Optimization of the spherical integrity for sustained-release alginate microcarriers-encapsulated doxorubicin by the Taguchi method.

This study aimed to develop biodegradable calcium alginate microcarriers with uniform particle size and spherical integrity for sustained-release targeting transarterial chemoembolization. To determine related parameters including the ratio of cross-linking volume (sodium alginate: CaCl2), concentrations of sodium alginate and CaCl2 solutions, collection distance, flow rate, stirring speed, syringe needle diameter and hardening time to fabricate the microcarriers, the Taguchi method was applied. Using different conditions, a total of 18 groups were prepared. The average size of microspheres from different groups was estimated as ~ 2 mm (range 1.1 to 1.6 mm). Signal-to-noise ratio analysis showed the optimal spherical integrity (F1) achieved when the above parameters were designed as 0.1, 2.5 wt%, 6 wt%, 8 cm, 30 mL/h, 150 rpm, 0.25 mm and 2 h, respectively. The best (F1), middle (F2) and worst (F3) groups were used for further experiments. Fourier-transform infrared spectroscopy spectrum showed that F1, F2 and F3 conformations were distinct from original sodium alginate. Drug-loaded calcium alginate microcarriers demonstrated rougher surfaces compared to microspheres without drug under transmission electron microscopy. Compared to pH 7.4, swelling rates in PBS were decreased at pH 6.5. Encapsulation and loaded efficiencies of the Dox-loaded microcarriers were estimated as ~ 40.617% and ~ 3.517%. In vitro experiments indicated that the F1 Dox-loaded microcarriers provide a well sustained-release efficacy for about two weeks at 37 °C in PBS. Treatments of calcium alginate microcarriers without the Dox in two distinct hepatocellular carcinoma-derived cell lines, Huh-7 and Hep-3B, indicated that these microcarriers were non-toxic. The Dox-loaded microcarriers displayed sustained-release capacity and reduced cell viabilities to ~ 30% in both cell lines on Day 12.

Effects of tibolone on osteoarthritis in ovariectomized rats: association with nociceptive pain behaviour.

BACKGROUND: To investigate the role of the synthetic steroid tibolone in the progression of osteoarthritis (OA) and in nociceptive behaviour in an experimental rat model of OA and ovariectomy (OVX)-induced osteoporosis. METHODS: OA was induced in Wistar rats by anterior cruciate ligament transection (ACLT) of the right knee. Osteoporosis was induced by bilateral OVX. Groups of animals were subjected to ACLT, OVX, sham or OVX + ACLT. In addition, two groups were subjected to OVX + ACLT surgeries and were orally administered 0.1 or 0.5 mg tibolone every other day for 14 consecutive weeks, starting 6 weeks after surgery. Nociceptive behaviours (secondary mechanical allodynia and weight-bearing distribution of the hind paws) were analysed prior to and every 3 weeks after surgery up to 24 weeks. At 24 weeks, histopathological studies were performed on the cartilage and synovial membranes of the knee joints, and bone metabolism was assessed by measuring serum concentrations of calcium, phosphorus and alkaline phosphatase. RESULTS: Rats undergoing ACLT or OVX + ACLT surgeries showed obvious OA changes in the joints. Animals subjected to ACLT + OVX and treated with tibolone had significantly less cartilage degeneration and synovitis and showed improved nociceptive tests compared with animals undergoing ACLT + OVX surgeries alone. OVX increased the severity of the ACLT-induced OA changes. There was a significant increase in serum alkaline phosphatase in the tibolone-treated ACLT + OVX groups. CONCLUSIONS: Treatment with tibolone attenuated the development of OA, concomitantly reduced nociception and increased serum alkaline phosphatase in ACLT + OVX rats.

Investigation on maternal physiological and psychological factors of cheilopalatognathus.

OBJECTIVE: Case-control study on mothers of cheilopalatognathus children was conducted, to investigate the maternal physiological and psychological factors for occurrence of cheilopalatognathus. MATERIALS AND METHODS: One hundred ten mothers of cheilopalatognathus children who were scheduled for one-stage surgery were selected as a research group, and 110 mothers of normal children served as a normal control group at the same time. Trait Anxiety Inventory (T-AI), Life Events Scale (LES), Trait Coping Style Questionnaire (TCSQ), Type C Behavior Scale (CBS), adult Eysenck Personality Questionnaire (EPQ), and homemade general questionnaire survey were employed for the investigation. RESULTS: Compared with the control group, the scores for negative event tension value, anxiety, and depressive factors were higher in the study group (p < 0.05); while the scores for positive event tension value, intellect, optimism, and social support factors were lower (p < 0.05). Regression analysis found that physiological factors included were five: education, changes in body weight during pregnancy, the intake amount of milk and beans, and intake of healthcare products, and supplementary folic acid taken or not, while the psychological factors included were four: positive event stimulation, negative event stimulation, the amount of social support, as well as introvert and extrovert personalities. CONCLUSION: The study results suggest that pregnant women's physiological and psychological factors can cause changes in cheilopalatognathus incidence, which is expected to be guidance for healthcare during pregnancy, to prevent the occurrence of cheilopalatognathus.

Intrathecal granulocyte colony-stimulating factor modulate glial cell line-derived neurotrophic factor and vascular endothelial growth factor A expression in glial cells after experimental spinal cord ischemia.

The hematopoietic growth factor, granulocyte colony-stimulating factor (G-CSF), has become one of the few growth factors approved for clinical use. It has therapeutic potential for numerous neurodegenerative diseases; however, at present the cellular effects of G-CSF on the central nervous system remain unclear and in need of investigation. In the present study, we used spinal cord ischemia, a neurodegenerative model, to examine the effects of intrathecal (i.t.) G-CSF on glial cell (microglia and astrocyte) activation and neuroprotective factor expression, including glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor A (VEGF-A) protein expression. Our results indicate that i.t. G-CSF could enhance ischemia-induced microglial activation and inhibit ischemia-induced astrocyte activation. Both GDNF and VEGF-A are upregulated after injury, and i.t. G-CSF could enhance GDNF and VEGF-A expressions after injury. Interestingly, our results indicate that performing i.t. G-CSF alone on normal animals could have the effect of microglial and astrocyte activation and enhanced GDNF and VEGF-A expressions. Furthermore, through laser scanning confocal microscopy, we found that astrocytes may contribute to the majority of GDNF and VEGF-A expressions of G-CSF after spinal cord ischemia. Overall, this G-CSF-induced upregulation suggests that activation of endogenous neuroprotective mechanisms could resist neurodegenerative insults. These observations demonstrate the cellular mechanism of i.t. G-CSF after spinal cord ischemia and confirm the neuroprotective effect of G-CSF after spinal cord ischemia injury.

Critical role of arachidonic acid-activated mTOR signaling in breast carcinogenesis and angiogenesis.

The mammalian target of rapamycin (mTOR) signaling pathway is upregulated in the pathogenesis of many cancers. Arachidonic acid (AA) and its metabolites play critical role in the development of breast cancer, but the mechanisms through which AA promotes mammary tumorigenesis and progression are poorly understood. We found that the levels of AA and cytosolic phospholipase A2 (cPLA2) strongly correlated with the signaling activity of mTORC1 and mTORC2 as well as the expression levels of vascular epithelial growth factor (VEGF) in human breast tumor tissues. In cultured breast cancer cells, AA effectively activated both mTOR complex 1 (mTORC1) and mTORC2. Interestingly, AA-stimulated mTORC1 activation was independent of amino acids, phosphatidylinositol 3-kinase (PI3-K) and tuberous sclerosis complex 2 (TSC2), which suggests a novel mechanism for mTORC1 activation. Further studies revealed that AA stimulated mTORC1 activity through destabilization of mTOR-raptor association in ras homolog enriched in brain (Rheb)-dependent mechanism. Moreover, we showed that AA-stimulated cell proliferation and angiogenesis required mTOR activity and that the effect of AA was mediated by lipoxygenase (LOX) but not cyclooxygenase-2 (COX-2). In animal models, AA-enhanced incidences of rat mammary tumorigenesis, tumor weights and angiogenesis were inhibited by rapamycin. Our findings suggest that AA is an effective intracellular stimulus of mTOR and that AA-activated mTOR plays critical roles in angiogenesis and tumorigenesis of breast cancer.

Effect of caffeine, norfloxacin and nimesulide on heartbeat and VEGF expression of zebrafish larvae.

The use of pharmaceuticals during pregnancy may causes abnormalities to the embryo. Sometime the drug also effect to the new born if the drug transferred through lactation. We have used zebrafish model to see the effect of some pharmaceuticals on embryos and larvae. Three drugs, caffeine, norfloxacin and nimesulide, were used for this study to see the effect mainly the hatching rate of eggs, heart beat rate and the vascular endothelial growth factor (VEGF) expression of the larvae. VEGF is an important signaling protein that involved generating the new blood vessels during embryonic development. We have used 10, 20, 50, 100 microg ml(-1) concentrations of all the drugs to see the effect. No significant mortality or malformations were observed in zebrafish embryos. Hatching was stared from 60 hr. In control group, 91% hatching rate was observed. Lowest hatching rate was observed using highest concentration of norfloxacin (100 microg ml(-1)) and nimesulide (100 microg ml(-1)) i.e. 55 and 56% respectively. In control group, 110 to 115 heart beat rate was counted per minute. Significantly higher heart beat was observed in caffeine treated group which is 125 to 140 min(-1) Lower heart beat was noted in nimesulide treated group which is 100 min(-1). We have tried to observe the possible effect of VEGF of the larvae by these three drugs. Expression of VEGF was very low in caffeine treated group. Almost no VGF expression was observe in 100 microg ml(-1) caffeine treated group. These studies suggest that there is a possibility that high dosage of caffeine can harm the unborn baby or new born babies, if the mothers use caffeine.

A hypothetical relationship between the nuclear reprogramming factors for induced pluripotent stem (iPS) cells generation--bioinformatic and algorithmic approach.

A hypothetical evolutionary relationship was generated between the nuclear reprogramming factors for induced pluripotent stem (iPS) cells generation. Utilizing bioinformatics techniques, sequence analyses and phylogenetic tree algorithms, a comparative study has been performed to understand the evolutionary relationship of human nuclear reprogramming factors of induced pluripotent stem cells (iPSCs) generation. Among the total six nuclear reprogramming factors, the four reprogramming factors (SOX2, C-MYC, KLF4, and LIN28) have significant evolutionary origin. Our study shows SOX2 and C-MYC have evolutionary relationship and common point of origin. Likewise, KLF4 and LIN28 are having evolutionary relationship and have common point of origin. Based on these evidences, we propose that our study may be a great help to the future researchers to understand the mechanism(s) as well as pathway of nuclear reprogramming process.

Suppressive effects of intrathecal granulocyte colony-stimulating factor on excessive release of excitatory amino acids in the spinal cerebrospinal fluid of rats with cord ischemia: role of glutamate transporters.

Recently, the hematopoietic factor, granulocyte colony-stimulating factor (G-CSF), has been shown to exhibit neuroprotective effects in CNS injuries. Our previous study demonstrated that intrathecal (i.t.) G-CSF significantly improved neurological defects in spinal cord ischemic rats. Considerable evidence indicates that the release of excessive amounts of excitatory amino acids (EAAs) plays a critical role in neuron injury induced by ischemic insult. In the present study, we used a spinal cord ischemia-microdialysis model to examine whether i.t. G-CSF exerted antiexcitotoxicity effects in a rat model of spinal cord ischemia. I.t. catheters and a microdialysis probe were implanted in male Wistar rats. The results revealed that spinal cord ischemia-induced neurological defects were accompanied by a significant increase in the concentration of EAAs (aspartate and glutamate) in the spinal dialysates from 30 min to 2 days after reperfusion. I.t administration of G-CSF immediately after the performance of surgery designed to induce ischemia led to a significant reduction in ischemia-induced increases in the levels of spinal EAAs. Moreover, i.t. G-CSF also brought about a significant reduction in the elevation of spinal EAA concentrations induced by exogenous i.t. administration of glutamate (10 microl of 500 mM). I.t. G-CSF attenuated spinal cord ischemia-induced downregulation of expression of three glutamate transporters (GTs), glial transporter Glu-Asp transporter (GLAST), Glu transporter-1 (GLT-1), and excitatory amino acid carrier 1 (EAAC1) protein 48 h after spinal cord ischemic surgery. Immunohistofluorescent staining showed that i.t. G-CSF significantly upregulated expression of the three GTs in the gray matter of the lumbar spinal cord from 3 to 24 h after injection. We propose that i.t. G-CSF possesses an ability to reduce the extent of spinal cord ischemia-induced excitotoxicity by inducing the expression of glutamate transporters.

Isoflurane attenuates dynorphin-induced cytotoxicity and downregulation of Bcl-2 expression in differentiated neuroblastoma SH-SY5Y cells.

BACKGROUND: It has been proposed that the volatile anesthetic isoflurane induces neuroprotection and that the endogenous opioid peptide dynorphin induces neurocytotoxicity in cells. The levels of dynorphin are often significantly elevated in neuropathophysiological conditions, and dynorphin can directly induce toxicity. However, the neuroprotective effects of isoflurane on dynorphin-induced cytotoxicity are still unclear. METHODS: In order to determine the effect of isoflurane on dynorphin-induced cytotoxicity in neuronal cells, we have designed a device wherein cultured human neuroblastoma SH-SY5Y cells can be exposed to isoflurane. Fully differentiated SH-SY5Y cells were obtained by treating the cells with retinoic acid for 6 days. We examined SH-SY5Y cell survival, apoptosis, and antiapoptotic protein expression by cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling stain, and Western blot analysis, respectively. RESULTS: After 16 h of dynorphin (10 microM) treatment, the SH-SY5Y cells showed significant cytotoxicity, apoptosis, and downregulation of the antiapoptotic Bcl-2 protein expression. These effects of dynorphin were significantly inhibited by isoflurane exposure for 32 h [pretreatment for 16 h and posttreatment (after dynorphin treatment) for 16 h]. CONCLUSION: Thus, our results suggest that isoflurane exerts neuroprotective effects in the case of dynorphin-induced pathophysiological disruption.

Increase in excitatory amino acid concentration and transporters expression in osteoarthritic knees of anterior cruciate ligament transected rabbits.

OBJECTIVE: The present study aimed to determine the role of excitatory amino acids (EAAs) and EAA transporters (EAATs) in an osteoarthritis (OA) model of rabbit knees. METHODS: OA was induced in New Zealand white male rabbits by anterior cruciate ligament transection (ACLT) in one knee of one hind limb; the other knee left unoperated. Rabbits that received ACLT of knee were assigned to the ACLT group (n=6), while a sham-operated group (n=6) underwent arthrotomy with no ACLT. Six naïve rabbits that received no surgery were used as normal control. The width of the knee joint was measured to determine the severity of joint inflammation. Before operation and at 10, 20, and 30 weeks after operation, knee joint dialysates were collected by microdialysis and assayed for EAAs by high-performance liquid chromatography. Gross morphology and histopathology and EAATs glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) expression in the articular cartilage of the knees were evaluated by immunohistochemistry and western blot analysis. RESULTS: In the ACLT knees, a significant increase in the joint width was observed (5.3+/-0.9 mm, P<0.05) at 30 weeks after operation, while the sham-operated and naïve knees showed no difference as compared with the basal values. The concentrations (microM) of aspartate and glutamate in knee dialysates at 30 weeks after ACLT in naïve, sham, and ACLT were 0.36+/-0.07 and 4.5+/-1.10; 0.38+/-0.09 and 4.61+/-1.11; 0.67+/-0.18 and 9.71+/-2.89, respectively. Levels of glutamate and aspartate in the dialysates obtained from the ACLT knees increased by 213.3+/-29.6% and 187.5+/-33.8% (P<0.05) when compared to those in the sham-operated knees. Both naïve and ACLT chondrocytes were positively stained by antibodies against GLAST and GLT-1. GLAST and GLT-1 protein expressions were significantly increased in the ACLT knees (P<0.05). CONCLUSION: Our findings indicate an involvement of EAAs and EAATs in the pathogenesis of OA in ACLT rabbits.

Intrathecally injected granulocyte colony-stimulating factor produced neuroprotective effects in spinal cord ischemia via the mitogen-activated protein kinase and Akt pathways.

Granulocyte colony-stimulating factor (G-CSF) is a potent hematopoietic factor. Recently, this factor has been shown to exhibit neuroprotective effects on many CNS injuries. Spinal cord ischemic injury that frequently results in paraplegia is a major cause of morbidity after thoracic aorta operations. In the present study, we examined the neuroprotective role of G-CSF on spinal cord ischemia-induced neurological dysfunctions and changes in the mitogen-activated protein kinase (MAPK) and Akt signaling pathways in the spinal cord. Spinal cord ischemia was induced in male Wistar rats by occluding the descending aorta with a 2F Fogarty catheter for 12 min 30 s. Immediately after ischemia surgery, the rats were administered G-CSF (10 mug) or saline by intrathecal (i.t.) injection. The rats were divided into four groups: control, ischemia plus saline, ischemia plus G-CSF and G-CSF alone. The neurological dysfunctions were assessed by calculating the motor deficit index after ischemia surgery. The expressions of MAPK and Akt were studied using Western blotting and double immunohistochemistry. First, we observed that ischemia plus i.t. G-CSF can significantly reduce the motor function defects and downregulate phospho-p38 and phospho-c-Jun N-terminal kinase protein expressions-this can be compared with the ischemia plus saline group. In addition, G-CSF inhibited the ischemia-induced activation of p38 in the astrocytes. Furthermore, we concluded that i.t. G-CSF produced a significant increase in phospho-Akt and phospho-ERK in the motor neurons and exhibited beneficial effects on the spinal cord ischemia-induced neurological defects.

Intra-articular injection of the cyclooxygenase-2 inhibitor parecoxib attenuates osteoarthritis progression in anterior cruciate ligament-transected knee in rats: role of excitatory amino acids.

OBJECTIVE: Our present study examined the effect of intra-articular cyclooxygenase-2 (COX-2) inhibitor parecoxib on osteoarthritis (OA) progression and the concomitant changes in excitatory amino acids' (EAAs) levels of the anterior cruciate ligament-transected (ACLT) knee joint dialysates. METHODS: OA was induced in Wistar rats by anterior cruciate ligament transection of the knee of one hindlimb, the other was left unoperated and untreated. Rats were placed into four groups: Group ACLT/P received intra-articular parecoxib injection (100 microg) in the ACLT knee once a week for 5 consecutive weeks starting at 8 weeks after surgery. Group ACLT/S received the same procedure as group ACLT/P with saline injection instead. Naïve (Naïve/P) rats received only intra-articular parecoxib injection in one knee once a week for 5 consecutive weeks without surgery. The sham-operated rats underwent arthrotomy only without treatment. Twenty weeks after surgery, knee joint dialysates were collected and EAAs' concentration was assayed by high-performance liquid chromatography, and gross morphology and histopathology (Mankin and synovitis grading) were examined on the medial femoral condyles and synovia. RESULTS: Parecoxib alone had no effect on cartilage and synovium of normal knees in Naïve/P rats. In ACLT/P rats, parecoxib treatment showed a significant inhibition of cartilage degeneration of the medial femoral condyle at both the macroscopic level (1.15+/-0.17 vs 2.55+/-0.12, P<0.05) and the Mankin scores (3.03+/-0.28 vs 8.82+/-0.43, P<0.05). Intra-articular parecoxib injection also suppressed the synovial inflammation of ACLT joint compared to the ACLT/S group (3.92+/-0.41 vs 9.25+/-0.32, P<0.05). Moreover, glutamate and aspartate levels were also significantly reduced in the ACLT/P group compared to the ACLT/S group by parecoxib treatment (91.2+/-9.4% vs 189.5+/-17.0%, P<0.05 and 98.2+/-11.6% vs 175.3+/-12.4%, P<0.05, respectively). CONCLUSION: This study shows that intra-articular injection of COX-2 inhibitor parecoxib inhibits the ACLT-induced OA progression; it was accompanied by a reduction of glutamate and aspartate concentration in the ACLT joint dialysates. From our present results, we suggested that intra-articular parecoxib injection, in addition to the anti-inflammatory effect, inhibiting the EAAs' release, may also play a role in inhibiting the traumatic knee injury induced OA progression.