Schisandrin Protects against Norepinephrine-Induced Myocardial Hypertrophic Injury by Inhibiting the JAK2/STAT3 Signaling Pathway

Aims. Heart failure is closely associated with norepinephrine-(NE-) induced cardiomyocyte hypertrophy. Schisandrin is derived from the traditional Chinese medicine Schisandra; it has a variety of pharmacological activities, and the mechanism of schisandrin-mediated protection of the cardiovascular system is not clear. Main Methods. NE was used to establish a cardiomyocyte hypertrophy model to explore the mechanism of action of schisandrin. An MTT assay was used for cell viability; Hoechst fluorescence staining was used to observe the cell morphology and calculate the apoptosis rate. The cell surface area was measured and the protein to DNA ratio was calculated, changes in mitochondrial membrane potential were detected, and the degree of hypertrophic cell damage was evaluated. WB, QRT-PCR, and immunofluorescence were used to qualitatively, quantitatively, and quantitatively detect apoptotic proteins in the JAK2/STAT3 signaling pathway. Key Findings. In the NE-induced model, schisandrin treatment reduced the apoptosis rate of cardiomyocytes, increased the ratio of the cell surface area to cardiomyocyte protein/DNA, and also, increased the membrane potential of the mitochondria. The expression of both JAK2 and STAT3 was downregulated, and the BAX/Bcl-2 ratio was significantly reduced. In conclusion, schisandrin may protect against NE-induced cardiomyocyte hypertrophy by inhibiting the JAK2/STAT3 signaling pathway and reducing cardiomyocyte apoptosis.

An in situ hybridization study of perlecan, DMP1, and MEPE in developing condylar cartilage of the fetal mouse mandible and limb bud cartilage

<p>The main purpose of this <em>in situ</em> hybridization study was to investigate mRNA expression of three bone/cartilage matrix components (<em>perlecan, DMP1</em>, and <em>MEPE)</em> in developing primary (tibial) and secondary (condylar) cartilage. <em>Perlecan</em> mRNA expression was first detected in newly formed chondrocytes in tibial cartilage at E13.0, but this expression decreased in hypertrophic chondrocytes at E14.0. In contrast, at E15.0, <em>perlecan</em> mRNA was first detected in the newly formed chondrocytes of condylar cartilage; these chondrocytes had characteristics of hypertrophic chondrocytes, which confirmed the previous observation that progenitor cells of developing secondary cartilage rapidly differentiate into hypertrophic chondrocytes. <em>DMP1</em> mRNA was detected in many chondrocytes within the lower hypertrophic cell zone in tibial cartilage at E14.0. In contrast, <em>DMP1</em> mRNA expression was only transiently detected in a few chondrocytes of condylar cartilage at E15.0. Thus, DMP1 <em>may</em> be less important in the developing condylar cartilage than in the tibial cartilage. Another purpose of this study was to test the hypothesis that MEPE may be a useful marker molecule for cartilage. <em>MEPE</em> mRNA was not detected in any chondrocytes in either tibial or condylar cartilage; however, MEPE immunoreactivity was detected throughout the cartilage matrix. Western immunoblot analysis demonstrated that MEPE antibody recognized two bands, one of 67 kDa and another of 59 kDa, in cartilage-derived samples. Thus MEPE protein may gradually accumulate in the cartilage, even though mRNA expression levels were below the limits of detection of <em>in situ</em> hybridization. Ultimately, we could not designate MEPE as a marker molecule for cartilage, and would modify our original hypothesis.</p>

Evidence That Estrogen Hastens Epiphyseal Fusion and Cessation of Longitudinal Bone Growth by Irreversibly Depleting the Number of Resting Zone Progenitor Cells in Female Rabbits

With age, growth plate cartilage undergoes programmed senescence, eventually causing cessation of bone elongation and epiphyseal fusion. Estrogen accelerates this developmental process. We hypothesized that senescence occurs because progenitor cells in the resting zone are depleted in number and that estrogen acts by accelerating this depletion. To test this hypothesis, juvenile ovariectomized rabbits received injections of estradiol cypionate or vehicle for 5 weeks, and then were left untreated for an additional 5 weeks. Exposure to estrogen accelerated the normal decline in growth plate height and in the number of proliferative and hypertrophic chondrocytes. Five weeks after discontinuation of estrogen treatment, these structural parameters remained advanced, indicating an irreversible advancement in structural senescence. Similarly, transient estrogen exposure hastened epiphyseal fusion. Estrogen also caused a more rapid decline in functional parameters of growth plate senescence, including growth rate, proliferation rate, and hypertrophic cell size. However, in contrast to the structural parameters, once the estrogen treatment was discontinued, the growth rate, chondrocyte proliferation rate, and hypertrophic cell size all normalized, suggesting that estrogen has a reversible, suppressive effect on growth plate function. In addition, estrogen accelerated the normal loss of resting zone chondrocytes with age. This decrease in resting zone cell number did not appear to be due to apoptosis. However, it was maintained after the estrogen treatment stopped, suggesting that it represents irreversible depletion. The findings are consistent with the hypothesis that estrogen causes irreversible depletion of progenitor cells in the resting zone, thus irreversibly accelerating structural senescence and hastening epiphyseal fusion. In addition, estrogen reversibly suppresses growth plate function.

An Immunohistochemistry Study of Sox9, Runx2, and Osterix Expression in the Mandibular Cartilages of Newborn Mouse

The purpose of this study is to investigate the spacial expression pattern and functional significance of three key transcription factors related to bone and cartilage formation, namely, Sox9, Runx2, and Osterix in cartilages during the late development of mouse mandible. Immunohistochemical examinations of Sox9, Runx2, and Osterix were conducted in the mandibular cartilages of the 15 neonatal C57BL/6N mice. In secondary cartilages, both Sox9 and Runx2 were weakly expressed in the polymorphic cell zone, strongly expressed in the flattened cell zone and throughout the entire hypertrophic cell zone. Similarly, both transcriptional factors were weakly expressed in the uncalcified Meckel’s cartilage while strongly expressed in the rostral cartilage. Meanwhile, Osterix was at an extremely low level in cells of the flattened cell zone and the upper hypertrophic cell zone in secondary cartilages. Surprisingly, Osterix was intensely expressed in hypertrophic chondrocytes in the center of the uncalcified Meckel’s cartilage while moderately expressed in part of hypertrophic chondrocytes in the rostral process. Consequently, it is suggested that Sox9 is a main and unique positive regulator in the hypertrophic differentiation process of mandibular secondary cartilages, in addition to Runx2. Furthermore, Osterix is likely responsible for phenotypic conversion of Meckel’s chondrocytes during its degeneration.

Matrix Gla Protein Is a Developmental Regulator of Chondrocyte Mineralization And, When Constitutively Expressed, Blocks Endochondral and Intramembranous Ossification in the Limb

Matrix GLA protein (MGP), a γ-carboxyglutamic acid (GLA)–rich, vitamin K–dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage–dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a γ-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.

Pituitary Adenylate Cyclase-Activating Polypeptide Is an Auto/Paracrine Stimulator of Acute Progesterone Accumulation and Subsequent Luteinization in Cultured Periovulatory Granulosa/Lutein Cells*

Recently, we have demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) is transiently expressed in steroidogenic ovarian cells during the periovulatory period. This prompted us to establish an in vitro system in which the potential local regulatory role of PACAP during periovulatory progesterone production could be examined. Granulosa/lutein cells from PMSG- and human CG (hCG)-stimulated immature rats were used. The cells were isolated from preovulatory follicles 4–6 h after the hCG injection, at which time the transient ovarian PACAP expression begins in vivo. By immunocytochemistry on intact cells and RIA on cell extracts and culture medium, granulosa/lutein cells were found to accumulate and secrete PACAP during incubation. Furthermore, the cells responded to exogenous PACAP 38 with a rapid (10−7m induced a peak value 20-fold higher than controls at 2 h) and dose-dependent accumulation of progesterone. PACAP 38 (5 × 10−9m), in combination with an approximately half-maximal dose of hCG (1 ng/ml), showed an additive effect on progesterone accumulation. Immunoneutralization of endogenously released PACAP was performed using the IgG fraction from a specific PACAP antiserum that dose-dependently inhibits the progesterone accumulating effect of exogenous PACAP 38. The acute effects of endogenously released PACAP were studied during 8 h of incubation of granulosa/lutein cells with anti-PACAP IgG (100 μg/ml). A significant reduction in progesterone accumulation was observed after 4, 6, and 8 h [38.7% (P &lt; 0.05), 41.2% (P &lt; 0.02), and 50% (P &lt; 0.002), respectively], compared with nonimmune IgG (100 μg/ml) treated cultures. The long-term effects on luteinization induced by endogenously released PACAP were studied after incubation of the cells with anti-PACAP IgG or nonimmune IgG for 24 h, followed by incubation for 9 days in serum-containing medium. Under these conditions, nonimmune IgG-treated cells assumed a luteal phenotype, accumulating large and stable amounts of progesterone and acquiring hypertrophic cell bodies with numerous lipid droplets and distinct nucleoli in the large nuclei. Anti-PACAP IgG-treated cells displayed morphological and functional signs of impaired luteinization being smaller and more irregular and with progesterone accumulation being significantly lower throughout the incubation period [56.4% (P &lt; 0.02), 69.2% (P &lt; 0.05), 43.8% (P &lt; 0.02), and 52.2% (P &lt; 0.02) at 1, 4, 7, and 10 days, respectively]. Together, these findings support an auto- or paracrine role for PACAP during gonadotropin-induced acute periovulatory progesterone production and subsequent luteinization in granulosa/lutein cells.

Correlations between Presence of Spontaneous Lesions of the Pituitary (Adenohypophysis) and Plasma Prolactin Concentration in Aged Wistar Rats

The predictive value of elevated plasma prolactin concentrations for the presence of spontaneous pituitary lesions was studied in 40 male and 38 female Wistar (Cpb:WU) rats, all 30 months old. The pituitaries were examined light microscopically and stained for prolactin using immunohistochemical methods. Plasma prolactin concentrations were measured by radioimmunoassay. Pituitary lesions were classified on the basis of their morphology in hematoxylin and eosin-stained sections as foci of hypertrophic or hyperplastic cells and hemorrhagic, pleomorphic, or spongiocytic adenomas; no carcinomas were found. There were significantly ( P = 0.001) more female than male rats with pituitary adenomas (58% females, 33% males) or without any pituitary lesions (21% females, 5% males); however, there were less female (21%) than male rats (63%) with foci of hyperplastic and/or hypertrophic cells but no adenomas in the pituitary ( P = 0.001). Elevation of plasma prolactin concentration above the upper 99th percentile value in age-matched rats without lesions was predictive, but not conclusively, of the presence of pituitary hemorrhagic adenomas in both sexes. It was, however, not predictive of the presence of foci of hypertrophic or hyperplastic cells. Elevation of plasma prolactin concentration above 10 ng/ml in male and 60 ng/ml in female rats was conclusive for the presence of hemorrhagic adenomas. Using multivariate analysis, significant positive correlations ( P < 0.01) were found between plasma prolactin concentration and presence and size of hemorrhagic adenomas and their prolactin staining intensity (correlation coefficients between 0.392 and 0.652). Foci of hyperplastic cells stained positively for prolactin, whereas hypertrophic cell foci and pleomorphic and spongiocytic adenomas did not stain for prolactin. There were no correlations (coefficients of less than ± 0.189) between plasma prolactin concentration and the presence of hypertrophic or hyperplastic cell foci and pleomorphic or spongiocytic adenomas in the pituitary. The morphologic criteria developed to distinguish spontaneous hypertrophic, hyperplastic, and neoplastic lesions of the rat pituitary corresponded well with their prolactin immunoreactivity and/or ability to elevate plasma prolactin concentration. These criteria constitute a biologically meaningful classification system for these rat pituitary lesions.