DMSO Group Research Articles

Experimental Lung Transplantation Related With HIF-1, VEGF, ROS. Assessment of HIF-1α, VEGF, and Reactive Oxygen Species After Competitive Blockade of Chetomin for Lung Transplantation in Rats

Primary graft failure occurs 15 to 30 % of the time after transplantation. Although there have been improvements in preserving the lungs in good condition, there have not been studies on the regulation of transcription factors. Methods: We carried out an experimental study involving lung transplantation to indirectly evaluate reactive oxygen species (ROS) production and VEGF expression by competitive blockade of HIF-1α with chetomin. There were 5 groups: Group-1: Lung blocks were perfused with 0.9 % SSF, immediately harvested, and preserved. Group-2 (I-T): Immediate transplantation and then reperfusion for 1 h. Group-3 (I-R): Lung blocks were harvested and preserved in LPD solution for 6 h and reperfused for 1 h. Group-4 (DMSO): Lung blocks were treated for 4 h with DMSO, preserved for 6 h and transplanted to a receptor treated with DMSO. Group-5 (chetomin): Lung blocks were treated for 4 h with chetomin, preserved for 6 h and transplanted to a receptor treated with chetomin. ROS, mRNA, and protein levels of HIF-1α and EG-VEGF were determined. Results: The DMSO and chetomin groups had significantly lower ROS levels. Compared with those in the I-R group, the chetomin group exhibited the lowest level of HIF-1α. Conclusions: Addition of chetomin to the donor and the receptor results in a significant reduction in HIF-1A, VEGF and ROS.

Potential nephroprotective effect of dapagliflozin against renal ischemia reperfusion injury in rats via activation of autophagy pathway and inhibition of inflammation, oxidative stress and apoptosis

Renal ischemia/reperfusion injury is a common cause of acute kidney injury (AKI), causing tissue damage, inflammation and oxidative stress. Autophagy, responsible for breakdown and recycling of cytoplasmic components. In hypoxic and ischemic renal damage, autophagy may serve as a survival strategy for the cells. Dapagliflozin has been shown to have protective effects against ischemia. Objective: The study investigates the potential nephro-protective effects of dapagliflozin on renal ischemia reperfusion injury, evaluating its antioxidant, anti-inflammatory, anti-apoptotic effects and activation of autophagy via biochemical parameters and histopathological changes in adult male rats. Material and method: In this study, twenty eight male rats weighing between 200-300g, were divided into 4 groups sham, RIRI, DMSO, Dapagliflozin pretreated groups. All groups subjected to 30 min. of ischemia then 24 h of reperfusion except sham undergo the same anesthesia and surgical procedures except for bilateral renal ischemia. Dapagliflozin 1mg/kg and DMSO was given 2 hours before the induction of ischemia. Results: The study found that blood urea and serum creatinine levels increased in RIRI and DMSO groups when compared with sham group. Renal tissue levels of IL-6, Kim-1, caspase-3, LC-3 and Akt were also elevated in RIRI while GSH levels decreased in the RIRI. Dapagliflozin group showed significant increases in GSH, LC-3 and Akt levels when compared to sham group. Dapagliflozin reduced serum urea and creatinine levels, and renal tissue levels of IL-6, Kim-1, and caspase-3 also reduced. Conclusion: Dapagliflozin significantly reduced renal damage in rat model due to its antioxidant, anti-inflammatory, anti-apoptotic, and activation autophagy.

SNHG14 promotes triple-negative breast cancer cell proliferation, invasion, and chemoresistance by regulating the ERK/MAPK signaling pathway.

The functional role and molecular mechanisms of small-nucleolar RNA host gene 14 (SNHG14) in triple-negative breast cancer (TNBC) progression remain unclear. The expression levels of SNHG14 in breast cancer samples and cell lines were determined using real-time quantitative polymerase chain reaction. Cell proliferation, migration, and invasion abilities were detected using MTS and transwell assays. By RNA sequencing, differentially expressed genes were identified between the SNHG14 siRNA and the negative control group. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to predict the targets and pathways regulated by SNHG14. pRAF, pMEK, and pERK expression were measured by western blot. The xenograft model was constructed to access the biological function of SNHG14 in vivo. A minimal patient-derived xenograft model was established to evaluate the sensitivity to chemotherapy drugs. Our data indicated that SNHG14 expression was increased in TNBC tissues and cell lines. SNHG14 knockdown attenuated the proliferation, migration, and invasion abilities of TNBC cells both in vivo and in vitro. High SNHG14 expression was associated with lymph node metastasis and a high Ki67 index. The targets of SNHG14 were mainly enriched in the MAPK signaling pathway. pRAF, pMEK, and pERK expression were downregulated after being transfected with SNHG14 siRNA. Compared with the negative control group, the expression of CACNA1I, DUSP8, FGF17, FGFR4, FOS, PDGFRB, and DDIT3 was increased, and the expression of MKNK1 was decreased in the SNHG14 siRNA group. Minimal patient-derived xenograft model demonstrated that knockdown of SNHG14 enhanced the sensitivity to Docetaxel in vivo. Compared with the DMSO group, the proliferation of Docetaxel-resistant MDA-MB-231 cells was decreased in Dabrafenib, PD184352, and FR180204 treatment groups. SNHG14 knockdown inhibits TNBC progression by regulating the ERK/MAPK signaling pathway, which provides evidence for SNHG14 as a potential target for TNBC therapy.

Effect of abemaciclib and curcumin administration on sex hormones, reproductive functions, and oxidative DNA expression in rats

ABSTRACT This study investigated whether abemaciclib (ABE) administration had any adverse effects on ovarian and sex hormones in female rats, and the protective effect of curcumin. Forty female rats were equally divided into the sham control, DMSO, curcumin (CMN), ABE, and ABE+CMN groups. Pharmaceuticals were administered by gavage daily for 28 days. Serum sex hormones were measured in an autoanalyzer operating with a microparticle immunoassay method. In addition, histopathological examination and 8-OHdG expression were performed on the ovarian tissue. Progesterone and testosterone levels were significantly decreased, while estradiol levels were significantly increased, in the ABE group compared to the sham and DMSO groups. In addition, there were significant differences in sex hormone levels in the CMN and/or CMN+ABE groups compared to the ABE group. There was decreased expression of 8-OHdG in the ABE+CMN group compared to the ABE or CMN only groups. This study exhibited that ABE administration can adversely affect functions and histology of the ovarian tissue, but CMN therapy may be protective against the adverse effects on ovarian in ABE-induced rats.

52 Ferroptosis Suppressor Protein 1 is a Potent and Targetable Inhibitor of Ferroptosis in Chromophobe Renal Cell

Abstract Background There are currently no proven therapies for metastatic or unresectable Chromophobe renal cell carcinoma (ChRCC). Ferroptosis is an iron-dependent form of cell death characterized by the peroxidation of membrane polyunsaturated fatty acids, leading to membrane damage and cell death. Glutathione, a potent cellular antioxidant, counters lipid peroxidation to prevent ferroptosis. Both reduced and oxidized glutathione are markedly elevated in ChRCC (PNAS, 2018). We have previously discovered that ChRCC is hypersensitive to ferroptotic cell death induced by the pharmacologic disruption of glutathione homeostasis via the cysteine transporter SLC7A11 or glutathione peroxidase (GPX4) inhibition (PNAS, 2022). Ferroptosis suppressor protein (FSP1) is a glutathione-independent suppressor of ferroptosis. FSP1’s role in ChRCC is unexplored. Methods Transcriptional data from DepMap (484 cell lines) and Cancer Therapeutics Response Portal (CTRP) (860 cell lines) were used to study genetic or pharmacologic inhibition of GPX4 and SLC7A11. Statistical analysis was performed in PRISM 10 using Mann-Whitney U and ANOVA tests. Statistical significance was defined as p < 0.05. Results DepMap data revealed that FSP1 is the top upregulated gene in cells resistant to RNAi inhibition of GPX4 (Achilles, DEMETER2) (Panel A). In CTRP data, FSP1 was the top upregulated gene in cells resistant to the GPX4 inhibitor RSL3 and the 19th most upregulated gene in cells resistant to the SCL7A11 inhibitor, Erastin (Panel B). In The Cancer Genomic Atlas (TCGA) KICH (ChRCC) dataset, FSP1 was 2-fold higher in ChRCC compared to normal kidney (mean RSEM = 818.9 vs 445.9, p-value <0.0001) (Panel C). In vitro studies of two ChRCC cell lines, UOK276 and RCJ-T2, revealed that while siRNA-mediated inhibition of GPX4 or FSP1 individually did not induce substantial cell death, their combination was synergistic and resulted in almost complete cell death (UOK276 mean viability 8% of siCTRL, p<0.0001; RCJ-T2 mean viability 13% of siCTRL, p<0.0001). Cell viability was completely rescued by the ferroptosis inhibitor ferrostatin-1 but not by the necroptosis inhibitor necrostatin-1 or the apoptosis inhibitor Z-Vad-FMK (Panel D). siRNA-mediated GPX4 knockdown combined with FSP1 pharmacologic inhibition (icFSP1 or FSEN1) induced extensive cell death (mean viability of siGPX4 + icFSP1 group = 21% of siCTRL + DMSO group, p<0.0001; mean viability of siGPX4 + FSEN1 group = 29% of siCTRL + DMSO group, p<0.0001) (Panel E). Combining RSL3 with FSEN1, or IKE with icFSP1, also demonstrated synergy, with the ChRCC cell lines showing increased overall sensitivity to these drug combinations compared to 786-0 cells (ccRCC-derived) and HeLa cells (Panel F). Snapshots from these experiments taken at [IKE] = 177nM and/or [icFSP1] = 20 mM for UOK276, and [RSL3] = 39.4 nM and/or [FSEN1] = 20 mM for RCJ-T2, show almost complete cell death when pharmacologic inhibitors of glutathione-dependent and -independent ferroptosis suppressors are combined, but no substantial cell death when each is used individually (7% and 10% viability in combination group compared to DMSO group for UOK276 and RCJ-T2 respectively, p<0.0001) (Panel G). Conclusions We show that in vitro genetic or pharmacologic inhibition of FSP1, a glutathione-independent inhibitor of ferroptosis, leads to ferroptosis in ChRCC-derived cells when combined with genetic or pharmacologic inhibition of the glutathione-dependent suppressors of ferroptosis GPX4 or SLC7A11.

Aryl Hydrocarbon Receptor Modulation and Resveratrol Influence on Dnah1 Expression: Implications for Male Reproductive Health

Male reproductive health is intricately regulated by molecular and physiological processes, with the aryl hydrocarbon receptor (AhR) playing a crucial role. AhR is activated by various ligands and influences the onset and progression of diseases. The aim of this study was to evaluate the role of AhR on spermatogenesis in adult male rats were affected by resveratrol (RES) and CH223191, an AhR antagonist. The study include forty rats were randomly divided into four equal groups: Control group, DMSO group, RES group and AhR‾ group, the rats received respective treatments intraperitoneally twice weekly for 60 days, and various parameters related to male reproductive health were evaluated. The AhR that activation by the RES treatment were enhanced the sperm morphology, sperm acrosomal integrity and luteinizing hormone. Moreover, the Dnah1 gene expression, is has play major role in sperm morphology, was upregulated and testosterone levels were elevated by RES. On the other hand, sperm morphology, sperm acrosomal integrity, testosterone hormone levels, and Dnah1 expression were all reduced in AhR‍ rats. The testicular tissue integrity was maintained by RES, according to a histopathological analysis. These findings highlight the role of AhR in male reproductive health and suggest RES as a potential therapeutic agent.

Human umbilical cord mesenchymal stem cells promoted tumor cell growth associated with increased interleukin-18 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is experiencing a concerning rise in both incidence and mortality rates. Current therapeutic strategies are limited in their effectiveness, largely due to the complex causes of the disease and significant levels of drug resistance. Given the latest developments in human umbilical cord mesenchymal stem cells (hUC-MSCs) research, there is a debate over the continued use of stem cell transplantation for treating tumors. Consequently, this study seeks to explore the role of hUC-MSCs in the management of HCC. HUC-MSCs increased the number (10.75 ± 1.50) in the DEN/TCPOBOP-induced mice hepatoma model, compared with DMSO group (7.25 ± 1.71). Moreover, the liver index in hUC-MSCs group (0.21 ± 0.06) was greater than that in DMSO group (0.09 ± 0.01). Immunohistochemical (IHC) analysis revealed that while hUC-MSCs did not alter Foxp3 expression, they significantly stimulated Ki67 expression, indicative of increased tumor cellular proliferation. Additionally, immunofluorescence (IF) studies showed that hUC-MSCs increased CD8+ T cell counts without affecting macrophage numbers. Notably, granzyme B expression remained nearly undetectable. We observed that serum IL-18 levels were higher in the hUC-MSCs group (109.66 ± 0.38 pg/ml) compared to the DMSO group (91.14 ± 4.37 pg/ml). Conversely, IL-1β levels decreased in the hUC-MSCs group (63.00 ± 0.53 pg/ml) relative to the DMSO group (97.38 ± 9.08 pg/ml). According to this study, hUC-MSCs promoted the growth of liver tumors. Therefore, we proposed that hUC-MSCs are not suitable for treating HCC, as they exhibit clinically prohibited abnormalities.

Sustained feeding of a diet high in fat resulted in a decline in the liver's insulin-degrading enzyme levels in association with the induction of oxidative and endoplasmic reticulum stress in adult male rats: Evaluation of 4-phenylbutyric acid

The current study explored the impact of high fat diet (HFD) on hepatic oxidative and endoplasmic reticulum (ER) stress and its insulin degrading enzyme (IDE) content with the injection of 4-phenyl butyric acid (4-PBA) in adult male rats.Following the weaning period, male offspring were distributed among six distinct groups. The corresponding diet was used for 20 weeks, subsequently 4-PBA was administered for three consecutive days. Plasma glucose and insulin levels, HOMA-β (homeostasis model assessment of β-cell), hepatic ER and oxidative stress biomarkers and IDE protein content were assessed.Long-term ingestion of HFD (31 % cow butter) induced oxidative and ER stress in the liver tissue. Accordingly, a rise in the malondialdehyde (MDA) content and catalase enzyme activity and a decrease in the glutathione (GSH) content were detected within the liver of the HFD and HFD + DMSO groups. Consumption of this diet elevated the liver expression of binding immunoglobulin protein (BIP) and C/enhancer-binding protein homologous protein (CHOP) levels while reduced its IDE content. The HOMA-β decreased significantly. The injection of the 4-PBA moderated all the induced changes.Findings from this study indicated that prolonged HFD consumption led to a reduction in plasma insulin levels, likely attributed to pancreatic β cell malfunction, as evidenced by a decline in the HOMA-β index. Also, the HFD appears to have triggered oxidative and ER stress in the liver, along with a decrease in its IDE content.

Lipid nephrotoxicity mediated by HIF-1α activation accelerates tubular injury in diabetic nephropathy

This study is intended to explore the effect of hypoxia-inducible factor-1α (HIF-1α) activation on lipid accumulation in the diabetic kidney. A type 1 diabetic rat model was established by STZ intraperitoneal injection. Cobalt chloride (CoCl2) and YC-1 were used as the HIF-1α activator and antagonist, respectively. CoCl2 treatment significantly increased HIF-1α expression, accelerated lipid deposition, and accelerated tubular injury in diabetic kidneys. In vitro, CoCl2 effectively stabilized HIF-1α and increased its transportation from the cytoplasm to the nucleus, which was accompanied by significantly increased lipid accumulation in HK-2 cells. Furthermore, results obtained in vivo showed that HIF-1α protein expression in the renal tubules of diabetic rats was significantly downregulated by YC-1 treatment. Meanwhile, lipid accumulation in the tubules of the DM + YC-1 group was markedly decreased in comparison to the DM + DMSO group. Accordingly, PAS staining revealed that the pathological injury caused to the tubular epithelial cells was alleviated by YC-1 treatment. Furthermore, the blood glucose level, urine albumin creatinine ratio, and NAG creatinine ratio in the DM + YC-1 group were significantly decreased compared to the DM + DMSO group. Moreover, the protein expression levels of transforming growth factor β1 (TGF-β1) and connective tissue growth factor (CTGF) in diabetic kidneys were decreased by YC-1 treatment. Our findings demonstrate that the activation of HIF-1α contributed to interstitial injury in a rat model of diabetic nephropathy and that the underlying mechanism involved the induction of lipid accumulation.

Histopathological and biochemical Effects of 18β-glycyrrhetinic acid application on lipopolysaccharide-induced kidney toxicity in rats

Lipopolysaccharide (LPS) is an endotoxin found in the wall of gram-negative bacteria and causes acute inflammation when it enters the tissues. 18β-glycyrrhetinic acid (18β-GA) is a substance found in licorice root and is responsible for this plant's antiallergic, antioxidant, and anti-inflammatory activity. This study aimed to examine the possible effects of 18β-glycyrrhetinic acid on the damage caused by LPS in kidney tissue. The study divided 40 Sprague Dawley adult male rats into 5 equal groups (n = 8). The groups were created as follows; the control group; the group that received 1cc physiological saline throughout the experiment was the DMSO group; DMSO, an intraperitoneal carrier substance, was given. LPS group; A single dose of 7.5 mg/kg intraperitoneal (i.p) LPS was administered. 18β-GA50+LPS group; 18β-glycyrrhetinic acid was given by gavage at 50 mg/kg daily for 10 days, followed by a single dose of 7.5 mg/kg i.p. LPS was administered. 18β-GA100+LPS group; 18β-glycyrrhetinic acid was administered by gavage at 100 mg/kg daily for 10 days, followed by a single dose of 7.5 mg/kg i.p. LPS was administered. 18β-GA100 group; 18β-glycyrrhetinic was given by gavage at 100 mg/kg daily for 10 days. 24 hours after LPS application to all groups, the kidney tissues of the rats were removed under anesthesia and placed in 10% formaldehyde. Histopathological and oxidative stress parameters analyses were performed in kidney tissue.

Nobiletin as a novel agent to enhance porcine in vitro embryo development and quality

In vitro embryo production (IVP) is of great importance to the porcine industry, as well as for basic research and biomedical applications. Despite the large efforts made in laboratories worldwide to address suboptimal culture conditions, porcine IVP remains inefficient. Nobiletin (Nob, 5,6,7,8,3′,4′ hexamethoxyflavone) supplementation to in vitro culture (IVC) medium, enhances in vitro embryo development in various species. However, its impact on the quality and developmental capacity of in vitro-produced pig embryos is yet to be established. This study evaluated the effects of different concentrations (2.5 and 5 μM) of Nob during the early culture of in vitro-produced pig embryos on embryo developmental competence, mitochondrial activity, lipid content, intracellular Reactive Oxygen Species (ROS) and Glutathione (GSH) content, Total Cell Number (TCN) per blastocyst, and expression of genes related to embryo development, quality and oxidative stress. Embryos cultured in medium without Nob supplementation and in medium supplemented with 0.01 % dimethyl sulfoxide (DMSO-vehicle for Nob) constituted the Control and DMSO groups, respectively. Embryo development rates were evaluated on Days 2, 6 and 7 of IVC. Additionally, a representative group of embryos was selected to assess mitochondrial activity, lipid, ROS and GSH content (on Days 2 and 6 of IVC), TCN assessment and gene expression analyses (on Day 6 of IVC). No significant differences were observed in any of the parameters evaluated on Day 2 of IVC. In contrast, embryos cultured under the presence of Nob 2.5 showed higher developmental rates on Days 6 and 7 of IVC. In addition, Day 6 embryos showed increased mitochondrial activity, with decreased levels of ROS and GSH in the Nob 2.5 group compared to the other groups. Both Nob 2.5 and Nob 5 embryos showed higher TCN compared to the Control and DMSO groups. Furthermore, Nob 2.5 and Nob 5 upregulated the expression of Superoxide dismutase type 1 (SOD1) and Glucose-6-phosphate dehydrogenase (G6PDH) genes, which could help to counteract oxidative stress during IVC. In conclusion, the addition of Nob during the first 48 h of IVC increased porcine embryo development rates and enhanced their quality, including the upregulation of relevant genes that potentially improved the overall efficiency of the IVP system.

Resveratrol prevents cognitive deficits induced by sleep deprivation via modulating sirtuin 1 associated pathways in the hippocampus.

Accumulating evidence confirms that sleep insufficiency is a high risk factor for cognitive impairment, which involves inflammation and synaptic dysfunction. Resveratrol, an agonist of the Sirt1, has demonstrated anti-inflammation and neuroprotective effects in models of Alzheimer's disease, Parkinson's disease, and schizophrenia. However, the beneficial effects of resveratrol on sleep deprivation-induced cognitive deficits and its underlying molecular mechanisms are unclear. In the present study, thirty-two male C57BL/6 J mice were randomly divided into a Control+DMSO group, Control+Resveratrol group, SD+DMSO group, and SD+Resveratrol group. The mice in the SD+Resveratrol group underwent 5 days of sleep deprivation after pretreatment with resveratrol (50 mg/kg) for 2 weeks, while the mice in the SD+DMSO group only underwent sleep deprivation. After sleep deprivation, we evaluated spatial learning and memory function using the Morris water maze test. We used general molecular biology techniques to detect changes in levels of pro-inflammatory cytokines and Sirt1/miR-134 pathway-related synaptic plasticity proteins. We found that resveratrol significantly reversed sleep deprivation-induced learning and memory impairment, elevated interleukin-1β, interleukin-6, and tumor necrosis factor-α levels, and decreased brain-derived neurotrophic factor, tyrosine kinase receptor B, postsynaptic density protein-95, and synaptophysin levels by activating the Sirt1/miR-134 pathway. In conclusion, resveratrol is a promising agent for preventing sleep deprivation-induced cognitive dysfunction by reducing pro-inflammatory cytokines and improving synaptic function via the Sirt1/miR-134 pathway.

Moexipril Improves Renal Ischemia/Reperfusion Injury in Adult Male Rats

Objective: The goal of this study is to see if moexipril can protect rats against renal ischemia/reperfusion injury. Methods: Overall twenty-eight males of rats were divided randomly into four groups (7 rat each group). Sham group: Except for ischemiainduction, these rats underwent IP anesthesia and surgery. Induced group: This group rats were anesthetized and given a midlinelaparotomy to induce bilateral renal ischemia for 30 min and 2 hours of reperfusion. DMSO group: Rats received DMSO IP injection 30 minbefore ischemia and subjected to 30 min bilateral ischemia and reperfusion for 2 hours, DMSO is a vehicle of moexipril and considered ascontrol. Moexipril group (pretreated group): moexipril was given in a dose 0.3 mg/kg I.P. injection 30 min before ischemia. Results: Renal IRI as indicated by a significant increase (P < 0.05) in urea, creatinine, NF-KB P65, IL-1β, and caspase-3 level, while GSH,SOD, and Bcl-2 levels significantly (P < 0.05) reduced in Renal tissues of rats in the induced group compared to sham group. Moexiprilpretreatment significantly (P < 0.05) ameliorate RIRI as suggested from significant lowering in urea, creatinine, and inflammatory markers(NF-KB P65 and IL-1β). The renal level of oxidative marker (SOD and GSH) and anti-apoptotic marker Bcl-2 were significant decreased(P < 0.05) and also significantly increase (P < 0.05) in caspase-3 level with moexipril group in comparison to induced group. Conclusion: By inhibiting oxidative stress, inflammation, and the apoptotic pathway, moexipril significantly protect from renal ischemiareperfusion in rats.

Exploring the Mechanisms of Sanguinarine in the Treatment of Osteoporosis by Integrating Network Pharmacology Analysis and Deep Learning Technology.

Sanguinarine (SAN) has been reported to have antioxidant, antiinflammatory, and antimicrobial activities with potential for the treatment of osteoporosis (OP). This work purposed to unravel the molecular mechanisms of SAN in the treatment of OP. OP-related genes and SAN-related targets were predicted from public databases. Differential expression analysis and VennDiagram were adopted to detect SAN-related targets against OP. Protein-protein interaction (PPI) network was served for core target identification. Molecular docking and DeepPurpose algorithm were further adopted to investigate the binding ability between core targets and SAN. Gene pathway scoring of these targets was calculated utilizing gene set variation analysis (GSVA). Finally, we explored the effect of SAN on the expressions of core targets in preosteoblastic MC3T3-E1 cells. A total of 21 candidate targets of SAN against OP were acquired. Furthermore, six core targets were identified, among which CASP3, CTNNB1, and ERBB2 were remarkably differentially expressed in OP and healthy individuals. The binding energies of SAN with CASP3, CTNNB1, and ERBB2 were -6, -6.731, and -7.162 kcal/mol, respectively. Moreover, the GSVA scores of the Wnt/calcium signaling pathway were significantly lower in OP cases than in healthy individuals. In addition, the expression of CASP3 was positively associated with Wnt/calcium signaling pathway. CASP3 and ERBB2 were significantly lower expressed in SAN group than in DMSO group, whereas the expression of CTNNB1 was in contrast. CASP3, CTNNB1, and ERBB2 emerge as potential targets of SAN in OP prevention and treatment.

Fat mass and obesity-associated protein (FTO) affects midpalatal suture bone remodeling during rapid maxillary expansion.

The fat mass and obesity-associated protein (FTO) is an RNA demethylase that contributes to several physiological processes. Nonetheless, the impact of FTO on bone remodeling in the midpalatal suture while undergoing rapid maxillary expansion (RME) remains unclear. First, to explore the expression of FTO in the midpalatal suture during RME, six rats were randomly divided into two groups: Expansion group and Sham group (springs without being activated). Then, suture mesenchymal stem cells (SuSCs) were isolated as in vitro model. The expression of FTO was knocked down by small interfering RNA to study the effect of FTO on the osteogenic differentiation of SuSCs. Finally, to evaluate the function of FTO in the process of bone remodeling in the midpalatal suture, ten rats were randomly divided into two groups: FB23-2 group (10 μM, a small molecule inhibitor of FTO) and DMSO group (control). Increased arch width and higher expression of OCN and FTO in the midpalatal area were observed in expansion group (P < .05). In the in vitro model, the mRNA expression levels of Runx2, Bmp2, Col1a1, Spp1, and Tnfrsf11b were decreased (P < .05) upon knocking down FTO. Additionally, the protein levels of RUNX2 and OPN were also decreased (P < 0.05). Adding FB23-2, a small-molecule inhibitor targeting FTO, to the medium of SuSCs caused a decrease in the mRNA expression levels of Runx2, Bmp2, Col1a1, Spp1, and Tnfrsf11b (P < 0.05). There was a statistically significant difference in evaluating the expression of OCN and OPN on the palatal suture between the FB23-2 and DMSO groups (P < .05). The molecular mechanisms by which FTO regulates SuSCs osteogenesis remain to be elucidated. The FTO conditional knock out mouse model can be established to further elucidate the role of FTO during RME. FTO contributes to the osteogenic differentiation of SuSCs and plays a promoting role in midpalatal suture bone remodeling during the RME.

Abstract WMP114: EZH2 Inhibitor Improves Cognitive Behavior in CAA

Introduction: Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid-β (Aβ) in the leptomeningeal and cortical vessels. CAA may lead to dementia and stroke; however effective options currently are very limited to treat or halt CAA progression. EZH2 (enhancer of zeste 2 polycomb repressive complex 2) is a histone methyltransferase, a key regulator of inflammation and microglia phagocytosis. The present study investigates the therapeutic potential of an EZH2 inhibitor (GSK343) in CAA mice. Methods: Brain samples were collected from a cohort of 4 WT and 4 TgSwDI (CAA model) male mice and lysed for immunoblot analysis to quantify EZH2 and H3K27me3 expression levels. A second cohort of 10 WT and 20 TgSwDI male mice (15 months old) were randomly assigned into DMSO (vehicle) and GSK343 groups. After performing baseline behavioral testing (Barnes maze and NORT), WT and TgSwDI-DMSO groups were injected with 0.1% DMSO while TgSwDI-GSK343 groups were injected with GSK343 (5 mg/kg) every other day intraperitoneally. After 6 weeks of treatment, behavioral testing was performed, and brain tissues were collected for IHC. Data are presented as Mean±SEM. Results: Immunoblot assays showed elevated expression of EZH2 and H3K27me3 in TgSwDI in comparison to WT mice. TgSwDI mice injected with GSK343 had improvements in the ability to recognize a novel object (58.36%±1.84) as compared to TgSwDI controls (45.04%±3.41) in the NORT ( p = 0.0006). The latency to escape (63.08±2.86) was significantly faster in GSK343-treated mice compared to TgSwDI control mice (77.34±3.43) in the Barnes maze ( p = 0.003). IHC results were in line with behavioral testing as GSK343 reduced the expression of EZH2 and Aβ in the hippocampus and frontal cortex. Conclusions: Our study revealed that EZH2 expression is upregulated in the brain of CAA-model mice. Pharmacological inhibition of EZH2 improved cognitive outcomes in CAA mice, suggesting a possible therapeutic target.

Mechanism of Shikonin on spinal cord injury in rats based on TNFR/RIPK1 pathway

To explore the effect of shikonin on the recovery of nerve function after acute spinal cord injury(SCI) in rats. 96 male Sprague-Dawley(SD)rats were divided into 4 groups randomly:sham operation group (Group A), sham operation+shikonin group (Group B), SCI+ DMSO(Group C), SCI+shikonin group (Group D).The acute SCI model of rats was made by clamp method in groups C and D . After subdural catheterization, no drug was given in group A. rats in groups B and D were injected with 100 mg·kg-1 of shikonin through catheter 30 min after modeling, and rats in group C were given with the same amount of DMSO, once a day until the time point of collection tissue. Basso-Beattie-Bresnahan(BBB) scores were performed on 8 rats in each group at 6, 12, and 3 d after moneling, and oblique plate tests were performed on 1, 3, 7 and 14 d after modeling, and then spinal cord tissues were collected. Eight rats were intraperitoneally injected with propidine iodide(PI) 1 h before sacrificed to detection PI positive cells at 24 h in each group. Eight rats were sacrificed in each group at 24 h after modeling, the spinal cord injury was observed by HE staining.The Nissl staining was used to observe survivor number of nerve cells. Western-blot technique was used to detect the expression levels of Bcl-2 protein and apoptosis related protein RIPK1. After modeling, BBB scores were normal in group A and B, but in group C and D were significantly higher than those in group A and B. And the scores in group D were higher than those in group C in each time point (P<0.05). At 12 h after modeling, the PI red stained cells in group D were significantly reduced compared with that in group C, and the disintegration of neurons was alleviated(P<0.05). HE and Nissl staining showed nerve cells with normal morphology in group A and B at 24h after operation. The degree of SCI and the number of neuronal survival in group D were better than those in group C, the difference was statistically significant at 24h (P<0.05). The expression of Bcl-2 and RIPK1 proteins was very low in group A and B;The expression of RIPK1 was significantly increased in Group C and decreased in Group D, with a statistically significant difference (P<0.05);The expression of Bcl-2 protein in group D was significantly higher than that in group C (P<0.05). Shikonin can alleviate the pathological changes after acute SCI in rats, improve the behavioral score, and promote the recovery of spinal nerve function. The specific mechanism may be related to the inhibition of TNFR/RIPK1 signaling pathway mediated necrotic apoptosis.

Plectin Promotes the Migration of Hepatocellular Carcinoma Cells Through Inducing F-actin Polymerization

To explore the relationship between the expression of plectin and the migration of hepatocellular carcinoma (HCC) cells and to elucidate the molecular mechanisms by which plectin expression affects the migration of HCC cells. First of all, Western blot was performed to determine the expression of plectin in normal hepatocytes and HCC cells. Secondly, a plectin-downregulated HCC cell strain was established and the control group (shNC group) and shPLEC group were set up. Each group was divided into a vehicle control group (shNC+DMSO group or shPLEC+DMSO group) and a F-actin cytoskeleton polymerization inducer Jasplakinolide group (shNC+Jasp group or shPLEC+Jasp group). Western blot was performed to determine the expression of plectin and epithelial-mesenchymal transition (EMT)-related proteins, including N-cadherin, vimentin, and E-cadherin. HCC cell migration was evaluated by Transwell assay. KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to analyze the signaling pathways related to plectin gene. The polymerization of F-actin was analyzed by immunofluorescence assay. Compared with the normal hepatocytes, HCC cells showed high expression of plectin. Compared with those in the shNC group, the expression of plectin in the shPLEC group was decreased (P<0.05), the migration ability of HCC cells was weakened (P<0.05), and the EMT process was inhibited (with the expression of N-cadherin and vimentin being decreased and the expression of E-cadherin being increased) (P<0.05). KEGG analysis showed that the regulation of cytoskeletal F-actin was most closely associated with plectin and cytoskeletal F-actin depolymerized in the shPLEC group. After treatment with Jasplakinolide, an inducer of F-actin cytoskeleton polymerization, the migration ability of HCC cells in the shPLEC+Jasp group was enhanced compared with that of shPLEC+DMSO group (P<0.05) and the EMT process was restored (with the expression of N-cadherin and vimentin being increased and the expression of E-cadherin being decreased) (P<0.05). In addition, the polymerization of cytoskeletal F-actin in HCC cells was also restored. Plectin is highly expressed in HCC cells. Plectin promotes the migration and the EMT of HCC cells through inducing F-actin polymerization.

Evaluation of the antiplatelet effect of generic ticagrelor 90 mg (ticaspan ® ) alone and in combination with aspirin 75 mg as compared to ticagrelor (innovator): An in vitro study.

To evaluate an in vitro antiplatelet effect of generic ticagrelor 90 mg (ticaspan) alone and in combination with aspirin 75 mg as compared to the innovator formulation of ticagrelor alone and in combination with aspirin among healthy Indian volunteers. 18 volunteers were enrolled and platelet viability was tested using lactate dehydrogenase (LDH) assay in six of 18 volunteers. In 12 volunteers, maximum platelet aggregation (MPA) and percentage inhibition of platelet aggregation (PI) were assessed using a platelet aggregometer in six study groups. There was no significant increase in LDH levels when platelets were incubated with an innovator or generic drug alone and in combination with aspirin as compared to the dimethyl sulfoxide [DMSO] group. All five study groups showed a significant reduction in the MPA values compared to the DMSO group ( P < 0.01). The extent of decrease in MPA observed with the generic drug was not significantly different from the innovator drug ( P = 0.325). Similarly, the MPA observed with the two combination groups did not differ from each other ( P = 1.000), but it was significantly different from the MPA observed with aspirin ( P = 0.039, each). The PI of platelet aggregation was significantly more in four study groups [generic drug alone; innovator alone; generic drug + aspirin; and innovator drug + aspirin] ( P < 0.01) as compared to the aspirin group. The generic ticagrelor and its combination with aspirin demonstrated an antiplatelet effect equivalent to the innovator drug and its combination with aspirin.

CNOT4 Knockout Induces Proteasome Inhibitor Resistance in Multiple Myeloma Cells

Background: The outcomes of multiple myeloma (MM) patients have been significantly improved with novel therapies including proteasome inhibitors (PI). However, many patients develop PI resistance and becoming refractory to PI. Although several studies have identified the mechanisms of PI resistance, such as mutation to the proteasome β5 subunit, ER stress suppression, autophagy activation, and activation of efflux drug pumps, it remains unclear yet. To investigate new genes that may contribute to PI resistance, we performed a genome-wide CRISPR dropout screening in an MM cell line. Materials and Methods: To identify the genes responsible for PI resistance, we used a CRISPR library (Kosuke Yusa, V3) targeting 18,740 human genes. The knockout cells, in which an CRISPR library lentivirus was introduced into a Cas9-expressing AMO1 MM cell line, were treated with ixazomib (IXA), carfilzomib (CFZ), and DMSO for two weeks. Next, we used next generation sequencing (NGS) to count sgRNA copy numbers of the pre-drug control group and the drug-treated group. NGS analysis of the treated cells showed that inactivated genes which induced higher levels of cell proliferation in the PI group than in the DMSO group were considered candidate genes for PI resistance. Results: We identified 35 genes for PI resistance including the genes encoding subunits of the proteasome 19S complex, consistent with previous reports that shows reducing 19S subunits protects MM cells from bortezomib. Among 35 candidate genes, we chose the top hit gene, CNOT4, to further investigate. CNOT4 is a component of CCR4-NOT complex which removes the mRNA poly(A) tail for degradation of mRNA. In addition, CNOT4 has E3 ubiquitin ligase activity and is reported to control the proteasome complex formation. To further validate the effect of PI resistance with inactivated CNOT4, we generated CNOT4 KO AMO1 cells and performed an MTS assay. We observed a decrease in PI sensitivity compared with AMO1 wildtype (WT) (IXA IC 50: 24.8 nM (KO) vs 13.0 nM (WT), CFZ IC 50: 7.45 nM (KO) vs 2.76 nM (WT)). To investigate the influence of CNOT4 on proteasome complex formation and its activity, we first measured the expression of ubiquitinated proteins by Western blotting. In CNOT4 KO AMO1 cells, the expression of ubiquitinated proteins is increased with or without PI treatment, suggesting that inactivated CNOT4 can suppress the function of degrade ubiquitinated proteins in proteasome 19S complex. To elucidate the mechanism underlying the resistance effect, we next checked the expression of ER stress marker proteins. In CNOT4 KO AMO1 cells, the expression of PERK and IRE1α proteins are decreased with PI treatment, suggesting that inactivated CNOT4 can suppress the ER stress levels in spite of the increases of ubiquitinated proteins. Conclusion: Our CRISPR screen revealed that CNOT4 inactivation induced PI resistance in human MM cells. CNOT4 inactivation suppresses the function of 19S proteasome, which is reported the influence of PI resistance, and downregulates the ER stress signal leading to avoid PI induced cell death. Our findings demonstrate that CNOT4 plays an important role in PI sensitivity.