BeM(CO)3- (M = Co, Rh, Ir) and BeM(CO)3 (M = Ni, Pd, Pt): Triply bonded terminal beryllium in zero oxidation state.

We perform detailed potential energy surface explorations of BeM(CO)3- (M = Co, Rh, Ir) and BeM(CO)3 (M = Ni, Pd, Pt) using both single-reference and multireference-based methods. The present results at the CASPT2(12,12)/def2-QZVPD//M06-D3/def2-TZVPPD level reveal that the global minimum of BeM(CO)3- (M = Co, Rh, Ir) and BePt(CO)3 is a C3v symmetric structure with an 1A1 electronic state, where Be is located in a terminal position bonded to M along the center axis. For other cases, the C3v symmetric structure is a low-lying local minimum. Although the present complexes are isoelectronic with the recently reported BFe(CO)3- complex having a B-Fe quadruple bond, radial orbital-energy slope (ROS) analysis reveals that the highest occupied molecular orbital (HOMO) in the title complexes is slightly antibonding in nature, which bars a quadruple bonding assignment. Similar weak antibonding nature of HOMO in the previously reported BeM(CO)4 (M = Ru, Os) complexes is also noted in ROS analysis. The bonding analysis through energy decomposition analysis in combination with the natural orbital for chemical valence shows that the bonding between Be and M(CO)3q (q = -1 for M = Co, Rh, Ir and q = 0 for M = Ni, Pd, Pt) can be best described as Be in the ground state (1S) interacting with M(CO)30/- via dative bonds. The Be(spσ) → M(CO)3q σ-donation and the complementary Be(spσ) ← M(CO)3q σ-back donation make the overall σ bond, which is accompanied by two weak Be(pπ) ← M(CO)3q π-bonds. These complexes represent triply bonded terminal beryllium in an unusual zero oxidation state.

[Ginsenoside Re regulates mitochondrial biogenesis through Nrf2/HO-1/PGC-1α pathway to reduce hypoxia/reoxygenation injury in H9c2 cells].

This article explored the mechanism by which ginsenoside Re reduces hypoxia/reoxygenation(H/R) injury in H9c2 cells by regulating mitochondrial biogenesis through nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)/peroxisome prolife-rator-activated receptor gamma coactivator-1α(PGC-1α) pathway. In this study, H9c2 cells were cultured in hypoxia for 4 hours and then reoxygenated for 2 hours to construct a cardiomyocyte H/R injury model. After ginsenoside Re pre-administration intervention, cell activity, superoxide dismutase(SOD) activity, malondialdehyde(MDA) content, intracellular reactive oxygen species(Cyto-ROS), and intramitochondrial reactive oxygen species(Mito-ROS) levels were detected to evaluate the protective effect of ginsenoside Re on H/R injury of H9c2 cells by resisting oxidative stress. Secondly, fluorescent probes were used to detect changes in mitochondrial membrane potential(ΔΨ_m) and mitochondrial membrane permeability open pore(mPTP), and immunofluorescence was used to detect the expression level of TOM20 to study the protective effect of ginsenoside Re on mitochondria. Western blot was further used to detect the protein expression levels of caspase-3, cleaved caspase-3, Cyto C, Nrf2, HO-1, and PGC-1α to explore the specific mechanism by which ginsenoside Re protected mitochondria against oxidative stress and reduced H/R injury. Compared with the model group, ginse-noside Re effectively reduced the H/R injury oxidative stress response of H9c2 cells, increased SOD activity, reduced MDA content, and decreased Cyto-ROS and Mito-ROS levels in cells. Ginsenoside Re showed a good protective effect on mitochondria by increasing ΔΨ_m, reducing mPTP, and increasing TOM20 expression. Further studies showed that ginsenoside Re promoted the expression of Nrf2, HO-1, and PGC-1α proteins, and reduced the activation of the apoptosis-related regulatory factor caspase-3 to cleaved caspase-3 and the expression of Cyto C protein. In summary, ginsenoside Re can significantly reduce I/R injury in H9c2 cells. The specific mechanism is related to the promotion of mitochondrial biogenesis through the Nrf2/HO-1/PGC-1α pathway, thereby increasing the number of mitochondria, improving mitochondrial function, enhancing the ability of cells to resist oxidative stress, and alleviating cell apoptosis.

[Tetrahydropalmatine inhibiting mitophagy through ULK1/FUNDC1 pathway to alleviate hypoxia/reoxygenation injury in H9c2 cells].

This study explored the specific mechanism by which tetrahydropalmatine(THP) inhibited mitophagy through the UNC-51-like kinase 1(ULK1)/FUN14 domain containing 1(FUNDC1) pathway to reduce hypoxia/reoxygenation(H/R) injury in H9c2 cells. This study used H9c2 cells as the research object to construct a cardiomyocyte H/R injury model. First, a cell viability detection kit was used to detect cell viability, and a micro-method was used to detect lactate dehydrogenase(LDH) leakage to evaluate the protective effect of THP on H/R injury of H9c2 cells. In order to evaluate the protective effect of THP on mitochondria, the chemical fluorescence method was used to detect intracellular reactive oxygen species, intramitochondrial reactive oxygen species, mitochondrial membrane potential, and autophagosomes, and the luciferin method was used to detect intracellular adenosine 5'-triphosphate(ATP) content. Western blot was further used to detect the ratio of microtubule-associated protein 1 light chain 3(LC3) membrane type(LC3-Ⅱ) and slurry type(LC3-Ⅰ) and activated cleaved caspase-3 expression level. In addition, ULK1 expression level and its phosphorylation degree at Ser555 site, as well as the FUNDC1 expression level and its phosphorylation degree of Ser17 site were detected to explore its specific mechanism. The results showed that THP effectively reduced mitochondrial damage in H9c2 cells after H/R. THP protected mitochondria by reducing the level of reactive oxygen species in cells and mitochondria, increasing mitochondrial membrane potential, thereby increasing cellular ATP production, enhancing cellular activity, reducing cellular LDH leakage, and finally alleviating H/R damage in H9c2 cells. Further studies have found that THP could reduce the production of autophagosomes, reduce the LC3-Ⅱ/LC3-Ⅰ ratio, and lower the expression of the apoptosis-related protein, namely cleaved caspase-3, indicating that THP could reduce apoptosis by inhibiting autophagy. In-depth studies have found that THP could inhibit the activation of the ULK1/FUNDC1 pathway of mitophagy and the occurrence of mitophagy by reducing the phosphorylation degree of ULK1 at Ser555 and FUNDC1 at Ser17. The application of ULK1 agonist BL-918 reversely verified the effect of THP on reducing the phosphorylation of ULK1 and FUNDC1. In summary, THP inhibited mitophagy through the ULK1/FUNDC1 pathway to reduce H/R injury in H9c2 cells.

Efficacy and safety of endoscopic submucosal dissection for early gastric cancer and precancerous lesions in elderly patients.

BACKGROUND: With advancements in the development of endoscopic technologies, the endoscopic submucosal dissection (ESD) has been one of the gold-standard therapies for early gastric cancer. AIM: To investigate the efficacy and safety ESD in the treatment of early gastric cancer and precancerous lesions in the elderly patients. METHODS: Seventy-eight elderly patients with early gastric cancer and precancerous lesions admitted to the Third Affiliated Hospital of Qiqihar Medical University were selected and classified into two groups according to the different surgical therapies they received between January 2021 and June 2022. Among them, 39 patients treated with ESD were included in an experimental group, and 39 patients treated with endoscopic mucosal resection (EMR) were included in a control group. We compared the basic intraoperative conditions, postoperative short-term recovery, long-term recovery effects and functional status of gastric mucosa between the two groups; the basic intraoperative conditions included lesion resection, intraoperative bleeding and operation time; the postoperative short-term recovery assessment indexes were length of hospital stay and incidence of surgical complications; and the long-term recovery assessment indexes were the recurrence rate at 1 year postoperatively and the survival situation at 1 year and 3 years postoperatively; and we compared the preoperative and predischarge serum pepsinogen I (PG I) and PG II levels and PG I/PG II ratio in the two groups before surgery and discharge. RESULTS: The curative resection rate and the rate of en bloc resection were higher in the experimental group than in the control group. The intraoperative bleeding volume was higher in the experimental group than in the control group. The operation time was longer in the experimental group than that in the control group, and the rate for base residual focus was lower in the experimental group than that of the control group, and the differences were all statistically significant (all P < 0.05). The length of hospital stay was longer in the experimental group than in the control group, and the incidence of surgical complications, 1-year postoperative recurrence rate and 3-year postoperative survival rate were lower in the experimental group than in the control group, and the differences were statistically significant (all P < 0.05). However, the difference in the 1-year postoperative survival rate was not statistically significant between the two groups (P > 0.05). Before discharge, PG I and PG I/PG II ratio were elevated in both groups compared with the preoperative period, and the above indexes were higher in the experimental group than those in the control group, and the differences were statistically significant (both P < 0.05). Moreover, before discharge, PG II level was lower in both groups compared with the preoperative period, and the level was lower in the experimental group than in the control group, and the differences were all statistically significant (all P < 0.05). CONCLUSION: Compared with EMR, ESD surgery is more thorough. It reduces the rate of base residual focus, recurrence rate, surgical complications, and promotes the recovery of gastric cells and glandular function. It is safe and suitable for clinical application.

A quasi-experimental evaluation of the association between implementation of Quality-Based Procedures funding for hip fractures and improvements in processes and outcomes for hip fracture patients in Ontario: an interrupted time series analysis.

PURPOSE: In 2013, Ontario introduced a patient-based funding model for hip fracture care (Quality-Based Procedures [QBP]). The association of QBP implementation with changes in processes and outcomes has not been evaluated. METHODS: We conducted a quasi-experimental study using linked health data for adult hip fractures as an interrupted time series. The pre-QBP period was from 2008 to 2012 and the post-QBP period was from 2014 to 2018; 2013 was excluded as a wash-in period. We used segmented regression analyses to estimate the association of QBP implementation with changes in processes (surgery in less than two days from admission, use of echocardiography, use of nerve blocks, and provision of geriatric care) and clinical outcomes (90-day mortality, 90-day readmissions, length of stay, and days alive at home). We estimated the immediate (level) change, trend (slope) postimplementation, and total counterfactual differences. Sensitivity analyses included case-mix adjustment and stratification by hospital type and procedure. RESULTS: We identified 45,500 patients in the pre-QBP period and 41,256 patients in the post-QBP period. There was a significant total counterfactual increase in the use of nerve blocks (11.1%; 95% confidence interval [CI], 6.2 to 16.0) and a decrease in the use of echocardiography (-2.5%; 95% CI, -3.7 to -1.3) after QBP implementation. The implementation of QBP was not associated with a clinically or statistically meaningful change in 90-day mortality, 90-day readmission, length of stay, or number of days alive at home. CONCLUSION: Evaluation of the QBP program is crucial to inform ongoing and future changes to policy and funding for hip fracture care. The introduction of the QBP Hip Fracture program, supported by evidence-based recommendations, was associated with improved application of some evidence-based processes of care but no changes in clinical outcomes. There is a need for ongoing development and evaluation of funding models to identify optimal strategies to improve the value and outcomes of hip fracture care. STUDY REGISTRATION: Open Science Framework ( https://osf.io/2938h/ ); first posted 13 June 2022.

RéSUMé: OBJECTIF: En 2013, l'Ontario a mis en place un modèle de financement axé sur les patient·es pour les soins suivant une fracture de la hanche (procédures fondées sur la qualité [PFQ]). L'association entre la mise en œuvre des PFQ et les changements dans les processus et les devenirs n'a pas été évaluée. MéTHODE: Nous avons mené une étude quasi expérimentale en utilisant des données de santé couplées pour les fractures de la hanche chez l'adulte comme une série chronologique interrompue. La période précédant les PFQ s'étendait de 2008 à 2012, et la période subséquente à l'implantation des PFQ allait de 2014 à 2018. L'année 2013 a été exclue en tant que période de rodage. Nous avons utilisé des analyses de régression segmentées pour estimer l'association entre la mise en œuvre des PFQ avec des changements aux processus (chirurgie en moins que deux jours suivant l'admission, utilisation de l'échocardiographie, utilisation de blocs nerveux et prestation de soins gériatriques) et des issues cliniques (mortalité à 90 jours, réadmissions à 90 jours, durée de séjour et jours de vie à domicile). Nous avons estimé le changement immédiat (niveau), la tendance (pente) après la mise en œuvre et les différences contrefactuelles totales. Les analyses de sensibilité comprenaient l'ajustement et la stratification de la combinaison de cas par type d'hôpital et par procédure. RéSULTATS: Nous avons identifié 45 500 patient·es dans la période pré-PFQ et 41 256 patient·es dans la période post-PFQ. Il y a eu une augmentation contrefactuelle totale significative de l'utilisation de blocs nerveux (11,1 %; intervalle de confiance [IC] à 95 %, 6,2 à 16,0) et une diminution de l'utilisation de l'échocardiographie (−2,5 %; IC 95 %, −3,7 à −1,3) après la mise en œuvre des PFQ. La mise en œuvre des PFQ n'a pas été associée à un changement cliniquement ou statistiquement significatif de la mortalité à 90 jours, de la réadmission à 90 jours, de la durée de séjour ou du nombre de jours de vie à domicile. CONCLUSION: L'évaluation du programme de PFQ est cruciale pour guider les changements actuels et futurs aux politiques et au financement des soins suivant une fracture de la hanche. La mise en place du programme de PFQ pour les fractures de la hanche, appuyée par des recommandations fondées sur des données probantes, a été associée à une meilleure application de certains processus de soins fondés sur des données probantes, mais à aucun changement dans les devenirs cliniques. Il est nécessaire d'élaborer et d'évaluer continuellement des modèles de financement afin de déterminer les stratégies optimales pour améliorer la valeur et les devenirs des soins suivant une fracture de la hanche. ENREGISTREMENT DE L'éTUDE: Open Science Framework ( https://osf.io/2938h/ ); première publication le 13 juin 2022.

Effects of 5-HT on the cold resistance of mangrove Kandelia obovata seedlings.

5-hydroxytryptamine (5-HT) participates in plant growth and development, and can also delay senescence and cope with abiotic stress. To explore the role of 5-HT in regulating the abilities of mangrove in cold resis-tance, we examined the effects of cold acclimation and the spraying of p-chlorophenylalanine (p-CPA, 5-HT synthesis inhibitor) on leaf gas exchange parameters and CO2 response curves (A/Ca), as well as the endogenous phytohormone content levels in the mangrove species Kandelia obovata seedlings under low temperature stress. The results showed that low temperature stress significantly reduced the contents of 5-HT, chlorophyll, endogenous auxin (IAA), gibberellin (GA), and abscisic acid (ABA). It weakened the CO2 utilization abilities of plants and reduced net photosynthetic rate, which ultimately reduced carboxylation efficiency (CE). Under low temperature stress, exogenous p-CPA reduced the contents of photosynthetic pigments, endogenous hormones, and 5-HT in the leaves, which aggravated the damages caused by low temperature stress on photosynthesis. By enhancing cold acclimation abilities, the endogenous IAA content in the leaves could was reduced under low temperature stress, promoted the production of 5-HT, improved the contents of photosynthetic pigments, GA, and ABA, as well as enhanced photosynthetic carbon assimilation abilities, which would increase photosynthesis in the K. obovata seedlings. Under cold acclimation conditions, the spraying of p-CPA could significantly inhibit the synthesis of 5-HT, promote the production of IAA, and reduce the contents of photosynthetic pigments, GA, ABA, and CE, which would weaken the effects of cold acclimation by improving the cold resistance of mangroves. In conclusion, cold acclimation could improve the cold resistance abilities of K. obovata seedlings by regulating photosynthetic carbon assimilation capacity and the contents of endogenous phytohormone. 5-HT synthesis is one of the necessary conditions for improving the cold resistance abilities of mangroves.

Fasudil alleviates cerebral ischemia­reperfusion injury by inhibiting inflammation and improving neurotrophic factor expression in rats.

The Rho kinase inhibitor fasudil exerts neuroprotective effects. We previously showed that fasudil can regulate M1/M2 microglia polarization and inhibit neuroinflammation. Here, the therapeutic effect of fasudil on cerebral ischemia­reperfusion (I/R) injury was investigated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model in Sprague­Dawley rats. The effect of fasudil on the phenotype of microglia and neurotrophic factors in the I/R brain and its potential molecular mechanism was also explored. It was found that fasudil ameliorated neurological deficits, neuronal apoptosis, and inflammatory response in rats with cerebral I/R injury. Fasudil also promoted the polarization of microglia into the M2 phenotype, in turn promoting the secretion of neurotrophic factors. Furthermore, fasudil significantly inhibited the expression of TLR4 and NF­κB. These findings suggest that fasudil could inhibit the neuroinflammatory response and reduce brain injury after I/R injury by regulating the shift of microglia from an inflammatory M1 phenotype to an anti­inflammatory M2 phenotype, which may be related to the regulation of the TLR4/ NF­κB signal pathway.

TMEM16F may be a new therapeutic target for Alzheimer's disease.

TMEM16F is involved in many physiological processes such as blood coagulation, cell membrane fusion and bone mineralization. Activation of TMEM16F has been studied in various central nervous system diseases. High TMEM16F level has been also found to participate in microglial phagocytosis and transformation. Microglia-mediated neuroinflammation is a key factor in promoting the progression of Alzheimer's disease. However, few studies have examined the effects of TMEM16F on neuroinflammation in Alzheimer's disease. In this study, we established TMEM16F-knockdown AD model in vitro and in vivo to investigate the underlying regulatory mechanism about TMEM16F-mediated neuroinflammation in AD. We performed a Morris water maze test to evaluate the spatial memory ability of animals and detected markers for the microglia M1/M2 phenotype and NLRP3 inflammasome. Our results showed that TMEM16F was elevated in 9-month-old APP/PS1 mice. After TMEM16F knockdown in mice, spatial memory ability was improved, microglia polarization to the M2 phenotype was promoted, NLRP3 inflammasome activation was inhibited, cell apoptosis and Aß plaque deposition in brain tissue were reduced, and brain injury was alleviated. We used amyloid-beta (Aß25-35) to stimulate human microglia to construct microglia models of Alzheimer's disease. The levels of TMEM16F, inducible nitric oxide synthase (iNOS), proinflammatory cytokines and NLRP3 inflammasome-associated biomarkers were higher in Aß25-35 treated group compared with that in the control group. TMEM16F knockdown enhanced the expression of the M2 phenotype biomarkers Arg1 and Socs3, reduced the release of proinflammatory factors interleukin-1, interleukin-6 and tumor necrosis factor-α, and inhibited NLRP3 inflammasome activation through reducing downstream proinflammatory factors interleukin-1ß and interleukin-18. This inhibitory effect of TMEM16F knockdown on M1 microglia was partially reversed by the NLRP3 agonist Nigericin. Our findings suggest that TMEM16F participates in neuroinflammation in Alzheimer's disease through participating in polarization of microglia and activation of the NLRP3 inflammasome. These results indicate that TMEM16F inhibition may be a potential therapeutic approach for Alzheimer's disease treatment.

[Acupuncture ameliorates neurological function by suppressing microglia polarization and inflammatory response after cerebral ischemia in rats].

OBJECTIVE: To observe the effect of acupuncture on microglia polarization and inflammatory reaction in rats with cerebral ischemia-reperfusion injury (CIRI), so as to explore its mechanisms underlying improvement of CIRI. METHODS: Thirty male SD rats were randomly divided into sham operation, model, and acupuncture groups, with 10 rats in each group. The CIRI model was established by occlusion of the middle cerebral artery (MCAO) for 1 h, followed by reperfusion. After modeling, rats in the acupuncture group received manual acupuncture stimulation of "Dazhui" (GV14), "Baihui"(GV20), "Shuigou" (GV26), bilateral "Zusanli" (ST36) and "Fengchi" (GB20) by twirling the needles rapidly for 10 s/acupoint every 10 min, with the needles retained for 20 min. The treatment was conducted once daily for successive 7 days. The neurological function was evaluated according to Longa's method. The state of CIRI was observed after Nissl staining, and the expression levels of Iba-1, iNOS, Arg1, BDNF, GDNF and NeuN in the ischemic cortex tissue were detected by immunofluorescence staining. The contents of TNF-α, IL-6 and IL-10 in the ischemic tissue were assayed by ELISA. The protein expression levels of BDNF, GDNF, TLR4, MyD88 and NF-κB in the ischemic tissues were detected by Western blot. RESULTS: The neurological deficit score on the 24 h and 7th day was considerably higher in the model group than in the sham operation group (P<0.01), and evidently lower on the 7th day in the acupuncture group than in the model group (P<0.01). The number of NeuN positive cells，the area of immunofluorescence dual labelling of Arg1, BDNF and GDNF positive staining, IL-10 content, BDNF and GDNF protein expressions were significantly decreased (P<0.01), and the immunofluorescence dual labelling area of Iba-1 and iNOS, TNF-α and IL-6 contents, the pretein expression levels of TLR4, MyD88 and NF-κB considerably increased (P<0.01) in the model group relevant to the sham operation group. In contrast to the model group, the acupuncture group had a significant increase in the number of NeuN positive cells, the immunofluorescence dual labelling area of Arg1, BDNF and GDNF positive staining, IL-10 content, and BDNF and GDNF protein expressions (P<0.05, P<0.01), and an evident decrease in Iba-1 and iNOS positive staining, contents of TNF-α and IL-6, and the protein expression levels of TLR4, MyD88 and NF-κB (P<0.01, P<0.05). Nissl staining showed a marked reduction in the number of neurons, the nucleus pyknosis and nissl bodies and loose arrangement of the neuronal cells in the model group, which was relatively milder in the acupuncture group. CONCLUSION: Acupuncture intervention can improve neurological function in CIRI rats, which may be related to its effects in regulating the polarization of microglia, reducing inflammatory reaction and increasing the secretion of neurotrophic factors in the brain, inhibiting TLR4/MyD88/NF-κB signaling pathway.

T cell exhaustion drives osteosarcoma pathogenesis.

BACKGROUND: Osteosarcoma (OS) is a rare cancer with a bimodal age distribution that peaks in children and young adults. It has been shown that the expression of programmed cell death protein 1 (PD-1) and programmed death ligand-1 (PD-L1) on tumor-infiltrating immune cells negatively correlates with prognosis of OS patients. However, a comprehensive assessment of the tumor-infiltrating immune cells in OS and their function has not been performed. METHODS: CD8+ T cells were isolated from biopsy tissue samples collected from OS patients and control subjects. Mass cytometry, Treg suppression assay, mixed lymphocyte reaction assay, and effector T cell functional assay were performed to analyze the function of tumor-infiltrating T cells. A xenograft metastasis model was established in BALB/c nude mice. RESULTS: Macrophages and CD3+ T cells comprised most of the tumor-infiltrating immune cells in OS, with a disproportionately higher number of helper CD4+ T cells than effector CD8+ T cells. Whereas the tumor-infiltrating regulatory T cells were functionally intact, the CD8+ T cells showed increased expression of the immune checkpoint receptor (ICR) PD-1 and T cell immunoglobulin and mucin-domain containing 3 (TIM3) and were functionally inactive. TIM3 blockade using a monoclonal antibody restored the T cell alloreactive function of the CD8+ T cells ex vivo. TIM3 blockade in a xenograft model of OS impaired tumor growth in vivo. TIM3 blockade decreased the number of tumor-infiltrating CD4+ T cells while increasing the numbers and functional activation of tumor-infiltrating CD8+ T cells in vivo. CONCLUSIONS: These results highlight that TIM3 blockade might be a viable therapeutic option and should be tested in additional preclinical models.

Effects of ER-resident and secreted AGR2 on cell proliferation, migration, invasion, and survival in PANC-1 pancreatic cancer cells.

BACKGROUND: Anterior gradient-2 (AGR2) is a proto-oncogene involved in tumorigenesis and cancer progression. AGR2, predominantly localized in the endoplasmic reticulum (ER), is also a secreted protein detected in the extracellular compartment in multiple cancers. However, the biological functions of intracellular and extracellular AGR2 remain to be elucidated. METHODS: Based on the biochemical structure of AGR2 protein, PANC-1 pancreatic cancer cells stably expressing ER-resident or secreted AGR2 were generated by a lentivirus-mediated stable overexpression system. The capacities of cell proliferation, migration, invasion and survival were assessed in PANC-1 stable cells. Moreover, EGFR expression and activation were determined to explore the possible mechanism of AGR2 roles in pancreatic cancer tumorigenesis. RESULTS: It was discovered that secreted AGR2, but not ER-resident AGR2, promotes cell proliferation, migration and invasion of PANC-1 cells. Moreover, the data indicated that both the ER-resident and the secreted AGR2 enhance the survival capacity of PANC-1 cells after tunicamycin-induced ER stress and gemcitabine treatment. However, EGFR expression and activation were not found to be involved in AGR2-dependent oncogenic phenotypes in PANC-1 cells. CONCLUSIONS: Secreted AGR2 is predominantly involved in cell proliferation, migration and invasion in PANC-1 pancreatic cancer cells. Both secreted and ER-resident AGR2 contribute to the survival of PANC-1 cells under the challenging conditions. These findings provide insight into how different localizations of AGR2 have contributed to pancreatic cancer growth, metastasis, and drug sensitivity.

Fasudil reduces ß-amyloid levels and neuronal apoptosis in APP/PS1 transgenic mice via inhibition of the Nogo-A/NgR/RhoA signaling axis.

Recent studies have shown that Nogo-A and the Nogo-A receptor affect ß-amyloid metabolism and the downstream Rho GTP enzyme signaling pathway, which may affect the levels of ß-amyloid and tau. Nogo-A may play a key role in the pathogenesis of Alzheimer's disease. However, the underlying molecular mechanisms of Fasudil treatment in Alzheimer's disease are not yet clear. Our results have found that Fasudil treatment for two months substantially ameliorated behavioral deficits, diminished ß-amyloid plaque and tau protein pathology, and alleviated neuronal apoptosis in APP/PS1 transgenic mice. More importantly, two well-established markers for synaptic function, growth-associated protein 43 and synaptophysin, were upregulated after Fasudil treatment. Finally, the levels of Nogo-A, Nogo-A receptor complex NgR/p75NTR/LINGO-1 and the downstream Rho/Rho kinase signaling pathway were significantly reduced. These findings suggest that Fasudil exerts its neuroprotective function in Alzheimer's disease by inhibiting the Nogo-A/NgR1/RhoA signaling pathway.

Fasudil inhibits the activation of microglia and astrocytes of transgenic Alzheimer's disease mice via the downregulation of TLR4/Myd88/NF-κB pathway.

Emerging evidence suggests an association of Alzheimer's Disease (AD) with microglial and astrocytic dysregulation. Recent studies have proposed that activated microglia can transform astrocytes to a neurotoxic A1 phenotype, which has been shown to be involved in the promotion of neuronal damage in several neurodegenerative diseases, including AD. In the present study, we observed an obvious microglial activation and A1-specific astrocyte response in the brain tissue of APP/PS1 Tg mice. Fasudil treatment improved the cognitive deficits of APP/PS1 Tg mice, inhibited microglial activation and promoted their transformation to an anti-inflammatory phenotype, and further shifted astrocytes from an A1 to an A2 phenotype. Our experiments suggest Fasudil exerted these functions by inhibing the expression of TLR4, MyD88, and NF-κB, which are key mediators of inflammation. Using in vitro experiments, we further validated in vivo findings. Our cell experiments indicated that Fasudil induces a shift of inflammatory microglia towards an anti-inflammatory phenotype. LPS-induced microglia-conditioned medium promotes A1 astrocytic polarization, but Fasudil treatment resulted in a direct transformation of A1 astrocytes to A2. To summarize, our results show that Fasudil inhibits the neurotoxic activation of microglia and shifts astrocytes towards a neuroprotective A2 phenotype, representing a promising candidate for AD treatment.

TRPV4 is involved in levonorgestrel-induced reduction in oviduct ciliary beating.

Previous studies revealed the increasing risk of tubal pregnancy following failure of levonorgestrel (LNG)-induced emergency contraception, which was attributed to the reduced ciliary motility in response to LNG. However, understanding of the mechanism of LNG-induced reduction in the ciliary beat frequency (CBF) is limited. The transient receptor potential vanilloid (TRPV) 4 channel is located widely in the female reproductive tract and generates an influx of Ca2+ following its activation under normal physiological conditions, which regulates the CBF. The present study aimed to explore whether LNG reduced the CBF in the Fallopian tubes by modulating TRPV4 channels, leading to embryo retention in the Fallopian tubes and subsequent tubal pregnancy. The study provided evidence that the expression of TRPV4 was downregulated in the Fallopian tubes among patients with tubal pregnancy and negatively correlated with the serum level of progesterone. LNG downregulated the expression of TRPV4, limiting the calcium influx to reduce the CBF in mouse oviducts. Furthermore, the distribution of ciliated cells and the morphology of cilia did not change following the administration of LNG. LNG-induced reduction in the CBF and embryo retention in the Fallopian tubes and in mouse oviducts were partially reversed by the progesterone receptor antagonist RU486 or the TRPV4 agonist 4α-phorbol 12,13-didecanoate (4α-PDD). The results indicated that LNG could downregulate the expression of TRPV4 to reduce the CBF in both humans and mice, suggesting the possible mechanism of tubal pregnancy. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Effects of Levonorgestrel and progesterone on Oviductal physiology in mammals.

BACKGROUND: Our previous study indicated that emergency contraception, including levonorgestrel and progesterone, could lead to ectopic pregnancy following contraception failure. However, our understanding of the effects of levonorgestrel and progesterone on oviductal physiology is limited. METHODS: The receptivity of the fallopian tubal epithelium after levonorgestrel and progesterone treatment was examined through western blots for receptivity markers and JAr-spheroid-fallopian tubal epithelial cell attachment assays. The ciliary beat frequency was analyzed using an inverted bright-field microscope. Furthermore, an in vivo animal model of embryo-tubal transplantation was also studied to determine the effects of levonorgestrel- and progesterone-induced ciliary beat reduction. RESULTS: Our results showed that levonorgestrel and progesterone did not change the levels of fallopian tubal epithelial cell receptive markers, including LIF, STAT3, IGFBP1, ITGB3, MUC1, and ACVR1B, or affect JAr-spheroid implantation. However, levonorgestrel and progesterone reduced the ciliary beat frequency in fallopian tubes in a dose-dependent manner. An in vivo model also showed that levonorgestrel and progesterone could lead to embryo retention in the oviducts. CONCLUSIONS: These findings show that levonorgestrel and progesterone can reduce the ciliary beat frequency without altering receptivity, indicating a possible mechanism for progesterone- or levonorgestrel-induced tubal pregnancy.

Association between Alzheimer's disease pathogenesis and early demyelination and oligodendrocyte dysfunction.

The APPSwe/PSEN1dE9 (APP/PS1) transgenic mouse model is an Alzheimer's disease mouse model exhibiting symptoms of dementia, and is commonly used to explore pathological changes in the development of Alzheimer's disease. Previous clinical autopsy and imaging studies suggest that Alzheimer's disease patients have white matter and oligodendrocyte damage, but the underlying mechanisms of these have not been revealed. Therefore, the present study used APP/PS1 mice to assess cognitive change, myelin loss, and corresponding changes in oligodendrocytes, and to explore the underlying mechanisms. Morris water maze tests were performed to evaluate cognitive change in APP/PS1 mice and normal C57BL/6 mice aged 3 and 6 months. Luxol fast blue staining of the corpus callosum and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for myelin basic protein (MBP) mRNA were carried out to quantify myelin damage. Immunohistochemistry staining for NG2 and qRT-PCR for monocarboxylic acid transporter 1 (MCT1) mRNA were conducted to assess corresponding changes in oligodendrocytes. Our results demonstrate that compared with C57BL/6 mice, there was a downregulation of MBP mRNA in APP/PS1 mice aged 3 months. This became more obvious in APP/PS1 mice aged 6 months accompanied by other abnormalities such as prolonged escape latency in the Morris water maze test, shrinkage of the corpus callosum, upregulation of NG2-immunoreactive cells, and downregulation of MCT1 mRNA. These findings indicate that the involvement of early demyelination at 3 months and the oligodendrocyte dysfunction at 6 months in APP/PS1 mice are in association with Alzheimer's disease pathogenesis.

[Effect of bone morphogenetic protein-4 overexpression on the biological activity of mouse induced pluripotent stem cells].

OBJECTIVE: This study aimed to construct the expression of bone morphogenetic protein-4 (BMP4) lentiviral vector gene and explore its influence on the biological activity of mouse induced pluripotent stem (iPS) cells. METHODS: iPS cell lines stably overexpressing BMP4 were constructed by lentivirus transfection (BMP4-overexpressing group). Cells without transfection served as the blank group, and cells with only vector transfection served as the empty-vector group. Cell proliferation was detected by CCK8, and the expression levels of ameloblastin (AMBN), cytokeratin (CK) 14, dentin sialophospho-protein (DSPP), bone sialoprotein (BSP), and Runx2 mRNA were detected by quantitative polymerase chain reaction. Alkaline phosphatase (ALP) activity was used to detect the degree of cell differentiation. RESULTS: Compared with blank and empty-vector groups, proliferation activity and ALP activity of BMP4-overexpressing group obvious increased (P<0.05), BMP4, AMBN, CK14, DSPP, BSP, Runx2 mRNA expression also increased (P<0.05). CONCLUSIONS: BMP4 can significantly promote the odontogenic differentiation of iPS.

Chronic corticosterone exposure reduces hippocampal glycogen level and induces depression-like behavior in mice.

Long-term exposure to stress or high glucocorticoid levels leads to depression-like behavior in rodents; however, the cause remains unknown. Increasing evidence shows that astrocytes, the most abundant cells in the central nervous system (CNS), are important to the nervous system. Astrocytes nourish and protect the neurons, and serve as glycogen repositories for the brain. The metabolic process of glycogen, which is closely linked to neuronal activity, can supply sufficient energy substrates for neurons. The research team probed into the effects of chronic corticosterone (CORT) exposure on the glycogen level of astrocytes in the hippocampal tissues of male C57BL/6N mice in this study. The results showed that chronic CORT injection reduced hippocampal neurofilament light protein (NF-L) and synaptophysin (SYP) levels, induced depression-like behavior in male mice, reduced hippocampal glycogen level and glycogen synthase activity, and increased glycogen phosphorylase activity. The results suggested that the reduction of the hippocampal glycogen level may be the mechanism by which chronic CORT treatment damages hippocampal neurons and induces depression-like behavior in male mice.

[Renal protection of Tangke Decoction on rats with diabetes and its effect on the expression of TGF-beta1/Smad4].

OBJECTIVE: To observe the effect of Tangke Decoction (TD) on the expression of TGF-beta1/Smad4 of rats with early diabetes and to explore the effect and mechanism of TD against the renal injury induced by diabetes. METHODS: SD rats were randomly divided into the normal control group (n = 12), the model group (n = 10), the Chinese herbs prevented group (n =10), the Chinese herbs treated group (n = 10), and the Western medicine control group (n = 10). TD (18 mg/kg) was given by gastrogavage to rats in the Chinese herbs prevented group immediately after successful modeling for 12 weeks, once daily. At the 4th week of successful modeling, rats in the rest 4 groups were administered by gastrogavage. Equal volume of normal saline was given to rats in the model group and the normal control group. Benazepril suspension (1 mg/kg) was administered by gastrogavage to rats in the Western medicine control group for 8 weeks, once daily. TD (18 mg/kg) was given by gastrogavage to rats in the Chinese herbs treated group for 8 weeks, once daily. The body weight, kidney weight, index of kidney weight, fasting blood sugar, 24 h urinary albumin excretion rate were examined after experiment. The pathological changes of the renal tissue were observed by HE staining, Masson staining, and electron microscope. The expression of renal transforming growth factor-beta1, (TGF-beta1) and Smad4 were detected using immunohistochemical assay. RESULTS: Compared with the normal control group, the body weight of rats decreased significantly; the kidney weight, index of kidney weight, blood sugar, 24 h urinary protein excretion, the urinary albumin excretion rate,TGF-beta1 and Smad4 expression increased significantly in the model group (all P < 0.01). Compared with the model group, the aforesaid indices were improved in each treatment group with statistical difference (P < 0.05, P < 0.01). Compared with the Western medicine control group, the kidney weight, index of kidney weight, blood sugar, 24 h urinary protein excretion, and the urinary albumin excretion rate were obviously improved in the Chinese herbs prevented group (P < 0.01). The renal pathological changes were most obvious in the model group significantly, but they were improved in all treatment groups. CONCLUSION: TD could obviously improve the symptoms of diabetes and down-regulate the expression of renal TGF-beta1 and Smad4 of early diabetic nephropathy rats, which suggested that TD had certain preventive effect on early diabetic nephropathy.