Evaluation on the Metabolic Activity of Two Carboxylesterase Isozymes in Mouse Liver Microsomes by a LC-MS/MS Method.

An applicable method for the precise measurement of major carboxylesterase (CESs) activity in liver still limited. Clopidogrel and irinotecan are specific substrates for CES1 and CES2, respectively. Clopidogrel is metabolized to the inactive metabolite clopidogrel carboxylate (CCAM) by CES1. Irinotecan is metabolized to the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38) by CES2. In the present study, the LC-MS/MS method for the determination of CCAM and SN-38 were separately developed to characterize the metabolic activities of CES1 and CES2 in mouse liver microsomal. CCAM was separated on a Ecosil ODS column with an isocratic mobile phase consisted of 5 mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile (15:85, V:V) at a flow rate of 0.4mL/min. SN-38 was separated on a Waters symmetry C18 column with an gradient mobile phase consisted of 5 mmol/L ammonium formate and 0.1% formic acid in water and acetonitrile at a flow rate of 0.3 mL/min. Calibration curves were linear within the concentration range of 100-20,000 ng/mL for CCAM and 1-200 ng/mL for SN-38. The results of method showed excellent accuracy and precision. The recovery rate, matrix effect and stability inspection results were within the acceptance criteria. The optimized incubation conditions were as follows: protein concentration of microsomes were all 0.1 mg/mL, incubation time was 60 min for clopidogrel and 30 min for irinotecan, respectively. This method was sensitive and applicable for the determination of the activity of CESs in the mouse liver microsomes.

Systematic analysis of critical genes and pathways identified a signature of neuropathic pain after spinal cord injury.

Spinal cord injury (SCI) damages sensory systems, producing chronic neuropathic pain that is resistant to medical treatment. The specific mechanisms underlying SCI-induced neuropathic pain (SCI-NP) remain unclear, and protein biomarkers have not yet been integrated into diagnostic screening. To better understand the host molecular pathways involved in SCI-NP, we used the bioinformatics method, the PubMed database and bioinformatics methods to identify target genes and their associated pathways. We reviewed 2504 articles on the regulation of SCI-NP and used the text mining of PubMed database abstracts to determine associations among 12 pathways and networks. Based on this method, we identified two central genes in SCI-NP: interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α). Adult male Sprague-Dawley rats were used to build the SCI-NP models. The threshold for paw withdrawal was significantly reduced in the SCI group, and TLR4 was activated in microglia after SCI. Enzyme-linked immunosorbent assay（ELISA) analysis of TNF-α and IL-6 levels was significantly higher in the SCI group than in the sham group. Western blot showed that expressions of the TLR4/MyD88/NF-κB inflammatory pathway protein increased dramatically in the SCI group. Using the TLR4 inhibitor TAK-242, the pain threshold and expressions of inflammatory factors and proteins of the proteins of the inflammatory signal pathway were reversed, TLR4 in microglia was suppressed, suggesting that SCI-NP was related to neuroinflammation mediated by the TLR4 signalling pathway. In conclusion, we found that TNF-α and IL-6 were the neuroinflammation-related genes involved in SCI-NP that can be alleviated by inhibiting the inflammatory pathway upstream of the TLR4/MyD88/NF-κB inflammatory pathway.

A novel protoapigenone analog RY10-4 induces apoptosis of breast cancer cells by exacerbating mitochondrial Ca2+ influx through mitochondrial calcium uniporter.

RY10-4, a novel protoapigenone analog with a specific nonaromatic B-ring, displayed enhanced cytotoxicity in various tumor cells, especially for breast cancer cells, but the underlying mechanism remains unclear. In the present study, we confirmed the pro-apoptotic effect of RY10-4 on breast cancer cells. Furthermore, mitochondrial calcium uniporter (MCU) was proved to be up-regulated in RY10-4-treated MDA-MB-231 cells, which resulted in the overload of mitochondrial calcium ([Ca2+]m) and subsequently disrupted mitochondrial functions (characterized by mitochondrial reactive oxygen species (mtROS) accumulation, membrane potential (ΔΨm) depolarization and permeability transition pore (mPTP) opening). And finally, the mitochondrial apoptosis was activated by the release of cytochrome C. Interestingly, knockdown of MCU attenuated the overload of [Ca2+]m and blocked the apoptosis of MDA-MB-231 cells induced by RY10-4, which was consistent with the in vivo results. Taken together, this study proved that RY10-4 could induce apoptosis of breast cancer cells by elevating [Ca2+]m through MCU. Our work contributed previously unknown insights into the mechanisms involving in the clinical efficacy of RY10-4 on breast cancer cells, which also advanced calcium homeostasis as a potential target for chemotherapeutic drugs.

Oxaliplatin-induced hepatic sinusoidal obstruction syndrome.

Oxaliplatin (OXA) is a third-generation platinum anticancer drug that is mainly used for the treatment of metastatic colorectal cancer (CRC). Of note, hepatic sinusoidal obstruction syndrome (HSOS) induced by OXA has become a key concern for patients with CRC receiving chemotherapy with OXA in recent years. Splenomegaly, thrombocytopenia, abnormal liver function, and portal hypertension are some of the main clinical characteristics seen in patients with OXA-induced HSOS. Previous studies have suggested that oxidative stress, inflammatory damage, liver fibrosis, and platelet aggregation and adhesion may be involved in the pathogenesis of OXA-induced HSOS. Currently, there are no specific drugs for prevention and treatment of OXA-induced HSOS. In this review, we summarized the epidemiology, pathological characteristics, clinical predictive indicators, related mechanisms, possible prevention and treatment of OXA-related HSOS.

The different mechanisms of peripheral and central TLR4 on chronic postsurgical pain in rats.

Chronic postsurgical pain (CPSP) is a common complication after surgery; however, the underlying mechanisms of CPSP are poorly understood. As one of the most important inflammatory pathways, the Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling pathway plays an important role in chronic pain. However, the precise role of the TLR4/NF-κB signaling pathway in CPSP remains unclear. In the present study, we established a rat model of CPSP induced by skin/muscle incision and retraction (SMIR) and verified the effects and mechanisms of central and peripheral TLR4 and NF-κB on hyperalgesia in SMIR rats. The results showed that TLR4 expression was increased in both the spinal dorsal horn and dorsal root ganglia (DRGs) of SMIR rats. However, the TLR4 expression pattern in the spinal cord was different from that in DRGs. In the spinal cord, TLR4 was expressed in both neurons and microglia, whereas it was expressed in neurons but not in satellite glial cells in DRGs. Further results demonstrate that the central and peripheral TLR4/NF-κB signaling pathway is involved in the SMIR-induced CPSP by different mechanisms. In the peripheral nervous system, we revealed that the TLR4/NF-κB signaling pathway induced upregulation of voltage-gated sodium channel 1.7 (Nav1.7) in DRGs, triggering peripheral hyperalgesia in SMIR-induced CPSP. In the central nervous system, the TLR4/NF-κB signaling pathway participated in SMIR-induced CPSP by activating microglia in the spinal cord. Ultimately, our findings demonstrated that activation of the peripheral and central TLR4/NF-κB signaling pathway involved in the development of SMIR-induced CPSP.

Vertical transmission and kidney damage in newborns whose mothers had coronavirus disease 2019 during pregnancy.

OBJECTIVES: Coronavirus disease 2019 (COVID-19) has become a worldwide pandemic. However, the hazard to newborns in pregnancy remains controversial. The aim of this study was to investigate the vertical transmission of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from mother to child and developmental toxicity in the fetus. METHODS: All clinical information was recorded on 22 neonates born to mothers with confirmed COVID-19 pneumonia in Tongji Hospital. RESULTS: The average birth weight of the 22 newborns (16 males and 6 females) was 2980 g, and the mean gestational week was 37W+3. The birth weight of three babies was <2500 g, and the gestational week of all three low-birth-weight neonates was less than 36W. Three newborns had minor lesions of infection in the lungs as shown by computed tomography (CT) scans. Furthermore, three newborns had elevated SARS-CoV-2-related immunoglobin M (IgM) antibodies, and 11 newborns (52.4%) had positive immunoglobin G (IgG) antibodies. Notably, both cystatin C and ß2-microglobulin were increased in all newborns. Five of the 21 tested newborns had leukocytosis, and 11 had increased neutrophil levels. In addition, the aspartate aminotransferase of 18 newborns and the γ-glutamyl transpeptidase of 19 newborns were increased. Total bilirubin was elevated in all newborns and serum albumin was reduced in 20 of 22 newborns. CONCLUSIONS: This study was the first to discover that COVID-19 infection in the third trimester of pregnancy could cause fetal kidney developmental injury, as indicated by increased cystatin C and ß2-microglobulin in all neonates. Furthermore, there is the possibility of maternal-fetal transmission of SARS-CoV-2.

MiR-30b-5p attenuates oxaliplatin-induced peripheral neuropathic pain through the voltage-gated sodium channel Nav1.6 in rats.

Oxaliplatin is a third-generation derivative of platinum that is effective in the treatment of multiple solid tumors. However, it can cause peripheral neuropathic pain, and the molecular mechanisms of this effect remain unknown. We induced a model of peripheral neuropathic pain in rats by intraperitoneally injecting them with oxaliplatin twice a week for 4.5 weeks. We found that both the mRNA and protein expression levels of Nav1.6 (encoded by the gene Scn8a) increased while the miR-30b-5p (shorthand for miR-30b) expression decreased in the dorsal root ganglion (DRG) of treated rats. Using TargetScan and miRanda predictive software, we discovered that Scn8a was a major target of miR-30b. Moreover, we found that miR-30b negatively regulated Scn8a by binding to the Scn8a 3'UTR in PC12 cells. In addition, Nav1.6 and miR-30b were colocalized in the DRG neurons of naive rats. Overexpression of miR-30b using an miR-30b agomir attenuated neuropathic pain induced by oxaliplatin and inhibited both the mRNA and protein expression levels of Nav1.6 both in vitro and in vivo. Conversely, the inhibition of miR-30b with an miR-30b antagomir resulted in neuropathic pain and an increase in the expression of Nav1.6. More importantly, overexpression of miR-30b inhibited the proliferation of LS-174t cells (Colorectal cancer cells). These data suggest that miR-30b contributes to oxaliplatin-induced chronic neuropathic pain through Nav1.6 downregulation and could be a novel therapeutic target for the treatment of oxaliplatin-induced neuropathic pain as a side effect of chemotherapy in cancer patients.

HDAC2, but not HDAC1, regulates Kv1.2 expression to mediate neuropathic pain in CCI rats.

The expression of potassium ion channel subunit 1.2 (Kv1.2) in the dorsal root ganglion (DRG) influences the excitability of neurons, which contributes to the induction and development of neuropathic pain (NPP); however, the molecular mechanisms underlying the downregulation of Kv1.2 in NPP remain unknown. Histone deacetylase (HDAC) inhibitors are reported to attenuate the development of pain hypersensitivity in rats with NPP. Whether HDAC inhibitors contribute to regulation of Kv1.2 expression, and which specific HDAC subunit is involved in NPP, remain unexplored. In this study we established a chronic constrictive injury (CCI) model and used western blot, quantitative real-time PCR, immunostaining, intrathecal injection, and siRNA methods to explore which HDAC subunit is involved in regulating Kv1.2 expression to mediate NPP. Our results demonstrated that nerve injury led to upregulation of HDAC1 expression in the DRG, and of HDAC2 in the DRG and spinal cord. Double-labeling immunofluorescence histochemistry showed that Kv1.2 principally co-localized with HDAC2, but not HDAC1, in NF200-positive large neurons of the DRG. Intrathecal injection with the HDAC inhibitor, suberoylanilide hydroxamic acid, attenuated mechanical and thermal hypersensitivity and reversed the decreased expression of Kv1.2 in rats with CCI. Furthermore, treatment with HDAC2, but not HDAC1, siRNA also relieved mechanical and thermal hypersensitivity and upregulated the Kv1.2 expression in this model. In vitro transfection of PC12 cells with HDAC2 and HDAC1 siRNA confirmed that only HDAC2 siRNA could regulate the expression of Kv1.2. These findings suggest that HDAC2, but not HDAC1, is involved in NPP through regulation of Kv1.2 expression.

Baicalin Protects Against 17α-Ethinylestradiol-Induced Cholestasis via the Sirtuin 1/Hepatic Nuclear Receptor-1α/Farnesoid X Receptor Pathway.

Estrogen-induced cholestasis (EIC) is characterized by impairment of bile flow and accumulated bile acids (BAs) in the liver, always along with the liver damage. Baicalin is a major flavonoid component of Scutellaria baicalensis, and has been used in the treatment of liver diseases for many years. However, the role of baicalin in EIC remains to be elucidated. In this study, we demonstrated that baicalin showed obvious hepatoprotective effects in EIC rats by reducing serum biomarkers and increasing the bile flow rate, as well as by alleviating liver histology and restoring the abnormal composition of hepatic BAs. In addition, baicalin protected against estrogen-induced liver injury by up-regulation of the expression of hepatic efflux transporters and down-regulation of hepatic uptake transporters. Furthermore, baicalin increased the expression of hepatic BA synthase (CYP27A1) and metabolic enzymes (Bal, Baat, Sult2a1) in EIC rats. We showed that baicalin significantly inhibited hepatic inflammatory responses in EIC rats through reducing elevated levels of TNF-α, IL-1ß, IL-6, and NF-κB. Finally, we confirmed that baicalin maintains hepatic BA homeostasis and alleviates inflammation through sirtuin 1 (Sirt1)/hepatic nuclear receptor-1α (HNF-1α)/farnesoid X receptor (FXR) signaling pathway. Thus, baicalin protects against estrogen-induced cholestatic liver injury, and the underlying mechanism involved is related to activation of the Sirt1/HNF-1α/FXR signaling pathway.

Assessment of dose-response relationship of 5-fluorouracil to murine intestinal injury.

5-Fluorouracil (5-FU) is the most frequently prescribed anti-tumor drug, but has been reported to result in intestinal injury. Although some progress has been made in understanding the intestinal toxicity of 5-FU, confusion remains about animal models of 5-FU-induced intestinal injury, especially the dosage of 5-FU. This study aims to assess the dose-response relationship between the severity of intestinal injury and different doses of 5-FU, and to determine a proper dosing for the murine model. We found that mice in the 5-FU groups gradually lost body weight over time. Increasing doses of 5-FU resulted in more severe diarrhea, with a concomitant increase in mortality. Histopathological damage was more severe in mice that received higher doses of 5-FU. In addition, plasma diamine oxidase (DAO) activity decreased in experimental mice with intestinal injury in a dose-dependent way. TUNEL and western blot analysis showed cell apoptosis in the ileum and colon related to 5-FU dosage. However, administration of 200 and 400 mg/kg 5-FU caused extremely high mortality, severe diarrhea and histopathological damage, but 25 mg/kg 5-FU did not result in significant intestinal injury. The severity of intestinal injury induced by 5-FU appeared to be dose-dependent and we concluded that the proper dosage of 5-FU to induce a murine model with intestinal mucositis ranged from 50 mg/kg to 100 mg/kg.

Glucocorticoid receptor inhibit the activity of NF-κB through p38 signaling pathway in spinal cord in the spared nerve injury rats.

AIMS: The glucocorticoid receptors (GRs) are an active regulator in inflammatory responses. The inflammatory reaction plays an important role in neuropathic pain, but the underlying mechanisms that GR regulates the inflammatory responses in neuropathic pain are still unknown. The activation of GRs has been shown to participate in the p38MAPK-mediated suppression of transcription activation. An unanswered question is whether GRs take part in inflammatory responses in neuropathic pain through p38MAPK signaling pathway. MAIN METHODS: The spared nerve injury (SNI) in rats was used as a model of neuropathic pain. Pain sensitivity was tested by von Frey filaments. The expression of GR, p-p38 and NF-κB were detected by Western blot and immunofluorescence. Elisa was used to examine the expression of IL-6 and TNF-α. KEY FINDINGS: Nerve injury led to p38 activation and GR expression decline in spinal cord of SNI rats. Intrathecal injection of the p38MAPK antagonist SB203580 activated GR and decreased NF-κB, resulting in pain relief since 3 days post-operation in SNI rats. Moreover, Intrathecal injection of the GR antagonist RU38486 counteracted the effect of SB203580 on NF-κB expression along with the release of IL-6 and TNF-α. On the contrary, activation of the GR by intrathecal administration of dexamethasone, a GR agonist, inhibited the expression of NF-κB and the release of IL-6 and TNF-α, resulting in pain relief. SIGNIFICANCE: Activation of p38MAPK in spinal cord could downregulate the GR expression and thereby activate NF-κB, thus promoting the release of IL-6 and TNF-α and participating in the development of neuropathic pain.

Meta-analysis of stem cell transplantation for reflex hypersensitivity after spinal cord injury.

Stem cells have been used in novel therapeutic strategies for spinal cord injury (SCI), but the effect of stem cell transplantation on neuropathic pain after SCI is unclear. The current meta-analysis evaluates the effects of stem cell transplantation on neuropathic pain after SCI. We first conducted online searches of PubMed, Web of Science, China Academic Journals Full-text Database, and Wanfang Data for randomized controlled trials that compared stem cell transplantation and vehicle treatments in rodent models of neuropathic pain after SCI. Quality assessment was performed using Cochrane Reviewer's Handbook 5.1.0, and meta-analysis was conducted with RevMan 5.3. Then, we developed a rat model of SCI and transplanted bone marrow mesenchymal stem cells to verify meta-analysis results. Twelve randomized, controlled trials (n=354 total animals) were included in our meta-analysis and divided by subgroups, including species, timing of behavioral measurements, and transplantation time after SCI. Subgroup analysis of these 12 studies indicated that stem cell-treated animals had a higher mechanical reflex threshold than vehicle groups, with a significant difference in both rats and mice. The thermal withdrawal latency showed the same results in mouse subgroups, but not in rat subgroups. In addition, mesenchymal stem cell transplantation was an effective treatment for mechanical, but not thermal reflex hypersensitivity relief in rats. Transplantation showed a positive effect when carried out at 3 or 7days post-SCI. Stem cell transplantation alleviates mechanical reflex hypersensitivity in rats and mice and thermal reflex hypersensitivity in mice after SCI.

Neuroprotective effect of three caffeic acid derivatives via ameliorate oxidative stress and enhance PKA/CREB signaling pathway.

This study was conducted to elucidate the neuroprotective effect of caffeic acid phenethyl ester (CAPE), (R)-2-Hydroxy-3-(3,4-dihydroxyphenyl) propionic acid (Danshensu) and Curcumin, three caffeic acid derivatives which are contained in fruits, grains and certain dietary supplements. Our results showed that these compounds significantly attenuated H2O2-induced toxicity in PC12 cells in a dose-dependent manner. Furthermore, these compounds significantly improved the behavioral performance of d-gal-treated mice in both step-down avoidance test and Morris water maze test. Biochemical examination and western blot analysis showed that these compounds could ameliorate oxidative stress and facilitate activation of the protein kinase A (PKA)-cyclic AMP response element-binding protein (CREB) pathway. Its beneficial effects may partly relate to enhancing the activity of endogenous antioxidant enzymes and modulating the PKA/CREB signaling pathway. Furthermore, our results also indicated that the presence of 3, 4-dihydroxyphenyl groups in A ring may enhance their neuroprotective activity.

Small Interfering RNA Targeting Dickkopf-1 Contributes to Neuroprotection After Intracerebral Hemorrhage in Rats.

Excessive Dickkopf-1 (Dkk-1) plays a vital role in secondary brain injury following ischemic stroke and psychotic disease. However, it is unclear whether an increased expression of Dkk-1 occurred after intracerebral hemorrhage (ICH). The present study examined the potential role of Dkk-1 after ICH. ICH was induced by a single injection of autologous blood into the basal ganglia of rats. Dkk-1 protein levels in brain tissue and serum were detected by enzyme-linked immunosorbent assay after ICH. Rats were treated with small interfering RNA targeting Dkk-1 (siDkk-1) or vehicle following ICH. Behavioral deficits and brain water content were examined. Blood-brain barrier (BBB) integrity was detected by Evans blue extravasation and observed by transmission electron microscopy. Wnt-1 was evaluated by real-time RT-PCR. The tight junction protein zonula occludens-1 (ZO-1) was investigated by immunohistochemistry and Western blot assays. Serum level of Dkk-1 did not differ between the ICH and sham groups. However, the level of Dkk-1 in brain tissue was significantly increased at 24 and 72 h after ICH. BBB disruption and brain edema, as well as neurological deficits, were remarkably ameliorated by administration of siDkk-1. Moreover, siDkk-1 treatment significantly increased the transcription of Wnt-1 mRNA and upregulated the expression of ZO-1. These results provide the first evidence that siDkk-1 treatment is neuroprotective against secondary injury including brain edema and BBB permeability following ICH; the mechanism of neuroprotection may be associated with improvement of BBB integrity.

MicroRNA-30b regulates expression of the sodium channel Nav1.7 in nerve injury-induced neuropathic pain in the rat.

Voltage-gated sodium channels, which are involved in pain pathways, have emerged as major targets for therapeutic intervention in pain disorders. Nav1.7, the tetrodotoxin-sensitive voltage-gated sodium channel isoform encoded by SCN9A and predominantly expressed in pain-sensing neurons in the dorsal root ganglion, plays a crucial role in nociception. MicroRNAs are highly conserved, small non-coding RNAs. Through binding to the 3' untranslated region of their target mRNAs, microRNAs induce the cleavage and/or inhibition of protein translation. Based on bioinformatics analysis using TargetScan software, we determined that miR-30b directly targets SCN9A To investigate the roles of Nav1.7 and miR-30b in neuropathic pain, we examined changes in the expression of Nav1.7 in the dorsal root ganglion by miR-30b over-expression or knockdown in rats with spared nerve injury. Our results demonstrated that the expression of miR-30b and Nav1.7 was down-regulated and up-regulated, respectively, in the dorsal root ganglion of spared nerve injury rats. MiR-30b over-expression in spared nerve injury rats inhibited SCN9A transcription, resulting in pain relief. In addition, miR-30b knockdown significantly increased hypersensitivity to pain in naive rats. We also observed that miR-30b decreased Nav1.7 expression in PC12 cells. Taken together, our results suggest that miR-30b plays an important role in neuropathic pain by regulating Nav1.7 expression. Therefore, miR-30b may be a promising target for the treatment of chronic neuropathic pain.

ADAM10 mediates the cell invasion and metastasis of human esophageal squamous cell carcinoma via regulation of E-cadherin activity.

A disintegrin and metalloprotease 10 (ADAM10) is involved in the tumorigenesis, invasion and metastasis of several types of solid tumors. However, the potential role of ADAM10 in human esophageal squamous cell carcinoma (ESCC) is not yet well understood. The present study showed that ADAM10 was overexpressed in human ESCC tissues in vivo, and positively associated with depth of tumor invasion, lymph node metastasis and TNM stage, contributing to tumor carcinogenesis, invasion and metastasis. Additionally, ADAM10 was overexpressed in 3 types of ESCC cell lines in vitro, as compared to that in normal esophageal epithelial cells (NEECs); and moreover, ESCC cells with high ADAM10 expression obtained enhanced invasion and migration ability. Subsequently, ADAM10 silencing by small interfering (si) RNA in ESCC cell line, EC-1, reduced cell invasion, migration and proliferation in vitro. Finally, ADAM10 negatively regulated E-cadherin in ESCC in vivo and in vitro. In conclusion, active ADAM10 promotes the carcinogenesis, invasion, metastasis and proliferation of ESCC and controls invasion and metastasis at least in part through the shedding of E-cadherin activity, which makes it a potential biomarker and a useful therapeutic target for ESCC.

The growth-inhibitory and apoptosis-inducing effect of deferoxamine combined with arsenic trioxide on HL-60 xenografts in nude mice.

OBJECTIVE: The aim of this study is to investigate the growth-inhibitory and apoptosis-inducing effect of deferoxamine (DFO) combined with arsenic trioxide (ATO) on the human HL-60 xenografts in nude mice and its mechanism. METHOD: The highly tumorigenic leukemia cell line HL-60 cells were inoculated subcutaneously into nude mice to establish a human leukemia xenograft model. The HL-60 xenograft nude mice models were randomly divided into four groups: control (Normal saline, NS), 50mg/kg DFO, 3mg/kg ATO, the combined treatment (50mg/kg DFO+1.5mg/kg ATO) once HL-60 cells were inoculated. Tumor sizes, growth curves, inhibitory rates, cell apoptosis, and the expression of apoptosis related markers were measured to evaluate the tumor growth. RESULTS: Xenografted tumors were observed in all nude mice since the 5th day of inoculation. The inhibitory rates of tumor weight were 2.67%, 10.69%, and 25.57% in DFO, ATO and combination therapy groups, respectively. The combination of DFO with ATO induces significantly more tumor cell apoptosis than either agent alone (p<0.05). The expression of NF-κBp65 and survivin proteins decreased significantly while the expression of Caspase-3 and Bax increased in the combination therapy group (p<0.05). Double immunofluorescence for Caspase-3 and NFκBp65 demonstrated an inverse relationship between Caspase-3-positive areas and NFκBp65-positive areas, as well as the co-localization of Bax and survivin in xenografted tumor cells. CONCLUSIONS: Combination of DFO and ATO has synergistic effects on tumor growth inhibition and apoptosis-inducing in vivo with no significant side effects. The DFO and ATO can up-regulate the expression of Caspase-3 and Bax, and down-regulate the expression of NF-κBp65 and survivin, especially for their combination.

Endoplasmic reticulum stress-mediated signaling pathway of gastric cancer apoptosis.

BACKGROUND/AIMS: To investigate ER stress-mediated CHOP-signaling pathway of gastric cancer apoptosis in vitro and in vivo. METHODOLOGY: Based on the dose-and time-response experiments about tunicamycin (TM),gastric cancer cell line BGC823 was treated with 10tg/mL of TM for 24h. BGC823 apoptosis was detected with TUNEL assay and ultrastructural changes in BGC823 cells under ER stress were observed with transmission electron microscopy (TEM). RT-PCR and western blot-ting were used to determine the expression of ERS-related proteins, glucose-regulated protein 78 (GRP78) and CHOP and apoptosis-associated protein B-cell lympho-ma 2 (Bcl-2). After the knockdown of CHOP, the changes were also observed in vitro and in vivo. RESULTS: TEM assay showed that after treatment with TM, BGC823 cell size became smaller with ER dilation, vacuolization and karyopyknosis. RT-PCR and western blotting indicated that TM up-regulated GRP78 and CHOP expression and down-regulated Bcl-2 expression. The knock-down of CHOP could convert Bcl-2 expression and reduce BGC823 apoptosis caused by ERS in vitro and in vivo, but failed to influence GRP78. CONCLUSIONS: TM can induce ESR and regulate downstream molecules CHOP up-regulation and Bcl-2 down-regulation which lead to BGC823 apoptosis. This study may provide a new theoretical basis for the pathogenesis of gastric cancer.

[Effects of estrogen on P-Tau, ChAT and nerve growth factor protein expressions in the brain tissue of rats with Alzheimer's disease].

OBJECTIVE: To examine the effect of estrogen on the expressions of phosphorylated Tau (P-Tau), ChAT and nerve growth factor (NGF) protein in the brain tissue of rat models of Alzheimer disease (AD). METHODS: Rat models of AD were established by injecting Aß1-42 protein fragments in the right lateral ventricle. Two weeks later, 17ß-estradiol tablets were implanted subcutaneously at the neck of the rats and maintained for 30 days. The pathological changes in the rats' brain neurons and alterations in the expressions of P-Tau, ChAT and NGF proteins were observed using HE staining and immunohistochemistry, respectively. RESULTS: In the AD rats, neurofibrillary tangles occurred in the brain tissue, and estrogen treatment significantly reduced the formation of neurofibrillary tangles. Estrogen treatment also resulted in lowered P-Tau expression and increased ChAT and NGF protein expressions in comparison with those in the AD model rats. CONCLUSION: Estrogen can up-regulate ChAT and NGF and down-regulate tau protein expression, thus producing obvious therapeutic effect on AD in rats.

[Effect of polyoxyl ether analogous surfactants on the activity of cytochromes P450 3A in rats in vivo].

To evaluate the effects of p-octyl polyethylene glycol phenyl ether (Triton X-100), polyoxyl 35 caster oil (EL35) and polyoxyl 40 hydrogenated caster oil (RH40) on the activity of Cytochrome P450 3A (CYP3 As) in vivo. Rats were administered with saline, ketoconazole (75 mg x kg(-1) x d(-1)), Triton X-100 (30 mg x kg(-1) x d(-1)), EL35 (150 mg x kg(-1) x d(-1)) and RH40 (150 mg x kg(-1) x d(-1)) intragastrically for 5 consecutive days, and then given midazolam 10 mg x kg(-1) 20 min after the last treatment of ketoconazole or three surfactants with the same dose through duodenal administration. Pharmacokinetics parameters for midazolam and its metabolite 1'-hydroxymidazolam were estimated from the plasma concentration-time data by a noncompartmental approach. The results showed that multiple dose administration of Triton X-100, EL35 and RH40 decreased the ratios of 1'-hydroxymidazolam and midazolam AUC0-infinity from 1.14 to 0.90, 1.03 and 0.64, respectively. In contrast, multiple dose administration of ketoconazole caused the ratios of 1'-hydroxymidazolam and midazolam a significant decrease to 0.50. This study indicated that Triton X-100 and EL35 would have no inhibition on CYP3A, while RH40 had significant inhibition on CYP3A. Therefore, RH40 might be used to prepare drug formulations in pharmaceutical industry and would increase the bioavailability of some drugs transformed by CYP3As and further lead to significant clinical pharmacologic effects.