Overall adjustment acupuncture improves osteoporosis and exerts an endocrine-modulating effect in ovariectomized rats.

Background: Acupuncture is a widely practiced, convenient, and safe treatment modality within complementary and integrative medicine. Increasing studies have revealed the efficacy of acupuncture for the treatment of osteoporosis in both human and non-human subjects. The aim of the present study was to assess the improvement of osteoporosis after overall adjustment acupuncture (OA) as well as its endocrine-modulating effect in an ovariectomized rat model. Methods: In total, 32 female Sprague-Dawley (SD) rats were randomly divided into the sham, model, ovariectomy+estrogen (OVX+E), and OVX+OA (OVX+A) groups with eight rats in each group. The postmenopausal osteoporosis (PMOP) rat model was induced by bilateral ovariectomy. At 12 weeks after surgery, rats in the OVX+E group received estradiol (0.2 mg/kg/i.g./qod) for 12 weeks, and rats in the OVX+A group were treated with acupuncture at Zusanli (ST36), Shenshu (BL23), and Dazhu (BL11) points (qod) for 12 weeks. At the end of the treatment, all rats were sacrificed, and the body weight, uterus index, bone mineral density (BMD), bone mineral content (BMC), bone trabeculae structural parameters, femoral biomechanical properties, femoral histomorphology, and several hormone levels were examined. Results: In OVX rats, OA abrogated the body weight gain and improved osteoporosis in terms of BMD, BMC, bone trabeculae structural parameters, bone strength, and bone tissue histomorphology. Moreover, OA modulated the serum levels of estradiol, corticotropin releasing hormone (CRH), adrenocorticotropic hormone (ACTH), and corticosterone (CORT). Conclusions: OA improves osteoporosis and exerts an endocrine-modulating effect in ovariectomized rats.

Brain lipid-binding protein promotes proliferation and modulates cell cycle in C6 rat glioma cells.

Gliomas are the most common primary brain tumors affecting adults. Four grades of gliomas have been identified, namely, grades I-IV. Brain lipid-binding protein (BLBP), which functions in the intracellular transport of fatty acids, is expressed in all grades of human gliomas. The glioma cells that are cultured in vitro are grouped into the BLBP-positive and BLBP-negative cell lines. In the present study, we found that C6 rat glioma cells was a distinct type of BLBP-negative cell line. Our results confirmed that in the C6 cells, the expression of exogenous BLBP increased the proliferation and percentage of cells in the S phase, in the culture medium containing 10 or 1% FBS. Moreover, exogenous BLBP was found to downregulate the tumor suppressors p21 and p16 in the 1% FBS culture medium, but only p21 in the 10% FBS culture medium. The results of the xenograft model assay showed that exogenous BLBP also stimulated tumor formation and downregulated p21 and p16. In conclusion, our study demonstrated that exogenous BLBP promoted proliferation of the C6 cells in vitro and facilitated tumor formation in vivo. Therefore, BLBP expression in glioma cells may promote cell growth by inhibiting the tumor suppressors.

IDH1 R132H Mutation Enhances Cell Migration by Activating AKT-mTOR Signaling Pathway, but Sensitizes Cells to 5-FU Treatment as NADPH and GSH Are Reduced.

AIM OF STUDY: Mutations of isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene were recently discovered in vast majority of World Health Organization (WHO) grade II/III gliomas. This study is to understand the effects of IDH1 R132H mutation in gliomagenesis and to develop new strategies to treat glioma with IDH1 R132H mutation. MATERIALS AND METHODS: Over expression of IDH1 R132H in U87MG cells was done by transfecting cells with IDH1 R132H plasmid. MTT assay, scratch repair assay and western blot were performed to study effects of IDH1 R132H mutation on cell proliferation, migration, regulating AKT-mTOR signaling pathway and cell death respectively. NADP+/NADPH and GSH quantification assays were performed to evaluate effects of IDH1 R132H mutation on the production of antioxidant NADPH and GSH. RESULTS: We found that over expression of IDH1 R132H mutation decreased cell proliferation consistent with previous reports; however, it increased cell migration and enhanced AKT-mTOR signaling pathway activation. Mutations in isocitrate dehydrogenase (IDH) 1 also change the function of the enzymes and cause them to produce 2-hydroxyglutarate and not produce NADPH. We tested the level of NADPH and GSH and demonstrated that IDH1 R132H mutant stable cells had significantly low NADPH and GSH level compared to control or IDH1 wild type stable cells. The reduced antioxidants (NADPH and GSH) sensitized U87MG cells with IDH R132H mutant to 5-FU treatment. CONCLUSION: Our study highlights the important role of IHD1 R132H mutant in up- regulating AKT-mTOR signaling pathway and enhancing cell migration. Furthermore, we demonstrate that IDH1 R132H mutation affects cellular redox status and sensitizes gliomas cells with IDH1 R132H mutation to 5FU treatment.

Applied anatomical study of the modified anconeus flap approach.

PURPOSE: Conventional surgical therapy for an intercondylar humerus fracture might result in multiple potential complications. Our study was conducted to evaluate the modified anconeus flap approach by adequately exposing the distal humeral articular surface, avoiding osteotomy of the olecranon and transection of the main part of the triceps brachial tendon from the olecranon. METHODS: Preparations of 20 upper limb specimens from adult cadavers were used in this study. We investigated the anatomical features of the distal tendon of the triceps brachii. Then, we designed a modified anconeus flap approach in cadaver specimens combined with the medial paratricipital approach, and we compared the extent of exposure of the distal humeral articular surface between the triceps-reflecting anconeus pedicle approach and this modified approach. RESULTS: The downward neurovascular bundles supplying the anconeus were located far from the intramuscular tendon of the triceps brachii. In addition, the medial head of the triceps was continuous with the anconeus near the lateral epicondyle of the humerus. These anatomical properties could assist in reducing adverse events in surgery. The percentage of the exposed humerus distal articular surface was 42.7% by applying the modified anconeus flap approach combined with the medial paratricipital approach. The modified anconeus flap approach can overcome the shortcomings of osteotomy or triceps transverse and fulfill reduction and internal fixation of most distal humerus intercondylar fractures. CONCLUSIONS: The present study has demonstrated a new approach for adequately exposing the distal humeral articular surface during surgery for an intercondylar humerus fracture. With this modified approach, osteotomy of the olecranon and the separation or transection of the main part of the triceps brachial tendon from the olecranon are not necessarily required. Therefore, we suggest that this novel approach could be applied as the primary surgical approach in intercondylar humerus fracture surgeries if the surgeons are familiar with the regional features of distal tendon of the triceps brachii and anconeus.

Differentiation of human embryonic germ cells and transplantation in rats with acute myocardial infarction.

Human embryonic germ cells (hEGCs) are stem cells cultured from primordial germ cells, which reside in human embryonic genital ridges in vivo. In this study, hEGCs were induced to differentiate into cardiomyocytes by treatment with ascorbic acid in vitro and the effects of hEGC transplantation on rat models of acute myocardial infarction (AMI) were investigated. hEGCs were incubated with differentiation medium containing ascorbic acid at various concentrations. Levels of GATA-4 expression were measured to identify the optimal concentration of the inductor. Immunofluorescence microscopy was used to detect the expression of Cx43 on the induced cells. The hEGCs were injected into the myocardium of rats with AMI. The expression levels of MAB1281 and GATA-4 were used to indicate the survival, migration, distribution and differentiation of transplanted cells. The results revealed the positive expression of GATA-4, Cx43 and cardiac troponin T (cTnT) in differentiated cells, and immunocytochemistry showed that transplanted cells highly expressed GATA-4 and MAB1281. hEGCs were successfully induced to differentiate into cardiomyocytes by ascorbic acid in optimal concentrations in vitro and the transplanted hEGCs survived and differentiated into cardiomyocytes.

Curcumin inhibits LPS-induced CCL2 expression via JNK pathway in C6 rat astrocytoma cells.

The important role of neuroinflammation in many chronic and acute pathological conditions of the central nervous system is widely recognized. Curcumin is a major component of turmeric and reportedly has anti-inflammatory and anti-oxidant effects. This study investigated the inhibitory effect of curcumin on lipopolysacharide (LPS)-induced chemokine CCL2 (or monocyte chemoattractant protein-1, MCP-1) production and whether the effect is mediated by mitogen-activated protein kinases (MAPKs) in the rat astrocytoma cell C6. We observed that LPS (1 µg/ml) induced the upregulation of CCL2 mRNA and protein in C6. Treatment with curcumin (2.5, 10, and 25 µM) decreased the expression of CCL2 mRNA and protein in a dose-dependent manner under treatment with LPS. Additionally, the c-jun N-terminal kinase (JNK) inhibitor (SP600125) dose-dependently inhibited LPS-induced CCL2 upregulation, whereas the MAPK kinase (MEK) inhibitor (PD98059) only had a mild effect and the p38 MAPK inhibitor (SB203580) had no effect. Finally, western blot showed that LPS induced rapid JNK activation and curcumin reduced LPS-induced phosphoJNK (pJNK) expression at 30 min after LPS stimulation. These data suggest that the anti-neuroinflammatory effect of curcumin relates to the downregulation of CCL2 expression through the JNK pathway in astrocytoma cells, which indicates a possible benefit from the use of curcumin in the treatment of neuroinflammation-associated disorders.

The role of TNF-alpha and its receptors in the production of beta-1,4-galactosyltransferase I mRNA by rat primary type-2 astrocytes.

beta-1,4-galactosyltransferase I (beta-1,4-GalT I) plays an important role in the synthesis of the backbone structure of adhesion molecules involved in leukocyte-endothelial cell interaction. The expression of beta-1,4-GalT I mRNA increased in primary human endothelial cells after exposure to tumor necrosis factor-alpha (TNF-alpha). In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-alpha when stimulated with Lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of beta-1,4-GalT I mRNA in type-2 astrocytes after exposure to TNF-alpha and LPS. Real-time PCR showed that TNF-alpha or LPS affected beta-1,4-GalT I mRNA expression in a time- and dose-dependent manner. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes, and that TNF-alpha, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-alpha or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes, and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes, and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed beta-1,4-GalT I mRNA expression induced by TNF-alpha or LPS. From these results, we conclude that TNF-alpha signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce beta-1,4-GalT I mRNA. In addition, we observed that not only exogenous TNF-alpha but also TNF-alpha produced by type-2 astrocytes affected beta-1,4-GalT I mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-alpha contributes to the production of beta-1,4-GalT I mRNA in response to inflammation.

The role of TNF-alpha and its receptors in the production of Src-suppressed C kinase substrate by rat primary type-2 astrocytes.

Src-suppressed C kinase substrate (SSeCKS), an in vivo and in vitro protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein which markedly upregulated in several organs, including brain, lung, heart, kidney, etc., indicating a possible role of SSeCKS in inflammatory process. In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-alpha when stimulated with lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of SSeCKS mRNA in type-2 astrocytes after exposure to TNF-alpha and LPS. Real-time PCR showed that TNF-alpha or LPS affected SSeCKS mRNA expression in a time- and dose-dependent manner. Now that LPS induces SSeCKS expression in type-2 astrocytes and type-1 astrocytes are well known to play a pivotal role in immunity, we compared SSeCKS mRNA expression in type-1 astrocytes with type-2 astrocytes after LPS stimulation. Real-time PCR showed that SSeCKS mRNA level was higher in normal untreated type-2 astrocytes than that in normal untreated type-1 astrocytes, increased significantly after 0.1-100 ng/ml LPS stimulation in type-2 astrocytes, but increased weakly after 10-100 ng/ml LPS stimulation in type-1 astrocytes. By using siRNA knockdown of SSeCKS expression, LPS-induced TNF-alpha synthesis and secretion in type-2 astrocytes were partly inhibited, which indicated that SSeCKS played a role in the TNF-alpha biosynthesis in type-2 astrocytes during the stimulation with LPS. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes and that TNF-alpha, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-alpha or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed SSeCKS mRNA expression induced by TNF-alpha or LPS. From these results, we conclude that TNF-alpha signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce SSeCKS mRNA. In addition, we observed that not only exogenous TNF-alpha but also TNF-alpha produced by type-2 astrocytes affected SSeCKS mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-alpha contributes to the production of SSeCKS mRNA in response to inflammation. In addition, SSeCKS production was also drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor), which indicated that type-2 astrocytes which regulated SSeCKS expression after LPS stimulation were via ERK, SAPK/JNK, and P38MAP kinase signal pathway.

The role of TNF-alpha and its receptors in the production of beta-1,4 galactosyltransferase I and V mRNAs by rat primary astrocytes.

Glycosylation is one of the most important post-translational modifications. It is clear that the single step of beta-1,4-galactosylation is performed by a family of beta-1, 4-galactosyltransferases (beta-1,4-GalTs), and that each member of this family may play a distinct role in different tissues and cells. beta-1,4-GalT I and V are involved in the biosynthesis of N-linked oligosaccharides. beta-1,4-GalT I and V mRNAs are present in control astrocytes and affected by TNF-alpha and lipopolysaccharide (LPS) stimuli. In this study, we examined the regulatory mechanisms of tumor necrosis factor-alpha (TNF-alpha)-affected production of beta-1,4-GalT I and V mRNAs. We show here that cultured astrocytes express TNF-alpha receptor 1 (TNFR1) and increased slightly after exposure to LPS. TNF-alpha and TNFR2 are not detected in control astrocytes and upregulated significantly with LPS stimulation and that activation of these receptors by TNF-alpha affects expressions of beta-1,4-GalT I and V mRNAs. In addition, we observed that not only exogenous TNF-alpha but also TNF-alpha produced by astrocytes affected beta-1,4-GalT I and V mRNAs production in astrocytes. These results suggest that an autocrine loop involving TNF-alpha contributes to the production of beta-1,4-GalT I and V mRNA in response to inflammation.

A2B5 lineages of human astrocytic tumors and their recurrence.

Astrocytomas are very common intracranial glial cell neoplasms with an inherent tendency to progress. However, the heterogeneity of the morphological features and clinical behavior of the tumors makes accurate prognosis based on the histopathological grading system very difficult. Studies demonstrated that astrocytes have two distinctive cell lineages, and tumors arisen from these two astrocytic lineages have been speculated to have different biological and clinical manifestations. The present study aimed to delineate these two astrocytic lineages in human astrocytomas by using different immunohistochemical markers and to correlate the cell lineages of the tumors with their recurrence. Three markers were used, namely the A2B5 antigen, which is present in type 2 astrocytes but absent in type 1 astrocytes, glial fibrillary acidic protein (GFAP), a marker for astrocytes, and galactocerebroside (GC), a marker for oligodendrocytes. It was found that astrocytomas sharing the A2B5+ lineage (A2B5 positive and GFAP positive) have a significantly higher recurrence rate than the tumors of the A2B5- lineage (A2B5 negative and GFAP positive). Immunohistochemical staining and PCR-single-stranded conformational polymorphism analysis showed that p53 overexpression and p53 mutations were closely associated with the recurrent astrocytomas, and p53 abnormalities were more frequently detected in astrocytomas of the A2B5+ lineage. Quantification of proliferation by counting argyrophil nucleolar organizer regions (AgNORs) indicated a higher AgNOR count in the A2B5+ lineage than the A2B5- lineage. Our findings thus suggest that astrocytomas share similar antigenicity with astrocytes, and that the A2B5+ lineage exhibited a higher recurrence rate than the A2B5- lineage. The higher recurrence rate of the A2B5+ tumors may be in part related to the higher frequency of p53 abnormalities found in the tumors and the higher proliferative activity as reflected by the higher AgNOR count of the tumors.