Preparation of etoposide liposomes for enhancing antitumor efficacy on small cell lung cancer and reducing hematotoxicity of drugs.

Etoposide (VP16) is commonly used in the treatment of small cell lung cancer (SCLC) in clinical practice. However, severe adverse reactions such as bone marrow suppression toxicity limit its clinical application. Although several studies on VP16 liposomes were reported, no significant improvement in bone marrow suppression toxicity has been found, and there was a lack of validation of animal models for in vivo antitumor effects. Therefore, we attempted to develop a PEGylated liposomal formulation that effectively encapsulated VP16 (VP16-LPs) and evaluated its therapeutic effect and toxicity at the cellular level and in animal models. First, we optimized the preparation process of VP16-LPs using an orthogonal experimental design and further prepared them into freeze-dried powder to improve storage stability of the product. Results showed that VP16-LPs freeze-dried powder exhibited good dispersibility and stability after redispersion. In addition, compared to marketed VP16 injection, VP16-LPs exhibited sustained drug release characteristics. At the cellular level, VP16-LPs enhanced the cellular uptake of drugs and exhibited strong cytotoxic activity. In animal models, VP16-LPs could target and aggregate in tumors and exhibit a higher anti-tumor effect than VP16-injection after intravenous injection. Most importantly, hematological analysis results showed that VP16-LPs significantly alleviated the bone marrow suppression toxicity of drug. In summary, our study confirmed that PEGylated liposomes could enhance therapeutic efficacy and reduce toxicity of VP16, which demonstrated that VP16-LPs had enormous clinical application potential.

Performance of field demonstration nanoscale zero-valent iron in groundwater remediation: A review.

Nanoscale zero-valent iron (nZVI) has gained widespread usage in groundwater remediation due to its exceptional reactivity. Since its initial deployment in field demonstrations in 2001, nZVI has proven to be an effective nanomaterial for addressing groundwater contaminants. Subsequent research has highlighted the versatility of nZVI, showcasing its potential to overcome critical limitations associated with conventional remediation technologies. The effectiveness of nZVI in remediation varies, contingent on factors such as the type of nZVI, contaminant nature, site conditions, and injection methodologies employed. This review aims to present a comprehensive progress report on the field application of nZVI spanning 22 years across eight countries. Drawing from a database encompassing 32 pilot or full-scale remediation sites, the study delineates the various types of nZVI, modification methods, demonstration sites, and primary contaminants targeted in field tests. Specific attention is given to the application effects and mechanisms of unmodified nZVI, Pd, surfactants, and carbon-modified nZVI in diverse field demonstrations. An analysis of the key factors influencing their performance is provided, and potential future applications of nZVI in groundwater remediation are discussed.

Sulforaphane reduces adipose tissue fibrosis via promoting M2 macrophages polarization in HFD fed-mice.

Adipose tissue fibrosis has been identified as a novel contributor to the pathomechanism of obesity associated metabolic disorders. Sulforaphane (SFN) has been shown to have an anti-obesity effect. However, the impact of SFN on adipose tissue fibrosis is still not well understood. In this study, obese mice induced by high-fat diets (HFD) were used to examine the effects of SFN on adipose tissue fibrosis. According to the current findings, SFN dramatically enhanced glucose tolerance and decreased body weight in diet-induced-obesity (DIO) mice. Additionally, SFN therapy significantly reduced extracellular matrix (ECM) deposition and altered the expression of genes related to fibrosis. Furthermore, SFN also reduced inflammation and promoted macrophages polarization towards to M2 phenotype in adipose tissue, which protected adipose tissue from fibrosis. Notably, SFN-mediated nuclear factor E2-related factor 2 (Nrf2) activation was crucial in decreasing adipose tissue fibrosis. These results implied that SFN had favorable benefits in adipose tissue fibrosis, which consequently ameliorates obesity-related metabolic problems. Our research provides new treatment strategies for obesity and associated metabolic disorders.

Polypeptides as alternatives to PEGylation of therapeutic agents.

INTRODUCTION: Due to the concerns raised by the extensive application of PEGylation, polypeptides have stood out as excellent candidates with adequate biocompatibility and biodegradability with tunable hydrophilicity. AREAS COVERED: In this review, polypeptides with the potential to replace PEGylation have been summarized and their application has been reviewed, including XTEN, PASylation, polysarcosine, zwitterion polypeptides, ELPylation, etc. Besides their strengths, the remaining challenges have also been discussed and the future perspectives have been provided. EXPERT OPINION: Polypeptides have been applied in the designing of peptide/protein drugs as well as nanomedicines, and some of the pharmaceutics have made it into the clinical trials and got approved. These polypeptides showed similar hydrophilic properties to PEGylation, which increased the hydrodynamic volumes of protein drugs, reduced kidney elimination, decreased protein-polymer interaction and potentially improved the drug delivery efficiency due to the extended circulation time in the system. Moreover, they demonstrated superior biodegradability and biocompatibility, compensating for the deficiencies for polymers such as PEG.

Isorhamnetin protects zearalenone-induced damage via the PI3K/Akt signaling pathway in porcine ovarian granulosa cells.

Zearalenone (ZEA) is widely derived from moldy cereal grain, which has adverse effects on animal reproduction. In particular, pigs are more sensitive to ZEA-induced toxicity than other animals. Isorhamnetin has extensive pharmacological activity. However, the role of isorhamnetin in ZEA-induced cytotoxicity remains unclear. This study was designed to investigate the therapeutic effect of isorhamnetin on ZEA-induced damage in porcine ovarian granulosa cells and elucidate its molecular mechanism. Two experiments were conducted, where a minimum of 3 biological replicates were used for each treatment. In Exp. 1, ovarian granulosa cells were treated with different concentrations of isorhamnetin (1, 5, 10, 20 and 30 µmol/L) and ZEA (0, 10, 30, 60, 90 and 120 µmol/L) for 24 h. Our results indicated that 60 µmol/L ZEA (half-maximal inhibitory concentration value) and 20 µmol/L isorhamnetin (the most effective concentration against ZEA-induced cytotoxicity) were optimum concentrations. In Exp. 2, ovarian granulosa cells were treated with isorhamnetin (20 µmol/L) for 2 h, before treatment with ZEA (60 µmol/L) for 24 h. Apoptosis, endoplasmic reticulum stress, oxidative stress, proliferation and hormone secretion of ovarian granulosa cells were detected. Our findings showed that isorhamnetin suppressed (P < 0.05) ZEA-induced apoptosis by altering mitochondrial membrane potential and apoptosis-related proteins (B-cell lymphoma-2 [Bcl-2], Bcl2-associated x [Bax] and cleaved caspase-3 [C-Casp3]). Changes in intracellular Ca2+ levels and C/EBP homologous protein (CHOP), recombinant activating transcription factor 6 (ATF6), glucose regulated protein78 kD (GRP78) indicated that isorhamnetin rescued (P < 0.05) ZEA-induced endoplasmic reticulum stress. Furthermore, isorhamnetin prevented (P < 0.05) ZEA-induced oxidative stress via the mitogen-activated protein kinase (P38) signaling pathway. Mechanistically, isorhamnetin stimulated (P < 0.05) the expression of proliferating cell nuclear antigen (PCNA) and cyclin D, thereby increasing the ratio of S phase cells in response to ZEA-induced apoptosis via phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling pathway. Isorhamnetin also recovered (P < 0.05) ZEA-induced steroidogenesis disorder by regulating steroidogenic enzyme gene and proteins (follicle-stimulating hormone receptor [FSHR] and cytochrome P450 family 19 subfamily a member 1 [CYP19A1]). Collectively, these findings show that isorhamnetin protects ovarian granulosa cells from ZEA-induced damage, which promotes proliferation, alleviates apoptosis, endoplasmic reticulum stress, oxidative stress, and steroidogenesis disorder.

T-17, a novel cyclin-dependent kinases/histone deacetylases dual inhibitor, induces cancer cells death through cell cycle arrest and apoptosis.

Combination of cyclin-dependent kinases (CDKs) and histone deacetylases (HDACs) inhibitors may have statistical synergy in suppressing cancer cell proliferation. Herein, a novel CDKs/HDACs dual inhibitor T-17 was rationally designed, synthesized, and evaluated. Our results demonstrated that T-17 concurrently exhibited potent and balanced inhibitory activity against CDKs (IC50 = 18.0 nM) and HDACs (IC50 = 6.6 nM) and also displayed good cell viability inhibitory effect on four cancer cell lines. Meanwhile, T-17 blocked the MDA-MB-231 and A549 cell cycle at G1 phase and S phase, respectively. In addition, T-17 induced MDA-MB-231 cells apoptosis and inhibited the HDACs and CDKs mediated signaling pathways. Finally, we also found that T-17 had good antitumor activity in vivo. In summary, these results indicated that T-17 would be a promising lead compound which deserves further research.

Computational Analysis of the Related Factors of Deep Vein Thrombosis (DVT) Formation in Patients Undergoing Hip Fracture Surgery.

A retrospective study was conducted on 51 patients undergoing hip fracture surgery to investigate the factors associated with the formation of deep venous thrombosis (DVT). The independent sample t-test and correlation analysis were used to sort out and analyze the data. The findings are as follows. (1) Different gender samples showed significant differences in the Caprini score and thrombus location. Most DVTs in females are located in the posterior tibial vein and intermuscular veins. The Caprini score of females was significantly higher than that of males. (2) Age displays a positive correlation with DVT, coronary heart disease, hypertension, and different surgical types, respectively. (3) There is a correlation between age and operation duration. (4) Hyperlipidemia and cerebrovascular disease show a positive correlation with DVT. (5) There was a significant negative correlation between the Caprini score and the quantification of D-dimer. This indicates that in this sample, the higher the patients' Caprini score is, the lower the quantitation of D-dimer will be. (6) Hyperlipidemia and cardiac insufficiency show a positive correlation with cerebrovascular disease. Patients with hyperlipidemia and cardiac insufficiency may also suffer from cerebrovascular diseases.

Statistical Analysis of IABP-Surgery Data with the Co-use of Anticoagulants, Pulse of Dorsalis Pedis Artery, D-Dimer Data, and Coagulation Function.

Data analysis was performed on IABP (intra-aortic balloon pump) patients for the use of anticoagulants, pulse of dorsalis pedis artery, D-dimer data, and coagulation function. According to the differential diagnosis of 52 patients admitted to hospital, data on the use of anticoagulants, dorsalis pedis artery pulsation, D-dimer data, and coagulation function were collected. These data were analyzed by using a nonparametric test, linear regression analysis, adjustment effect analysis, and chi-square test. Some findings of the analysis included: (1) There were differences in the dorsalis pedis artery pulsation of samples from different sexes, all of which were significant. (2) Coagulation function has a significant positive relationship with D-dimer. (3) When the D-dimer affects the prognosis, the regulatory variable (dorsalis pedis artery pulse) is at different levels, and the influence amplitude has significant differences. (4) Samples taken with different anticoagulants all showed significant differences in the dorsalis pedis artery pulsation.

Systematic review and meta-analysis of ultrasonic elastography in the diagnosis of benign and malignant thyroid nodules.

BACKGROUND: In recent years, ultrasound elastography (USE) has been the main diagnostic technique for benign and malignant thyroid nodules. However, it cannot display the blood flow signals of nodules with smaller diameters clearly, which decreases its diagnostic accuracy. METHODS: Chinese and English databases were searched using "ultrasonic elastography", "benign and malignant thyroid nodules", and "diagnose" as the search terms. RevMan 5.3 software was used for meta-analysis. RESULTS: A total of 11 randomized controlled trials (RCTs) were included. Eight articles analyzed clinical symptoms, and the heterogeneity test results were Chi2 =7.46, df=7, I2=6%, and P=0.38; and Z=11.44, OR =14.58, 95% confidential interval (CI): 9.21-23.07, and P<0.01. The diagnostic efficacy of USE was analyzed in 8 articles, and the heterogeneity test results were Chi2 =3.03, df=7, I2=0%, and P=0.88; and Z=6.33, OR =7.47, 95% CI: 4.01-13.93, and P<0.01. The pathological diagnosis results of randomized controlled trials were analyzed in 10 literatures, and the heterogeneity test results were Tau2 =0.78, Chi2 =20.97, df=9, I2=57%, and P=0.01; and Z=7.27, OR =14.67, 95% CI: 7.11-30.27, and P<0.01. After meta-analysis, the combined sensitivity was 72.26% (95% CI: 0.625-0.764) and the combined specificity was 95.35% (95% CI: 0.815-0.943). The area under the receiver operating characteristic curve (AUC) was 0.857. CONCLUSIONS: This meta-analysis confirmed that USE shows high sensitivity and specificity in diagnosing benign and malignant thyroid nodules, which could reduce the false negative rate (FNR) and false positive rate (FPR), showing high clinical diagnostic value.

17ß-Estradiol Enhances Porcine Meiosis Resumption from Autophagy-Induced Gap Junction Intercellular Communications and Connexin 43 Phosphorylation via the MEK/ERK Signaling Pathway.

Estrogen and its analogues are ubiquitous in agricultural environments, with large biological functions of oocyte development. Gap junction intercellular communications (GJICs) are the structural basis in cumulus-oocyte complexes (COCs) and regulate oocyte maturation and developmental material transport through a number of pathways. This study mainly determines the effect and potential mechanism of estrogen (17ß-estradiol) in regulating GJICs in porcine COCs. In our study, 17ß-estradiol increased porcine nuclear maturation in a time-dependent manner. The analysis revealed that 17ß-estradiol upregulated the autophagy in COCs during in vitro maturation. In contrast with the control, 17ß-estradiol decreased GJICs in a time-dependent manner between cumulus cells and oocytes, while it was consistent with the control group at 24 h. Carbenoxolone (CBX) blocks GJICs as a negative control group used in our system. Autophagy inhibitor autophinib decreased oocyte maturation, and the reduced nuclear maturation treated with autophinib was abolished by 17ß-estradiol. Besides, the upregulation effect of autophinib on GJICs and transzonal projections (TZPs) was decreased by 17ß-estradiol. 17ß-Estradiol could reduce serine 368 phosphorylation of connexin 43 (Cx43) protein by autophinib in porcine COCs. These results were dependent upon the MEK/ERK signaling pathway. Furthermore, 17ß-estradiol-induced GJICs and Cx43 phosphorylation were inhibited by autophinib or the MEK/ERK pathway inhibitors (Trametinib and FR 180204), indicating that 17ß-estradiol regulated GJICs through the MEK/ERK signaling pathway. In conclusion, 17ß-estradiol improves the autophagy-mediated nuclear maturation with downregulating GJICs and TZPs in porcine COCs. Such an effect occurs by phosphorylation of Cx43, which was regulated via the MEK/ERK signaling pathway.

Isorhamnetin Promotes Estrogen Biosynthesis and Proliferation in Porcine Granulosa Cells via the PI3K/Akt Signaling Pathway.

Isorhamnetin is a natural flavonoid widely distributed in fruits and vegetables. However, the roles of isorhamnetin involved in steroidogenesis, proliferation, and apoptosis in ovarian granulosa cells (GCs) are poorly understood. We found that isorhamnetin promoted the secretion of estrogen and inhibited the secretion of progesterone and testosterone by modulating steroidogenesis-associated proteins and mRNA such as CYP19A1, StAR, and 3ß-HSD in ovarian GCs. Mechanistically, isorhamnetin stimulated the expression of the proliferating cell nuclear antigen and C-myc and promoted the proliferation of GCs via the PI3K/Akt signaling pathway. Furthermore, isorhamnetin increased the protein expression of CyclinB, CyclinD, CyclinE, and CyclinA, thereby raising the ratio of S-phase cells in response to GC proliferation. Changes in the expression of apoptosis-associated proteins (Bcl2, Bax, and cytochrome c) and intracellular reactive oxygen species levels showed that isorhamnetin inhibited GC apoptosis. Collectively, these findings indicate that isorhamnetin regulates steroidogenesis through the activation of PI3K/Akt, which promotes proliferation, inhibits apoptosis, and alleviates oxidative stress.

Effect of TIMP2/TIMP3 genes on the risk of osteosarcoma in Zhejiang population.

ABSTRACT: Osteosarcoma is a malignant tumor that develops from a mesenchymal cell line and is caused by gene-environment interactions. This study aimed to explore whether TIMP2/TIMP3 polymorphisms influenced the osteosarcoma risk.The expression of the TIMP2 and TIMP3 genes in osteosarcoma histiocytes was analyzed by immunohistochemistry. In this case-control study, which includes samples from 499 patients and 500 healthy controls, 10 single-nucleotide polymorphisms (SNPs) in TIMP2 and TIMP3 were selected. Furthermore, we used the Agena MassARRAY platform for genotyping. The statistical analysis was performed using χ2 test/Fisher exact test, and logistic regression analysis.The immunohistochemistry results showed that the expression of TIMP2 is obvious higher in osteosarcoma histiocytes than in the normal histiocytes. The association study indicated that the allele of rs2277698 and rs4789936 were protective SNPs reducing the risk of osteosarcoma (odds ratios  > 1, P < .05) by the χ2 test. In the genetic model, logistic regression analyses revealed that the rs2277698 and rs4789936 were associated with decreasing the risk of osteosarcoma under the codominant model, dominant model, and log-additive model. Stratification analysis revealed that 2 SNPs (rs2277698 and rs4789936) were significantly associated with a reduced risk of osteosarcoma in allele and genetic model after stratification by gender or age (P < .05). In addition, the haplotype "Trs2277698Crs2009169Crs7342880" of TIMP2 was associated with decreasing the osteosarcoma risk. The "Ars9609634Trs11547635" of TIMP3 was associated with reducing the osteosarcoma risk.This finding shed new light on the high expression of TIMP2 polymorphisms may contribute to decreasing the osteosarcoma risk in Zhejiang populations.

17ß-estradiol improves the developmental ability, inhibits reactive oxygen species levels and apoptosis of porcine oocytes by regulating autophagy events.

OBJECTIVES: Estrogen plays a critical role in the development and apoptosis of oocytes. Autophagy is an evolutionarily conserved and exquisitely regulated self-eating cellular process with important biological functions including the regulation of reproduction. This study aimed to determine the effect of autophagy regulated by the biologically active form of estrogen (17ß-estradiol) in porcine oocyte maturation in vitro. MATERIALS AND METHODS: We measured the effects of oocyte developmental competencies and autophagic activity in the porcine oocyte regulated by 17ß-estradiol using autophagic inhibitor (Autophinib). In addition, we studied the role of autophagy in reactive oxygen species (ROS) levels, mitochondrial distribution, Ca2+ production, mitochondrial membrane potential (ΔΨm), and early apoptosis by caspase-3, -8 activity in the mature oocytes. RESULTS: The results showed that the oocyte meiotic progression and early embryonic development were gradually decreased with Autophinib treatment, which was improved by 17ß-estradiol. Immunofluorescence experiments revealed that 17ß-estradiol primarily could promote the autophagy in the mature oocytes, and block the reduced-autophagic events by Autophinib. Moreover, 17ß-estradiol improved the Autophinib induced high ROS levels, abnormal mitochondrial distribution and low Ca2+ production in mature oocytes. Analyses of early apoptosis and ΔΨm showed that autophagy inhibition was accompanied by increased cellular apoptosis, and 17ß-estradiol reduced apoptosis rates of mature oocytes. Importantly, autophagy was downregulated by treatment with Autophinib, an activation of caspase-8 and cleaved caspase-3 increased. Those effects were abolished by 17ß-estradiol, which could upregulate autophagy. CONCLUSIONS: Our study have showed important implications that 17ß-estradiol could promote efficacy of the development of porcine oocytes, enhance the autophagy, reduce ROS levels and apoptosis activity in vitro maturation.

Development and characterization of in vitro self-assembled recombinant human follicle stimulating hormone originated from goat mammary epithelial cells.

Follicle stimulating hormone (FSH), composed of FSHα and FSHß subunits, is essential for female follicle development and male spermatogenesis. The recombinant human FSH (rhFSH) products on the market are mainly generated from mammalian cells and are expensive. Large animal mammary gland bioreactors are urgently needed to produce large amounts of rhFSH. However, there are currently no effective methods to prepare rhFSH by large animals mainly due to the fact that excessive accumulation of FSH might cause many adverse effects in animals. We herein report the development and characterization of functional self-assembled rhFSH produced in goat mammary epithelial cells (GMECs). FSHα and FSHß stably expressed in Chinese hamster ovary (CHO) cell lines were secreted into culture medium and well glycosylated. Importantly, FSHα and FSHß expressed apart were able to assemble into functional FSH. We next inserted human FSHα or FSHß gene separately into goat ß-Lactoglobulin locus in GMECs by CRISPR/Cas9. Inactive FSHα and FSHß subunits expressed from GMECs assembled into rhFSH as analyzed by His-tag pull down assay. Functional assessment of rhFSH by cAMP induction assay, mouse ovulation induction and rat ovarian weight gain experiments showed that the bioactivity of self-assembled rhFSH expressed by GMECs was comparable to that of Gonal-F both in vitro and in vivo. Our study demonstrated that FSHα and FSHß can be separately expressed and assembled into functional rhFSH, and provided the basis for future preparing FSH by goat mammary gland bioreactor with less health problems on the producing animals.

A novel self-coated polydopamine nanoparticle for synergistic photothermal-chemotherapy.

The combination of photothermal therapy (PTT) and chemotherapy is a promising strategy to overcome the shortcomings of monotherapy. For the first time, we designed a self-coated nanoparticle formed by mesoporous polydopamine (MPDA) core and polydopamine (PDA) shell, which was used to load docetaxel and modified with hyaluronic acid (HA). The obtained nanoparticle can achieve targeted drug delivery and further exert the synergistic effect of PTT and chemotherapy. The MPDA core has high drug loading due to mesopores, and the PDA shell can prevent the drug from releasing in the non-target-site because of the pH-sensitivity of the PDA. Compared with other PDA coated nanoparticle, self-coated nanoparticle has a simpler composition and can avoid the potential toxicity caused by the introduction of other materials. Experimental results showed that it had good photothermal conversion ability both in vivo and in vitro, and could be actively targeted into tumor cells through HA-mediated targeting. Under laser irradiation, it ablated the tumors. Simple ingredient and preparation, good compatibility and obvious therapeutic effect make it have a broad application prospect in tumor therapy.

In vitro and in vivo evaluation of liposomes modified with polypeptides and red cell membrane as a novel drug delivery system for myocardium targeting.

Ischemic cardiac disease (ICD) is a cardiovascular disease with high morbidity and mortality. In this study, a novel myocardial targeted drug delivery system was developed represented by co-modified liposomes consisting of red cell membrane (RCM), and the peptides TAT and PCM. Liposomes were prepared using a membrane dispersion-ultrasonic method; the prepared 1% TAT and 3% PCM micelles were mixed with liposomes and under overnight stirring to form polypeptid-modified liposomes. RCM was isolated from mice blood, and the mechanical force facilitated RCM adhesion to the lipid bilayer. The characteristics of liposomes such as the morphology, particle size, zeta-potential, and RCM-conjugation to lipsomes were evaluated. Uptake efficiency and cellular toxicity of liposomes were evaluated in vitro on myocardial cells (MCs). As regard the experiments in vivo, liposomes were intravenously injected into mice, and the blood and organs were collectedat different times to analyze the pharmacokinetics profile of liposomes. The cellular uptake and intracellular distribution of liposomes of different composition into MCs demonstrated that RCM-modified liposomes had the best delivery capability. The pharmacokinetics study further demonstrated that RCM-modified liposomes had prolonged mean residence time (MRT) and more accumulation in the heart. This study indicated that RCM can be used to modify liposomes in combination with polypeptides, because such modification increases the myocardial targeting of liposomes. Therefore, this system constructed in this study might be a potentially effective myocardial drug delivery system.

Facile preparation of hyaluronic acid-based quercetin nanoformulation for targeted tumor therapy.

In this study, a novel hyaluronic acid-based quercetin nanoformulation (QUT/HA-NF) was constructed for targeted tumor therapy. QUT/HA-NFs have the advantages of simple operation steps, low production cost and good industrialization prospects. Moreover, surfactants, solvents and chemicals are not involved in the preparation process, which avoid the cumbersome extraction of surfactants and the side effects caused by residual solvents or chemicals. The QUT/HA-NFs we prepared have small particle size and high drug loading. In vitro studies have shown that QUT/HA-NFs can significantly enhance tumor cell uptake and tumor cell killing through CD44 receptor mediation. In a xenograft mouse model of 4T1 tumor, QUT/HA-NFs showed stronger inhibition of tumor growth compared with quercetin alone. The QUT/HA-NFs proposed in this study are expected to provide a potential candidate for the treatment of tumors.

Phospholipase C inhibits apoptosis of porcine primary granulosa cells cultured in vitro.

Phospholipase C (PLC) can participate in cell proliferation, differentiation and aging. However, whether it has a function in apoptosis in porcine primary granulosa cells is largely uncertain. The objective of this study was to examine the effects of PLC on apoptosis of porcine primary granulosa cells cultured in vitro. The mRNA expression of BAK, BAX and CASP3, were upregulated in the cells treated with U73122 (the PLC inhibitor). The abundance of BCL2 mRNA, was upregulated, while BAX and CASP3 mRNA expression was decreased after treatment with m-3M3FBS (the PLC activator). Both the early and late apoptosis rate were maximized with 0.5 µM U73122 for 4 h. The rate of early apoptosis was the highest at 4 h and the rate of late apoptosis was the highest at 12 h in the m-3M3FBS group. The protein abundance of PLCß1, protein kinase C ß (PKCß), calmodulin-dependent protein kinaseII α (CAMKIIα) and calcineurinA (CalnA) were decreased by U73122, and CAMKIIα protein abundance was increased by m-3M3FBS. The mRNA expression of several downstream genes (CDC42, NFATc1, and NFκB) was upregulated by PLC. Our results demonstrated that apoptosis can be inhibited by altering PLC signaling in porcine primary granulosa cells cultured in vitro, and several calcium-sensitive targets and several downstream genes might take part in the processes.

The effects of phospholipase C on oestradiol and progesterone secretion in porcine granulosa cells cultured in vitro.

Granulosa cells play important roles in the regulation of ovarian functions. Phospholipase C is crucial in several signalling pathways and could participate in the molecular mechanisms of cell proliferation, differentiation and ageing. The objective of this study was to identify the effects of phospholipase C on the steroidogenesis of oestradiol and progesterone in porcine granulosa cells cultured in vitro. Inhibitor U73122 or activator m-3M3FBS of phospholipase C was added to the in vitro medium of porcine granulosa cells, respectively. The secretion of oestradiol decreased after 2 hr, 8 hr, 12 hr, 24 hr and 48 hr of treatment with 500 nM U73122 (p < .05) and decreased after 2 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The secretion of progesterone increased after 4 hr of treatment with 500 nM U73122 (p < .05) and increased after 2 hr and 8 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The ratio of oestradiol to progesterone decreased at each time point, except 8 hr after the addition of 500 nM U73122 (p < .05). The ratio of oestradiol to progesterone decreased after 2 hr (p < .05) of treatment with 500 nM m-3M3FBS. In genes that regulate the synthesis of oestradiol or progesterone, the mRNA expression of CYP11A1 was markedly increased (p < .05), and the mRNA expression of other genes did not change significantly in the U73122 treatment group, while the addition of m-3M3FBS did not change those genes significantly despite the contrary trend. Our results demonstrated that phospholipase C can be a potential target to stimulate the secretion of oestradiol and suppress progesterone secretion in porcine granulosa cells cultured in vitro, which shed light on a novel biological function of phospholipase C in porcine granulosa cells.

Interplay between Alzheimer's disease and global glucose metabolism revealed by the metabolic profile alterations of pancreatic tissue and serum in APP/PS1 transgenic mice.

Increasing evidence suggests that there is a correlation between type 2 diabetes mellitus (T2D) and Alzheimer's disease (AD). Increased Aß polypeptide production in AD patients would promote metabolic abnormalities, insulin signaling dysfunction and perturbations in glucose utilization, thus leading to the onset of T2D. However, the metabolic mechanisms underlying the interplay between AD and its diabetes-promoting effects are not fully elucidated. Particularly, systematic metabolomics analysis has not been performed for the pancreas tissues of AD subjects, which play key roles in the glucose metabolism of living systems. In the current study, we characterized the dynamic metabolic profile alterations of the serum and the pancreas of APP/PS1 double-transgenic mice (an AD mouse model) using the untargeted metabolomics approaches. Serum and pancreatic tissues of APP/PS1 transgenic mice and wild-type mice were extracted and subjected to NMR analysis to evaluate the functional state of pancreas in the progress of AD. Multivariate analysis of principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were conducted to define the global and the local (pancreas) metabolic features associated with the possible initiation of T2D in the progress of AD. Our results showed the onset of AD-induced global glucose metabolism disorders in AD mice. Hyperglycemia and its accompanying metabolic disorders including energy metabolism down-regulation and oxidative stress were observed in the serum of AD mice. Meanwhile, global disturbance of branched-chain amino acid (BCAA) metabolism was detected, and the change of BCAA (leucine) was positively correlated to the alteration of glucose. Moreover, increased level of glucose and enhanced energy metabolism were observed in the pancreas of AD mice. The results suggest that the diabetes-promoting effects accompanying the progress of AD are achieved by down-regulating the global utilization of glucose and interfering with the metabolic function of pancreas. Since T2D is a risk factor for the pathogenesis of AD, our findings suggest that targeting the glucose metabolism dysfunctions might serve as a supplementary therapeutic strategy for Alzheimer's disease.