Hyperoside inhibits proinflammatory cytokines in human lung epithelial cells infected with Mycoplasma pneumoniae.

Mycoplasma pneumoniae pneumonia (MPP) is the most common respiratory infection in young children and its incidence has increased worldwide. In this study, high expression of chemokine ligand 5 (CCL5) was observed in the serum of MPP patients, and its expression was positively correlated to DNA of M. pneumoniae (MP-DNA). In vitro, M. pneumoniae (MP) infection to A549 cells induced the expression of CCL5, chemokines receptor 4 (CCR4), nuclear factor-κB (NF-κB) nuclear protein, and phosphorylation of NF-κB-p65 (p-NF-κB-p65), whereas NF-κB cytoplasmic protein was decreased. On the contrary, treatment of hyperoside counteracted the induction of MP infection and promoted the proliferation of MP-infected A549 cells. Similarly, MP-induced IL-8 and TNF-α production was also markedly reduced by hyperoside. And CCR4 inhibitor AZD2098 had a better effect than hyperoside. In addition, CCL5 recombinant protein inhibited the effect of hyperoside to promote IL-8 and TNF-α production and CCR4 expression. These results indicated that CCL5 may be involved in the progression of MPP, and hyperoside was beneficial for MPP probably through CCL5-CCR4 interactions, which may provide a potential effective therapy for MPP.

Functionalized graphene oxide NPs as a nanocarrier for drug delivery system in quercetin/ lurbinectedin as dual sensitive therapeutics for A549 lung cancer treatment.

Functionalized graphene oxide nanoparticles (NPs) have emerged as promising nanocarriers for drug delivery in lung cancer therapy. Quercetin and lurbinectedin encapsulated in graphene oxide (GO) NPs are tested for treating A549 lung cancer cells. Spectroscopic analyses show that graphene oxide functionalization creates a transparent, smooth surface for drug loading. Treatment with quercetin/lurbinectedin-loaded GO NPs induces notable cytotoxic effects in lung cancer cells, as evidenced by distinct morphological alterations and confirmed apoptotic cellular death observed through fluorescence microscopy. Additionally, our study highlights the impact of this approach on lung cancer metastasis, supported by qRT-PCR analysis of relative gene expression levels, including p53, Bax, Caspase-3, and Bcl 2, revealing robust molecular mechanisms underlying therapeutic efficacy against A549 and PC9 cell lines. Flow cytometric analyses further confirm the induction of cellular death in lung cancer cells following administration of the nanoformulation. Our findings show that quercetin/lurbinectedin-loaded GO NPs may be a promising lung cancer treatment, opening new avenues for targeted and effective therapies.

NOX1 triggers ferroptosis and ferritinophagy, contributes to Parkinson's disease.

The progressive loss of dopaminergic neurons in the midbrain is the hallmark of Parkinson's disease (PD). A newly emerging form of lytic cell death, ferroptosis, has been implicated in PD. However, it remains unclear in terms of PD-associated ferroptosis underlying causative genes and effective therapeutic approaches. This research explored the underlying mechanism of ferroptosis-related genes in PD. Here, Firstly, we found NOX1 associated with ferroptosis differently in PD patients by bioinformatics analysis. In vitro and in vivo models of PD were constructed to explore the underlying mechanism. qPCR, Western blot analysis, immunohistochemistry, immunofluorescence, Ferro orange, and BODIPY C11 were utilized to analyze the levels of ferroptosis. Transcriptomics sequencing was to investigate the downstream pathway and the analysis of immunoprecipitation to validate the upstream factor. In conclusion, NOX1 upregulation and activation of ferroptosis-related neurodegeneration, therefore, might be useful as a clinical therapeutic agent.

The Oral Delivery System of Modified GLP-1 by Probiotics for T2DM.

The glucagon-like peptide-1 (GLP-1) is a peptide with incretin activity and plays an important role in glycemic control as well as the improvement of insulin resistance in type 2 diabetes mellitus (T2DM). However, the short half-life of the native GLP-1 in circulation poses difficulties for clinical practice. To improve the proteolytic stability and delivery properties of GLP-1, a protease-resistant modified GLP-1 (mGLP-1) was constructed with added arginine to ensure the structural integrity of the released mGLP-1 in vivo. The model probiotic Lactobacillus plantarum WCFS1 was chosen as the oral delivery vehicle with controllable endogenous genetic tools driven for mGLP-1 secretory constitutive expression. The feasibility of our design was explored in db/db mice which showed an improvement in diabetic symptoms related to decreased pancreatic glucagon, elevated pancreatic ß-cell proportion, and increased insulin sensitivity. In conclusion, this study provides a novel strategy for the oral delivery of mGLP-1 and further probiotic transformation.

CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila.

Amycolatopsis is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some Amycolatopsis species and has accomplished the deletion of single gene or two genes. The goal of this study was to develop a high-efficient CRISPR/Cas9-mediated genome editing system in vancomycin-producing strain A. keratiniphila HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of gapdhp and ermE*p which drove the expressions of scocas9 and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene gtfD and inserted the gene eGFP with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of cds13-17 (7.7 kb), cds23 (12.7 kb) and cds22-23 (21.2 kb) in ECO-0501 biosynthetic gene cluster (BGC) with high efficiencies of 81%-97% by selecting the sgRNAs with a suitable PAM sequence. Finally, a larger fragment of cds4-27 (87.5 kb) in ECO-0501 BGC was deleted by a dual-sgRNA strategy. The deletion of the ECO-0501 BGCs revealed a noticeable improvement of vancomycin production, and the mutants, which were deleted the ECO-0501 BGCs of cds13-17, cds22-23 and cds4-27, all achieved a 30%-40% increase in vancomycin yield. Therefore, the successful construction of the CRISPR/Cas9-mediated genome editing system and its application in large fragment deletion in A. keratiniphila HCCB10007 might provide a powerful tool for other Amycolatopsis species.

Construction of a multifunctional 3D nanofiber aerogel loaded with ZnO for wound healing.

Bacterial infection and severe wound inflammation are two primary harmful problems that bring harm to the human body and may cause death when a large-scale skin defect occurs. Thus, developing an effective and quick wound healing strategy for curing skin damage and trauma is vital. This study has developed a multifunctional PLA/gelatin/ZnO nanofiber aerogel with a three-dimensional structure through electrospinning and freeze-drying technology for wound healing. It has validated that the nanofiber aerogel has an excellent antibacterial property and biocompatibility. Meanwhile, benefiting from its three-dimensional nanofiber structure, the PLA/gelatin/ZnO nanofiber aerogel possesses good water absorption and air permeability. In vivo experiments have determined that the PLA/gel/ZnO nanofiber aerogel scaffolds effectively promote skin infection's wound healing and enhance angiogenesis that is practical with increasing ZnO concentration.

LncRNA MACC1-AS1 Promotes Lung Adenocarcinoma Cell Proliferation by Downregulating PTEN.

Background: MACC1-AS1 is an oncogenic lncRNA in gastric cancer, which interacts with AMP-activated protein kinase (AMPK) to promote cancer development. AMPK is known to interact with phosphatase and tensin homolog (PTEN). Therefore, MACC1-AS1 may also have associations with PTEN. This study aimed to investigate the interactions between MACC1-AS1 and PTEN in lung adenocarcinoma (LUAD). Materials and Methods: This study recruited 64 LUAD patients admitted to The First People's Hospital of Wenling City. Gene and protein expression levels were determined by qPCR and western blot, respectively. Cell transfections were performed to assess gene interactions. Cell proliferation was evaluated by CCK-8 assay. Results: MACC1-AS1 was upregulated in LUAD and inversely correlated with the expression of PTEN. High expression levels of MACC1-AS1 in LUAD tissues were closely correlated with poor survival rate of LUAD patients. In LUAD cells, overexpression of MACC1-AS1 led to decreased expression of PTEN and increased proliferation rate of LUAD cells, while MACC1-AS1 silencing led to increased expression of PTEN and decreased proliferation rate of LUAD cells. Furthermore, overexpression of PTEN attenuated the effects of overexpressing MACC1-AS1. Conclusions: The authors' results demonstrated that MACC1-AS1 promoted cell proliferation by downregulating PTEN in LUAD cells.

Clinical Significance of Has_circ_0060937 in Bone Metastasis of NSCLC.

BACKGROUND: Bone metastasis can induce multiple types of bone diseases which reduce the non-small-cell lung cancer (NSCLC) patient's quality-of-life. Due to the difficulty of finding bone metastases and lack of effective early diagnosis, it is easy to miss the best treatment. Therefore, the study of serum tumor biomarkers is of great significance in the diagnosis of NSCLC bone metastasis. METHODS: The qRT-PCR assay was performed to assess has_circ_0060937 expression in 100 NSCLC patients. Furthermore, the small interfering RNAs si-has_circ_0060937 or si-NC were transfected into NSCLC bone metastasis cells. CCK8 assay was exercised to detect cell proliferation, and cell invasion assays were used to detect cell invasion in NSCLC bone metastasis cells. RESULTS: In this study, we firstly found that the expression of has_circ_0060937 in boneless metastasis NSCLC tissues and bone metastasis NSCLC tissues was significantly increased compared to normal tissues, and the expression of has_circ_0060937 was highest in bone metastasis. Expression of serum has_circ_0060937 in bone metastasis group from Grade I to Grade III NSCLC was drastically higher than boneless metastasis group or healthy group. In the Grade I to Grade III bone metastasis group, the expression of serum has_circ_0060937 gradually boosted with the increase of bone metastasis grades. Additionally, knockdown of has_circ_0060937 inhibited cell proliferation and cell invasion in NSCLC bone metastasis cell line. CONCLUSION: The results suggestthat has_circ_0060937 is closely associated with bone metastasis in NSCLC, and the circRNAs we inspected may be a potential biomarker of bone metastasis in NSCLC.

Micro/Nanorobot: A Promising Targeted Drug Delivery System.

Micro/nanorobot, as a research field, has attracted interest in recent years. It has great potential in medical treatment, as it can be applied in targeted drug delivery, surgical operation, disease diagnosis, etc. Differently from traditional drug delivery, which relies on blood circulation to reach the target, the designed micro/nanorobots can move autonomously, which makes it possible to deliver drugs to the hard-to-reach areas. Micro/nanorobots were driven by exogenous power (magnetic fields, light energy, acoustic fields, electric fields, etc.) or endogenous power (chemical reaction energy). Cell-based micro/nanorobots and DNA origami without autonomous movement ability were also introduced in this article. Although micro/nanorobots have excellent prospects, the current research is mainly based on in vitro experiments; in vivo research is still in its infancy. Further biological experiments are required to verify in vivo drug delivery effects of micro/nanorobots. This paper mainly discusses the research status, challenges, and future development of micro/nanorobots.

Antidiabetic effects of selenium-enriched Bifidobacterium longum DD98 in type 2 diabetes model of mice.

Both selenium and probiotics have shown antidiabetic effects in a type 2 diabetes model. The objective of this study is to investigate the alleviating effects of selenium-enriched Bifidobacterium longum DD98 (Se-B. longum DD98) on diabetes in mice and explore the possible underlying mechanism. A type 2 diabetes model was established using a high-fat diet and streptozotocin (STZ) injection in mice. To investigate the beneficial effects of Se-B. longum DD98, diabetic mice were then treated with B. longum DD98, Se-B. longum DD98, or sodium selenite (Na2SeO3) for three weeks. The results suggested that all three treatments could reduce the levels of fasting blood glucose (FBG), glycated hemoglobin (HbA1c), insulin and leptin, improve glucose tolerance, regulate lipid metabolism, and protect against the impairment of the liver and pancreas, while Se-B. longum DD98 showed a greater effect on relieving the above mentioned symptoms of type 2 diabetes in mice. Furthermore, this effect was associated with butyrate production and inflammatory response. Se-B. longum DD98 better increased the level of butyrate in feces and decreased the levels of proinflammatory cytokines in the pancreas compared with B. longum DD98 and Na2SeO3, leading to ameliorative insulin resistance. Se-B. longum DD98 also improved the glucagon like peptide-1 (GLP-1) level in serum and intestinal cells, which protected the pancreatic ß-islet cells from damage induced by type 2 diabetes. These results demonstrated that Na2SeO3, B. longum DD98, or Se-B. longum DD98 could alleviate the progression of type 2 diabetes in mice. Se-B. longum DD98 showed greater antidiabetic effects than the other two treatments, and could be considered as a promising candidate for treating type 2 diabetes.

Tumor-Microenvironment- Responsive Size-Shrinkable Drug-Delivery Nanosystems for Deepened Penetration Into Tumors.

Over the years, the manipulation and clinical application of drug-delivery nanosystems for cancer diseases have attracted a rapid growth of academic research interests, and some nanodrugs have been approved for clinic application. Although encouraging achievements have been made, the potency of nanomedicines in cancer treatment is far from satisfaction, and one significant reason is the inefficient penetration of nanoparticles into solid tumors. Particle size is one of the most significant features that influence diffusion ability of the drug-delivery system in tumors. Size-shrinkable drug-delivery nanosystems possess a size-switchable property that can achieve passive targeting via the enhanced permeability and retention (EPR) effect and transform into ultrasmall particles in tumors for deep penetration into tumors. The tumor microenvironment is characterized by acidic pH, hypoxia, upregulated levels of enzymes, and a redox environment. In this review, we summarize and analyze the current research progresses and challenges in tumor microenvironment responsive size-shrinkable drug-delivery nanosystems. We further expect to present some meaningful proposals and enlightenments on promoting deep penetration into tumors of nanoparticles.

[Early morphological changes in the mouse testis induced by 5-fluorouracil].

OBJECTIVE: To propose a simple and practical method for preparing a mouse model of oligo-astheno-terato-spermia/azoospermia, and to offer a methodological suggestion for the studies on the related mechanism of spermatogenesis and evaluation of medication efficacy by observing the early changes of testis morphology after 5-fluorouracil treatment. METHODS: Mice were injected with a single dose of 5-flourouracil at 250 mg/kg via the tail vein, and their testes were harvested and paraffin sections prepared before and 3, 7, 11 and 14 d after the injection to be observed for the morphological changes by hematoxylin and eosin staining. RESULTS: The numbers of spermatocytes/spermatids were progressively reduced inside the testis seminiferous tubules of the mice at 3, 7 and 11 d after the 5-fluorouracil injection, and the tubule walls became thinner, which reached the nadir at 11 d, with evident swelling and crazing of the seminiferous tubules. At 14 d, the swelling almost disappeared and spermatocytes became repopulated, while the flaws still existed in the seminiferous tubules and no mature sperm were seen. CONCLUSION: One-dose injection of 5-fluorouracil via the tail vein might be a simple and effective method for preparing the animal model of reproductive function damage induced by chemotherapeutic medication.

TREM1: A positive regulator for inflammatory response via NF-κB pathway in A549 cells infected with Mycoplasma pneumoniae.

Herein, we found that serum content of the triggering receptor expressed on myeloid cells-1 (TREM1) was increased, and positively correlated with Mycoplasma pneumoniae (MP)-DNA in children with MP infection. In this study, A549 cells, known as human lung epithelial cells, were co-cultured with 107 CCU/ml of MP to established in vitro model of MP infection. We studied the roles of TREM1 in inflammatory response of A549 cell by regulating the secretions of cytokine interleukin (IL)-8 and tumor necrosis factor (TNF)-α in cell culture supernatants. Moreover, transcriptional activity of nuclear factor kappa B (NF-кB) was assessed by measuring protein levels of NF-кB in the cytoplasm and nuclear. Our data suggested that sanguinarine chloride significantly decreased TREM1 expression, and alleviated inflammatory response of MP-infected A549 cells via preventing NF-кB nuclear translocation. To study the roles of TREM1 in inflammatory regulation in MP-infected A549 cells and the underlying mechanisms, we established TREM1 overexpression transfected A549 cells. PDTC was used for inhibiting NF-кB nuclear translocation. We found that TREM1 overexpression induced server inflammatory response of A549 cells. Besides, TREM1 overexpression attenuated anti-inflammatory effects of sanguinarine chloride in MP-infected cells. More importantly, pro-inflammatory effects of TREM1 overexpression was significantly reversed with additional PDTC treatment in MP-infected cells treated with sanguinarine chloride, suggesting that TREM1 was a pro-inflammatory factor via regulating NF-кB nuclear translocation in MP-infected A549 cells.

[Interrelationship of abnormal family history in the first degree relatives and clinical phenotype of patients with polycystic ovary syndrome].

OBJECTIVE: To study the relationship of abnormal family history in the first degree relatives and the clinical phenotype of patients with polycystic ovary syndrome (PCOS). METHODS: Clinical data of first degree relatives of 139 women with PCOS were collected by questionnaires, including body mass index (BMI), waist hip ratio (WHR), and hursutism score. Follicle stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), testosterone (T), androstenedione (A), oral glucose tolerance test (OGTT) and insulin releasing test were measured. RESULTS: (1) Compared with patients with a negative family history of diabetes mellitus, for women with a positive family history, WHR (0.99 +/- 0.10 vs 0.79 +/- 0.08) and score of hirsutism (1.9 +/- 1.2 vs 1.8 +/- 1.2) were increased, the duration of menstruation was longer [(108 +/- 10) vs (92 +/- 19) days]; A [(11 +/- 6) vs (8 +/- 5) nmol/L], homeostasis model assessment of insulin resistance (HOMA-IR, 3.5 +/- 2.0 vs 2.7 +/- 1.6), area under curve (AUC) glucose [(836 +/- 245) vs (748 +/- 139) nmol.L(-1).min(-1)], AUC insulin [(9670 +/- 4582) vs (7330 +/- 4311) mIU.L(-1).min(-1)], fasting glucose [(5.0 +/- 1.1) vs (4.8 +/- 0.5) mmol/L] and fasting insulin [(15 +/- 8) vs (11 +/- 8) mIU/L] were increased, while early insulin secretion function index (DeltaI60/DeltaG60, 32 +/- 22 vs 52 +/- 30), insulin sensitive index (ISI, 0.019 +/- 0.011 vs 0.033 +/- 0.014) and disposition index (DI, 18 +/- 10 vs 30 +/- 22; P < 0.05) were decreased. (2) For women with a positive family history of menstrual disorder, WHR and score of hirsutism (0.99 +/- 0.09 vs 0.80 +/- 0.10 and 1.9 +/- 1.0 vs 1.6 +/- 1.1) were increased respectively, the duration of menstruation [(105 +/- 28) vs (84 +/- 31) days] was longer, HOMA-IR (3.6 +/- 2.4 vs 2.5 +/- 1.7) and fasting insulin level [(15 +/- 14) vs (12 +/- 11) mIU/L] were increased, while HOMA-beta (178 +/- 134 vs 207 +/- 175), ISI (0.017 +/- 0.009 vs 0.033 +/- 0.012) and DI (23 +/- 18 vs 28 +/- 19, P < 0.05) were decreased. (3) For women with a positive family history of premature balding, BMI and the score of hirsutism [(26 +/- 4) vs (23 +/- 5) kg/m(2) and 2.1 +/- 1.1 vs 1.7 +/- 1.3] were increased respectively, while DI (20 +/- 11 vs 30 +/- 23, P < 0.05) was decreased. (4) DeltaI60/DeltaG60 (34 +/- 27 vs 50 +/- 30) was decreased and fasting insulin [FINS, (13 +/- 10) vs (10 +/- 9) mIU/L] was increased in PCOS women with a family history of hypertension (P < 0.05). (5) For women with or without a family history of coronary heart disease, they did not have any difference in every parameter mentioned before. CONCLUSIONS: The family history of diabetes mellitus has the most effect on the clinical phenotype in women with PCOS. The family history of other diseases such as menstrual disorder, premature balding and hypertension play less significant roles. A family history of positive coronary heart disease does not affect the clinical phenotype of such patients.

Neuroprotection by intravenous transplantation of bone marrow mononuclear cells from 5-fluorouracil pre-treated rats in a model of ischemic stroke.

Our previous studies showed that bone marrow mononuclear cells (BMMNCs) from 5-fluorouracil (5-FU) pre-treated rats (named BMRMNCs) had a better therapeutic efficacy in ischemia/reperfusion rats as compared to BMMNCs from untreated rats. This study was undertaken to further explore the potential mechanisms underlying the neuroprotective effects of BMRMNCs in the same model. Rats were intravenously pre-treated with 5-FU, and BMRMNCs were collected 7 days later and subjected to flow cytometry for detection of CD34, CD45 and CD90. Middle cerebral artery occlusion (MCAO) was induced in rats, and BMMNCs and BMRMNCs were independently transplanted via the tail vein at 24 h after MCAO. NISSL staining was performed 14 days after cell transplantation and the viable cells in the hippocampus were counted. Stromal cell-derived factor 1 (SDF-1) mRNA expression was detected in the penumbra at 7 and 14 days after treatment. The contents of pro-inflammatory cytokines and growth factors as well as microvessel density (MVD) were determined at 14 days. Results showed more BMRMNCs were positive for CD34, CD45 and CD90. After transplantation, more viable cells were observed in the hippocampus of BMRMNCs treated rats. In addition, BMRMNCs transplantation significantly increased MVD, reduced pro-inflammatory cytokines and raised growth factors in the penumbra. However, the SDF-1 mRNA expression was comparable between BMRMNCs group and BMMNCs group. Our results indicate that BMRMNCs are likely to more effectively improve the local microenvironment to increase viable cells and elevate angiogenesis, exerting neuroprotective effects on cerebral ischemia in rats.

Total flavonoid aglycones extract in Radix scutellariae inhibits lung carcinoma and lung metastasis by affecting cell cycle and DNA synthesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Scutellariae (Scutellaria baicalensis Georgi, RS), a traditional herbal medicine commonly used to treat inflammation, hypertension, cardiovascular disease, bacterial and viral infections, is reported to treat lung cancer by supplements of modern medicine. The total flavonoid aglycones extract (TFAE) from RS is the most important composition for the pharmacodynamic effects. The present study was designed to evaluate the anti-lung tumor effect of TFAE on A549 cells and A549 cell nude mice xenografts. The aim of the study is to investigate the effect and mechanism of TFAE treating non-small cell lung cancer both in vitro and in vivo. MATERIALS AND METHODS: The anti-tumor activity of TFAE in vitro was investigated using the MTT assay. The changes of cell invasion and migration were detected by Transwell assay and tube formation experiments were used to detect the anti-angiogenic effect. The anti-tumor effects of TFAE in vivo were evaluated in A549 cell nude mice xenografts. The mechanism of TFAE was detected by flow cytometry technology, western blot assay and immuno-histochemistry assay. RESULTS: In vitro, TFAE inhibited the proliferation, invasion and migration of A549 cells in a dose- and time-dependent manner. In vivo, TFAE by oral administration at 100mg/kg for 30 days decreased the tumor volume and tumor weight in A549 cell xenograft by 25.5% with no statistical significance (P<0.05) compared to the cis-platinum positive control group (30.0%). The cell cycle and DNA synthesis experiment illustrated that TFAE could induce A549 cell cycle to arreste in S phase and DNA synthesis in A549 cells be inhibited, while TFAE had no influence on apoptosis of A549 cells. Western Blot assay demonstrated that the treatment of TFAE could make Cyclin D1 decrease and p53 increase both in vitro and in vivo. CONCLUSION: TFAE displayed the inhibition effects of non-small cell lung cancer both in vitro and in vivo and the underlying mechanism might be related to the increased p53 protein expression and decreased Cyclin D1 expression, leading to cell cycle arrested in S phase and the decrease of DNA synthesis.

Are bone marrow regenerative cells ideal seed cells for the treatment of cerebral ischemia?

Bone marrow cells for the treatment of ischemic brain injury may depend on the secretion of a large number of neurotrophic factors. Bone marrow regenerative cells are capable of increasing the secretion of neurotrophic factors. In this study, after tail vein injection of 5-fluorouracil for 7 days, bone marrow cells and bone marrow regenerative cells were isolated from the tibias and femurs of rats, and then administered intravenously via the tail vein after focal cerebral ischemia. Immunohistological staining and reverse transcription-PCR detection showed that transplanted bone marrow cells and bone marrow regenerative cells could migrate and survive in the ischemic regions, such as the cortical and striatal infarction zone. These cells promote vascular endothelial cell growth factor mRNA expression in the ischemic marginal zone surrounding the ischemic penumbra of the cortical and striatal infarction zone, and have great advantages in promoting the recovery of neurological function, reducing infarct size and promoting angiogenesis. Bone marrow regenerative cells exhibited stronger neuroprotective effects than bone marrow cells. Our experimental findings indicate that bone marrow regenerative cells are preferable over bone marrow cells for cell therapy for neural regeneration after cerebral ischemia. Their neuroprotective effect is largely due to their ability to induce the secretion of factors that promote vascular regeneration, such as vascular endothelial growth factor.

A rapid fluorescent screening method for cellular sensitivity to anti-cancer compound.

The tetrazolium salts (MTT, XTT, MTS, WST) based colorimetric assay or resazurin based fluorimetric assay are currently typical methods for cell sensitivity determination to anticancer compounds. We presented here a new rapid method for this purpose. This method uses a fluorescent dye named DCFH-DA which is previously taken as a intracellular probe for measurement of H(2)O(2) levels within a cell. The application basis for this method lies in two facts: the membrane permeable feature of the final metabolite of DCFH-DA inside a cell, and the linearity relationship between cell number and H(2)O(2) level. The results showed that there was a perfect association between cell number and fluorescent intensity determined by the DCFH-DA method, no matter whether using resuspended or adherent cells, and further 50% concentration of inhibition (IC(50)) comparison between data obtained by DCFH-DA method or MTT method using a positive known anticancer compound Baicalin showed that there were no significant differences in cellular sensitivity determination to compound Baicalin though there existed a relatively higher coefficient of variation of IC(50) by the DCFH-DA method than that by the MTT method. Thus our data indicate that DCFH-DA might not only be a fine reagent for determination of H(2)O(2) levels in cells but also an ideal fluorescent dye for cellular sensitivity test of anti-cancer compounds, and may be suitable for primary high-throughput drugs screening.

Overexpression of 5-lipoxygenase increases the neuronal vulnerability of PC12 cells to Aß42.

5-Lipoxygenase (5-LOX), which is believed to be a major source of oxidative stress, participates in somatostatin-receptor transmembrane signaling in the central nervous system. We used the Tet-On inducible expression system in PC12 cells to obtain cell lines with reproducible, stable 5-LOX expression levels to study its function. Cell apoptosis rates induced by Aß(42) were determined using an apo-BrdDU kit. Lipid peroxide, antioxidant enzyme, and caspase-3 activities were evaluated with respective commercial kits. The expression of 5-LOX, bcl-2, and bax were detected by immunoblotting. A subclone of PC18 with Tet-On inducible expression of 5-LOX was selected from PC12 transfectants. Expression of 5-LOX had no significant inhibitory effect on the cell viability of the PC18 clone. In contrast, compared with the control group, the cell viability of clone PC18 was significantly reduced after the induction of 5-LOX during Aß exposure. The differences in cell viability before and after the induction of 5-LOX during Aß insult were significantly offset by AA861. Overexpression of 5-LOX only slightly improved the activities of superoxide dismutase (SOD). The levels of intracellular peroxides, SOD and caspase-3 activity, and Bax expression were significantly upregulated, and the levels of glutathione peroxidase and catalase were downregulated correspondingly in clone PC18 during Aß exposure. These results indicate that constitutive expression of 5-LOX is not detrimental per se, but overexpression of 5-LOX may become problematic during Aß exposure.

Panaxydol inhibits the proliferation and induces the differentiation of human hepatocarcinoma cell line HepG2.

Panaxydol, a polyacetylene compound isolated from Panax ginseng, exerts anti-proliferative effects against malignant cells. No previous study, however, has been reported on its effects on hepatocellular carcinoma cells. Here, we investigated the effects of panaxydol on the proliferation and differentiation of human hepatocarcinoma cell line HepG2. We studied by electronic microscopy of morphological and ultrastructural changes induced by panaxydol. We also examined the cytotoxicities of panaxydol against HepG2 cells using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and the effect of panaxydol on cell cycle distributions by flow cytometry. We investigated the production of liver proteins in panaxydol-treated cells including alpha-fetoprotein and albumin and measured the specific activity of alkaline phosphatase and gamma-glutamyl transferase. We further investigated the effects of panaxydol on the expression of Id-1, Id-2, p21 and pRb by RT-PCR or immunoblotting analysis. We found that panaxydol inhibited the proliferation of HepG2 cells and caused morphological and ultrastructural changes in HepG2 cells resembling more mature forms of hepatocytes. Moreover, panaxydol induced a cell cycle arrest at the G(1) to S transition in HepG2 cells. It also significantly decreased the secretion of alpha-fetoprotein and the activity of gamma-glutamyl transferase. By contrast, panaxydol remarkably increased the secretion of albumin and the alkaline phosphatase activity. Furthermore, panaxydol increased the mRNA content of p21 while reducing that of Id-1 and Id-2. Panaxydol also increased the protein levels of p21, pRb and the hypophosphorylated pRb in a dose-dependent manner. These findings suggest that panaxydol is of value for further exploration as a potential anti-cancer agent.