factor for trophoblastic syncytialization, but also formed a transcriptional heterodimer with glial cell missing-a to transactivate syncytin-1, a trophoblastic fusogen, and promote trophoblastic syncytialization. Taken together, this study uncovered a so far uncharacterized role of Hedgehog/GLI2 signaling in trophoblastic fusion, implicating that Hedgehog signaling, through GLI2, could be required for human placental development and pregnancy maintenance.