[Inhibition of glutaminolysis alleviates myocardial fibrosis induced by angiotensin II].

The present study was aimed to investigate the role and mechanism of glutaminolysis of cardiac fibroblasts (CFs) in hypertension-induced myocardial fibrosis. C57BL/6J mice were administered with a chronic infusion of angiotensin II (Ang II, 1.6 mg/kg per d) with a micro-osmotic pump to induce myocardial fibrosis. Masson staining was used to evaluate myocardial fibrosis. The mice were intraperitoneally injected with BPTES (12.5 mg/kg), a glutaminase 1 (GLS1)-specific inhibitor, to inhibit glutaminolysis simultaneously. Immunohistochemistry and Western blot were used to detect protein expression levels of GLS1, Collagen I and Collagen III in cardiac tissue. Neonatal Sprague-Dawley (SD) rat CFs were treated with 4 mmol/L glutamine (Gln) or BPTES (5 µmol/L) with or without Ang II (0.4 µmol/L) stimulation. The CFs were also treated with 2 mmol/L α-ketoglutarate (α-KG) under the stimulation of Ang II and BPTES. Wound healing test and CCK-8 were used to detect CFs migration and proliferation respectively. RT-qPCR and Western blot were used to detect mRNA and protein expression levels of GLS1, Collagen I and Collagen III. The results showed that blood pressure, heart weight and myocardial fibrosis were increased in Ang II-treated mice, and GLS1 expression in cardiac tissue was also significantly up-regulated. Gln significantly promoted the proliferation, migration, mRNA and protein expression of GLS1, Collagen I and Collagen III in the CFs with or without Ang II stimulation, whereas BPTES significantly decreased the above indices in the CFs. α-KG supplementation reversed the inhibitory effect of BPTES on the CFs under Ang II stimulation. Furthermore, in vivo intraperitoneal injection of BPTES alleviated cardiac fibrosis of Ang II-treated mice. In conclusion, glutaminolysis plays an important role in the process of cardiac fibrosis induced by Ang II. Targeted inhibition of glutaminolysis may be a new strategy for the treatment of myocardial fibrosis.

[Effect of qubi recipe on changes of oxygen free radical metabolism and hypoxia-inducible factor-1alpha in collagen-induced arthritis rats].

OBJECTIVE: To observe the effect of Qubi Recipe (QR) on the expression of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and hypoxia-inducible factor (HIF)-1alpha in rats with type II collagen-I induced arthritis (CIA), and to explore its therapeutic roles and mechanism. METHODS: Totally 72 male SD rats of SPF grade were recruited. Twelve were randomly selected as the blank control group. The CIA model was established in the rest 60 rats by subcutaneously injecting type II collagen of bovine emulsion from the tail root and induction of incomplete Freund's adjuvant. On day 15 after primary immunization rats were randomly divided into four groups, i.e., the CIA model group, the Tripterygium Glycosides (TG) group (at the daily dose of 9.68 mg/kg body weight), the high dose QR group (at the daily dose of 6.66 g/kg body weight), and the low dose QR group (at the daily dose of 3.33 g/kg body weight), 15 in each group. Corresponding medication was given to rats in all groups by gastrogavage once daily for 4 successive weeks. An equal volume of pure water was given to rats in the blank control group and the CIA model group by gastrogavage, once daily for 4 successive weeks. The swelling degree of the joints was measured. Rats were sacrificed after 4-week treatment. Plasma levels of SOD, MDA, and GSH-Px were measured with colorimetric method. The expression of HIF-1alpha was detected by immunohistochemistry. RESULTS: (1) Compared with the CIA model group, the swelling degree of the joints was significantly alleviated in the TG group and the high dose QR group (P < 0.01, P < 0.05), and it was obviously milder in the high dose QR group than in the TG group (P < 0.05). (2) Compared with the CIA model group, the activities of GSH-Px could be obviously elevated and activities of MDA lowered in the TG group, the high dose QR group, and the low dose QR group (P < 0.05). Plasma activities of SOD could be obviously elevated in the high dose QR group and the TG group (P < 0.05). (3) Compared with the CIA model group, the expression of HIF-1alpha obviously decreased in the TG group and the high dose QR group (P < 0.05), and it showed a decreasing tendency in the low dose QR group with no statistical difference (P > 0.05). CONCLUSIONS: QR could markedly alleviate the swelling degree of ankle joints in CIA model rats. Its therapeutic efficacy was superior to that of TG. Its mechanism might be achieved through down-regulating expression of HIF-1alpha in the joint, and regulating activities of SOD, MDA and GSH-Px in the plasma.

Effects of zuogui pill (see text) on Wnt singal transduction in rats with glucocorticoid-induced osteoporosis.

OBJECTIVE: To reveal the mechanism of Zuogui Pill (see text) in treatment of glucocorticoid-induced osteoporosis from the angle of the Wnt signal transduction pathway and to provide further experimental evidence for expounding the scientific connotation of "the kidney dominating the bones" in TCM. METHODS: Forty-two male Wistar rats were selected and randomly divided into three groups, control group (n = 12), model group (n = 15) and Zuogui Pill group (n = 15). Form the beginning, The rats were injected dexamethasone for eight weeks to make the model of osteoporosis, and the Zuogui Pill were administered intragastrically to the rats of Zuogui Pill group for eight weeks. The relative morphological parameters were measured in the undecalcified tibial slices. And the protein expression levels of Wnt1, LRP-5 and beta-catenin in rat tibial osteoblasts (OB) and bone marrow stromal cells (BMC) were detected by immunohistochemistry. RESULTS: Compared with the control group, TBV% and TFS% decreased significantly, while TRS% increased significantly, and the protein expression of Wnt1, LRP-5 and beta-catenin in OB and BMC decreased significantly in the model group. And compared with the model group, TBV% and TFS% increased significantly, and expression levels of Wnt1, LRP-5 and beta-catenin proteins increased significantly in the Zuogui pill group. CONCLUSION: Zuogui Pill can prevent and treat glucocorticoid-induced osteoporosis in rats by up-regulating the expression of the key signal molecules Wnt1, LRP-5 and beta-catenin in Wnt signal transduction pathway.

[Establishment of a rat model of rheumatoid arthritis with kidney deficiency syndrome].

OBJECTIVE: To establish a rat model of rheumatoid arthritis (RA) with kidney deficiency syndrome. METHODS: A total of 110 six-week-old specific pathogen-free male and female Sprague-Dawley rats were randomly divided into normal control group, sham-operated group, collagen-induced arthritis (CIA) control group, castration plus CIA group and hydroxyurea plus CIA group. Testiculus or ovary of rats in the castration plus CIA group was cut off, respectively. Rats in the hydroxyurea plus CIA group were given 375 mg/(kg·d) hydroxyurea by gavage administration for 17 d. Then rats in the CIA control group, castration plus CIA group and hydroxyurea plus CIA group were subcutaneously injected with mixture of type II collagen and incomplete Freund's adjuvant to induce rheumatoid arthritis. General state, arthritis index and joint swelling of the rats were observed to evaluate the onset of CIA. Contents of anti-type II collagen antibody, interleukin-6 (IL-6), IL-10, interferon-γ (IFN-γ) and corticosterone (CORT) in plasma were detected by enzyme-linked immunosorbent assay, and adrenal cyclic adenylic acid (cAMP) and cyclic guanylic acid (cGMP) levels were detected by radioimmunoassay. RESULTS: Compared with the CIA control group, the degrees of joint swelling and joint damage were significantly increased in the kidney-deficiency CIA rats (castration plus CIA group and hydroxyurea plus CIA group), with kidney deficiency syndrome similar to human clinical symptoms, such as depressed, bowed back, dullness, reduced diet and perianal contamination; the rats in those two groups were noted with a significantly decreased ratio of cAMP/cGMP; the content of CORT was increased in male rats while decreased in female rats, with an obvious increase in the content of anti-type II collagen antibody; the contents of IFN-γ, IL-6 and IL-10 were obviously increased in the castration plus CIA group. CONCLUSION: The rat model of RA with kidney deficiency syndrome has both obvious kidney deficiency syndrome and characteristics of RA and can reflect part of the patient's characteristics. However, castration is more suitable for inducing RA with kidney deficiency syndrome in rats.

Aerobic exercise improves cardiac function in rats with chronic heart failure through inhibition of the long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1).

BACKGROUND: To explore the beneficial effects and underlying mechanisms of aerobic exercise on chronic heart failure (CHF). METHODS: A CHF rat model was induced via left anterior descending coronary artery ligation. Four weeks post-surgery, CHF rats received aerobic exercise training over an 8-week period and cardiac function indexes including xxx were analyzed. To investigate the mechanisms involved in the aerobic exercise-induced benefits on CHF, overexpression of the long non-coding RNA MALAT1 was examined both in vivo and in vitro. Furthermore, the interaction between MALAT1 and the microRNA miR-150-5p and the downstream PI3K/Akt signaling pathway was investigated. RESULTS: Compared to the control group, the CHF rats showed evidence of left ventricular dysfunction including aggravated cardiac function indexes and lung to body weight ratio. The Masson staining demonstrated a significant degree of blue-stained fibrotic myocardial tissue in CHF rats compared to control rats. Furthermore, the levels of collagen I and collagen II were also markedly increased in CHF rats. Aerobic exercise improved cardiac function and left ventricular remodeling in rats with CHF. There was a significant reduction in the levels of the reactive oxygen species (ROS), inflammatory cytokines including TNF-α, IL-6, and IL-1ß, and inflammatory mediums containing the matrix metalloproteinases (MMPs) MMP-2 and MMP-9. Moreover, CHF rats receiving aerobic exercise showed decreased myocardial apoptosis and increased expression of autophagy-related proteins including beclin-1 and LC3B-II. Overexpression of the lncRNA MALAT1 eliminated all the beneficial effects related to aerobic exercise in CHF rats. Subsequent investigations demonstrated that miR-150-5p expression was up-regulated in CHF-Tr rats and down-regulated in CHF-Tr-MALAT1 rats. Furthermore, the downstream PI3K/Akt signaling pathway was re-activated in CHF-Tr-MALAT1 rats. In vitro experiments revealed that overexpression of MALAT1 reduced the miR-150-5p levels, resulting in increased cellular apoptosis and less autophagy. In addition, overexpression of MALAT1 suppressed the downstream PI3K/Akt signaling pathway. Restoring miR-150-5p level with a miR-150-5p mimic decreased the cellular apoptosis and increased autophagy, and the downstream PI3K/Akt signaling pathway was re-activated. CONCLUSIONS: Aerobic exercise improved cardiac function through inhibition of the lncRNA MALAT1 in CHF, and the potential mechanisms may be mediated via the miR-150-5p/PI3K/Akt signaling pathway.

Synthesis of a novel class of substituted benzothiophene or benzofuran derivatives as BMP-2 up-regulators and evaluation of the BMP-2-up-regulating effects in vitro and the effects on glucocorticoid-induced osteoporosis in rats.

The BMP pathway is a promising new target for the design of therapeutic agents for the treatment of low bone mass. To enrich our understanding of SAR and based on our previously concluded structure-effect relationship, 23 derivatives were prepared in this work. The synthesis, up-regulating activities on BMP-2 expression, and bone loss prevention efficacies of these compounds in rats with glucocorticoid-induced osteoporosis are presented. The bone histology of the tested rats assessed through light microscopy showed that compounds 1, 21, 35, and 38 significantly increased the trabecula compared with the model group, and the trabecula of the groups treated with 8a was similar to that obtained with raloxifene and alfacalcidol. The compounds exhibited potential for development as anabolic agents.

[Temperature dependence of collagen by Raman spectra].

Raman spectra of collagen I at different temperature were obtained. There was no change at 1003 cm(-1) line, 1302 cm(-1) line moved to a higher wave number, but other lines moved to lower wave number when temperature increased. In addition, the authors observed the temperature dependence of Raman intensity and four denaturation points at 0, 40, 68 and 90 degrees C respectively. The points at 40 and 68 degrees C are in agreement with the experimental data by DSC and SHG. The point at 0 degrees C might be frozen transition; the point at 90 degrees C might be related to the damage of secondary structure. When heated to 150 degrees C, the Raman intensity of all bands decreased rapidly and many lines disappeared.

Therapeutic effect of aqueous extract from Ecliptae herba on bone metabolism of ovariectomized rats.

OBJECTIVE: Ecliptae herba (EH) has long been used in China to strengthen bones. Accumulating evidence indicates that EH may have antiosteoporotic effects. The aim of this study was to evaluate the effects of aqueous EH extract (EHE) on rats that had osteoporosis-like features induced by ovariectomy, using aqueous Fructus Ligustri Lucidi extract as positive control agent. METHODS: Three-month-old female rats that underwent ovariectomy were treated with EHE (1.4 g/kg per day). After 12 weeks, bone mineral density and bone histomorphometric indices of tibiae were measured. Protein and messenger RNA expressions of osteoprotegerin and receptor activator of nuclear factor κ-B ligand (RANKL) in tibiae were evaluated by immunohistochemistry and in situ hybridization. In addition, serum concentrations of osteocalcin, interleukin-1ß, interleukin-6 (IL-6), calcitonin (CT), and parathyroid hormone were determined by enzyme-linked immunosorbent assay. RESULTS: EHE treatment prevented body weight gain and loss of uterine wet weight in ovariectomized rats. It remarkably increased bone mass in ovariectomized rats compared with ovariectomized controls. EHE treatment significantly down-regulated RANKL expression in tibiae from ovariectomized rats compared with controls; however, it had no significant effect on osteoprotegerin expression. In addition, EHE treatment significantly reduced serum IL-6 levels and remarkably increased CT levels but had no effect on parathyroid hormone. CONCLUSIONS: EHE increases bone mass in ovariectomized rats by inhibiting bone loss: down-regulated RANKL expression in tibiae and IL-6 level in serum, and up-regulated CT level in serum. This suggests that EHE may be developed as an alternative therapeutic agent for osteoporosis induced by postmenopause.