Therapeutic effects of modified Bushen Huoxue granules on ovariectomized osteoporosis rats

AIM: To observe the therapeutic effect of modified Bushen Huoxue granules (MBHG) on ovariectomized osteoporosis rats. METHODS: Female SD rats were randomly given sham operation (n=10) and ovariectomy, and then the model rats were further randomly divided into model group, MBHG treatment groups at doses of 200 mg/kg, 100 mg/kg and 50 mg/kg respectively, and positive control (estradiol valerate) group, with 10 rats in each group by intragastric administration for 12 weeks. The morphology, area, thickness, spacing and area percentage of trabecular bone in the rats were observed. The serum levels of calcium (Ca), phosphorus (P) and alkaline phosphatase (ALP) were mea-sured by automatic analyzer. Bone mineral density (BMD) was analyzed. Serum estradiol (E₂), osteocalcin (BGP), osteoprotegerin (OPG), receptor activator of nuclear factor-κB (RANK) and receptor activator of nuclear factor κB ligand (RANKL) levels were detected by ELISA. RESULTS: Compared with model group, trabecular bone significantly widened in all treatment groups with large number, and net-like structure restored partially. The thickness, area and area percentages of trabecular bone in treatments groups were higher than those in model group,and trabecular spacing was less than that in model group (P<0.05). The serum Ca, P, E₂ and OPG, and femoral BMD were significantly higher in treatment groups than those in model group, and the levels of ALP, BGP, RANK and RANKL were significantly lower than those in model group (P<0.01).CONCLUSION: MBHG has a significant therapeutic effect on ovariectomized osteoporosis rats. The mechanism may be related to the regulation of OPG and inhibition of RANKL secretion.     

Effect of urantide on liver function and histomorphology in atherosclerotic rats

AIM:To investigate the effect of urantide on the liver function and histomorphology in the rats with atherosclerosis (AS).METHODS:The AS Wistar rat model was induced by intraperitoneal injection of vitamin D₃ (VD₃) and feeding with high-fat diet. The rats were randomly divided into normal control group, AS model group, positive medicine group and urantide group. The liver function indexes of the rats were measured by biochemical test, and the pathological changes of the aorta and liver of the rats were observed by hematoxylin-eosin (HE) staining. The mRNA expression of urotensin Ⅱ (UⅡ) and GPR14 at mRNA and protein levels in rat livers was determined by RT-qPCR and Western blot. RESULTS:The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyltransferase (γ-GT), lactate dehydrogenase (LDH), total bilirubin (TBIL), indirect bilirubin (IBIL) and alkaline phosphatase (ALP) in AS model group were significantly increased compared with normal control group (P<0.05). The above indexes in urantide group were remarkably decreased compared with AS model group (P<0.05). No change of the levels of direct bilirubin (DBIL), total protein (TP), globulin (GLB) and albumin (ALB) in each group was observed. Urantide postponed hepatocyte fatty degeneration and repaired hepatocyte injury in the AS rats. Compared with normal control group, the mRNA and protein levels of UⅡ and GPR14 in the liver were significantly increased in AS model group (P<0.05). With the prolongation of dosing time, the mRNA and protein levels of UⅡ and GPR14 in the liver were significantly decreased in urantide group compared with AS model group (P<0.05). CONCLUSION:Urantide significantly attenuates the liver damage caused by liver fatty degeneration in AS rats.     

Effects of icariin on expression of transforming growth factor β1 and type Ⅳ collagen in diabetic rat testis

AIM: To determine the beneficial effects of icariin on streptozotocin (STZ)-induced diabetic testopathy in rats. METHODS: The diabetic animal model was induced in male Sprague-Dawley rats by an injection of streptozotocin (40 mg/kg, iv). The rats were randomly divided into 3 groups: control group, model group and icariin (80 mg/kg, ig) group. Twelve weeks after injected with streptozotocin, all rats were anaesthetized and killed to remove the testes from scrotum. Serum concentrations of glucose and testosterone, and the levels of succinate dehydrogenase (SDH), acid phosphatase (ACP), γ-glutamyl transpeptidase (γ-GT) and lactate dehydrogenase (LDH) in testes were measured. The morphology of the testicular tissues was observed under light microscope. Immunohistochemistry was employed to determine the protein levels of TGF-β₁ and type Ⅳ collagen. RESULTS: Compared with control group, the content of serum glucose increased while the serum level of testosterone and the activitiy of SDH, ACP, γ-GT and LDH in testis decreased in model group (P<0.01). The histopathological examination showed that the diameters of seminiferous tubules and various grades of spermatocytes in the testis were markedly decreased. Compared with control group, the expression of TGF-β₁ and collagen Ⅳ was significantly increased in model group. These alterations were significantly attenuated in icariin group (P<0.01). CONCLUSION: Icariin evidently relieves testicular damage in rats with diabetic testopathy by improving the secretion of testosterone and reducing the expression of TGF-β₁ and collagen Ⅳ at protein level.     

Effect of traditional Chinese medicine Muniziqi on gonad axis in rats with chronic stress premature ovarian failure

AIM: To investigate the effects of traditional Chinese medicine Muniziqi on chronic premature ovarian failure (POF) by observing the histomorphological changes of pituitary, hypothalamus and ovary in rats with chronic POF. METHODS: Mature Sprague-Dawley rats (female, n=90) were randomly divided into normal group (n=10), and stress model group (n=80). After the model was established, the rats with POF were screened. The model rats were divided into POF group, and POF with high-, medium- and low-dose Muniziqi groups. HE staining and Masson staining were used. The morphological changes of pituitary, hypothalamic and ovarian tissues were observed, and the ovarian and uterus indexes were calculated. The serum levels of estradiol (E₂), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the rats were measured by ELISA. RESULTS: The traditional Chinese medicine Muniziqi had certain effects on the morphological changes and hormone levels of pituitary, hypothalamic and ovarian tissues in the rats with chronic POF. Compared with POF group, each drug intervention group had different degrees of improvement. Compared with normal group, the E₂ level in POF group was significantly decreased (P<0.05), and the levels of LH and FSH were significantly increased (P<0.05). CONCLUSION: Chronic stress results in the occurrence of POF. The traditional Chinese medicine Muniqizi has the effects on prevention and treatment of POF and improvement of histomorphological changes and hormone levels of the gonaol axis.     

Effects of drynaria total flavonoids on serum levels of leptin,interleukin 6, prostaglandin E2 and expression of bone β2-adrenergic receptor in ovariectomized osteoporosis rats

AIM: To investigate the effects of drynaria total flavonoids on serum levels of leptin (LEP), interleukin 6(IL-6), prostaglandin E₂(PGE₂) and the expression of bone β₂-adrenergic receptor (ADRB2) in a rat model of ovariectomized osteoporosis(OP). METHODS: The osteoporosis model was established by ovariectomy. Twelve weeks after modeling,bone mineral density (BMD) was determined by dual-energy X-ray absorptiometry to verify successful modeling.Enzyme-linked immunosorbent assay was applied to detect the concentrations of LEP, IL-6 and PGE₂ in serum. The expression of ADRB2 was determined by immunohistochemical technique. RESULTS: Compared with sham group,BMD of the rats in model group significantly decreased in multiple regions 12 weeks after modeling(P<0.01). The serum levels of LEP, IL-6 and PGE₂ in model group were significantly higher than those in sham group(P<0.05). The levels of LEP, IL-6 and PGE₂ in drynaria total flavonoids group were significantly lower than those in model group(P<0.01). No significant difference of PGE₂ between these 2 groups was observed. The ADRB2 expression in sham group and treatment group was significantly different from that in model group, and no significant difference between sham group and treatment group was found. CONCLUSION: The serum levels of LEP, IL-6 and PGE₂ and the expression of bone ADRB2 increased in OP rats.Drynaria total flavonoids reduce the production of LEP, IL-6 and the expression of ADRB2, and suppress the bone absorption, which may be one of the mechanisms in treating OP.     

Alteration of myocardial sarcoplasmic reticulum Ca2+ transport protein expression in neonatal hypothyroid rats

AIM: To investigate the alteration of sarcoplasmic reticulum (SR) Ca²⁺ transport proteins including sarcoplasmic reticulum Ca²⁺-ATPase 2a(SERCA2a) and phospholamban(PLB) mRNA expression as well as the alteration of myocardial SR Ca²⁺-ATPase activity in neonatal hypothyroid rats, and to explore the effect of levothyroxine(L-T₄) substitution therapy on the above indexes.METHODS: Hypothyroidism was induced by the administration of propylthiouracil (PTU, 50 mg/d) to the pregnant SD rats by gavage beginning on embryonic day 15 and continuing throughout the lactational period. A subgroup of neonatal hypothyroid rats were intraperitoneally injected with L-T₄ levothroxine (20 μg/kg BW daily), starting from the day of birth. Other pregnant SD rats received normal saline instead of PTU. The samples of the rats in all 3 groups were harvested at postnatal day 3, 5 and 7 respectively (n=10). After measurement of serum thyroid hormone levels, the hearts were removed and the ventricles were weighed (HW). The concentration of calcium in ventricular myocardium(ventricular myoCa²⁺) was detected by fluorospectrophotometry and the activity of SR Ca²⁺-ATPase was determined by the inorganic phosphorus method. The mRNA expression of SERCA2a and PLB was also detected by real-time PCR. RESULTS: Neonatal hypothyroid rats had a significant lower level of SERCA2a mRNA (P<0.05) and a higher level of PLB mRNA (P<0.01), and subsequent lower SERCA2a/PLB at each postnatal day (P<0.01) was observed. Compared with hypothyroid group, the mRNA expression of SERCA2a significantly increased (P<0.05) and that of PLB significantly decreased (P<0.05) in L-T₄ treatment group. The concentration of ventricular MyoCa²⁺ in hypothyroid group was significantly higher than that in control group (P<0.01), and that in L-T₄ treatment group showed a significant decrease as compared with hypothyroid group (P<0.05). The activity of sarcoplasmic reticulum Ca²⁺-ATPase in hypothyroid group was significantly lower than that in control group (P<0.01), and that in L-T₄ treatment group showed a significant increase as compared to hypothyroid group (P<0.05). CONCLUSION: The deficiency of thyroid hormone, resulting in decreased expression of SERCA2a mRNA as well as increased PLB mRNA, contributes to the reduction of SR Ca²⁺-ATPase activity in neonatal rats. This may be one of the most important mechanisms of myocardial systolic and diastolic dysfunctions.     

Role of P2Y1 receptor in activation of astrocytes induced by Aβ25-35

AIM: To study the role of P2Y₁ receptor in the activation of astrocytes induced by Aβ_25-35.METHODS: Astrocytes were isolated and cultured from newborn Wistar rats and divided into control group, Aβ_25-35 group, MRS2179(P2Y₁receptor inhibitor)+Aβ_25-35 group and MRS2179 group by treating the cells with the corresponding reagents. The expression levels of GFAP and P2Y₁ were determined by the methods of immunohistochemistry, immunofluorescence and Western blotting.RESULTS: No significant change of the astrocyte numbers in all groups was observed. Compared with the control cells, the fluorescence intensity of GFAP significantly increased in Aβ_25-35 group and decreased in both MRS2179+Aβ_25-35 group and MRS2179 group. The expression level of GFAP determined by Western blotting and immunofluorescence showed the similar trend of change in each group. Compared with control group, the expression of P2Y₁ in Aβ_25-35 group was significantly increased (P<0.05), and no significant change between MRS2179+Aβ_25-35 group and MRS2179 group was found (P>0.05).CONCLUSION: Aβ_25-35 activates astrocytes by activation of P2Y₁ receptor.     

Changes of histone modification levels in rats with liver fibrosis

AIM: To observe the changes of histone modifications in the liver of rats with hepatic fibrosis and its possible role in the development of hepatic fibrosis. METHODS: Male Wistar rats (n=20) were randomly divided into liver fibrosis group and normal control group. The liver fibrosis model was established by hypodermic injection of CCl₄, and the rats in normal control group were injected with vegetable oils. At the end of the 8th week, all rats were killed. Liver function indexes including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and liver fibrosis indexes including haluronic acid (HA), laminin (LN), collagen type IV (Col Ⅳ) and procollagen type Ⅲ (PCⅢ) were determined by biochemical and RIA methods. The liver index was analyzed, and the liver fibrosis degree and the morphological change of the liver were detected by HE and Masson staining. The levels of α-smooth muscle actin (α-SMA), collagen type Ⅰ (ColⅠ), H3K4me2, H3K9me2, acH3K9 and acH4K12 were detected by Western blot. RESULTS: After hypodermic injection of CCl₄ for 8 weeks, the liver index, ALT, AST, HA, LN, Col Ⅳ and PCⅢ of the rats in liver fibrosis group were higher than those in control group (P<0.05). Compared with control group, the level of acH4K12 was decreased (P<0.05), while acH3K9, H3K9me2, α-SMA and ColⅠ were increased (P<0.05), but H3K4me2 had no significant change.CONCLUSION: The levels of acH4K12, acH3K9 and H3K9me2 may play essential roles in the pathogenesis of liver fibrosis, and these histone modifications may regulate gene expression associated with extracellular matrix metabolism.     

Effects of gypenosides on PCSK9 gene expression and blood lipids lowered by simvastatin

AIM: To explore the effect of gypenosides (GPs) on PCSK9 gene expression in hyperlipidemic rat liver and the blood lipids lowered by simvastatin.METHODS: Healthy male SD rats (n=60) were randomized into 5 groups:normal control group, hyperlipidemic model group, simvastatin group, GPs group and GPs combined with simvastatin group (combined group). The rats in all groups were fed high-fat diet except normal control group which were fed with ordinary diet. The rats in control group and hyperlipidemic model group were gavaged with 0.3% CMC-Na every day. The rats in GPs group were gavaged with GPs at 160 mg·kg^-1·d^-1. The rats in simvastatin group were gavaged with simvastatin at 5 mg·kg^-1·d^-1. The rats in combined group were gavaged with GPs and simvastatin. The experiment lasted for 8 weeks. The rats were anesthetized with chloral hydrate, and abdominal arterial blood samples were collected to detect the total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C). The body weight and the wet weight of the livers were measured, and the liver index was calculated. The pathological changes of the livers were observed under microscope with HE staining. The expression of PCSK9 and low-density lipoprotein receptor (LDLR) at mRNA and protein levels was determined by real-time PCR and Western blot. RESULTS: The model of hyperlipidemia rats was established successfully. Compared with model group, the levels of TC, TG and LDL-C in simvastatin group, GPs group and combined group were obviously decreased (P<0.05), and the HDL-C levels were obviously upregulated (P<0.05). Compared with model group, the liver indexes in simvastatin group, GPs group and combined group were obviously decreased (P<0.05). The pathological changes of the liver tissues showed that hepatic adipose appeared in model group, and that in simvastatin group and GPs group had different degrees of relief, especially in combined group. Compared with model group, the mRNA expression levels of PCSK9 and LDLR in simvastatin group were obviously increased, while the mRNA expression levels of PCSK9 in GPs group and combined group were obviously decreased (P<0.05), and the mRNA expression of LDLR in combined group was obviously increased (P<0.05). Compared with model group, the protein expression of PCSK9 and LDLR in simvastatin group was obviously increased, while the protein expression levels of PCSK9 in GPs group and combined group were obviously reduced, and the LDLR protein levels were obviously increased (P<0.05). CONCLUSION: Gypenosides inhibit the expression of PCSK9 and increase the expression of LDLR in the liver. The combination of gypenosides and simvastatin promotes the lipid-lowering effect of simvastatin and attenuates hepatic steatosis, which may be related to inhibiting the expression of PCSK9 in the liver.     

Effects of urocortin-I preconditioning on myocardial mitochondrial respiratory function and enzyme activity in rats

ATM: To investigate the influence of urocortin-I (Ucn I) preconditioning on the myocardial mitochondrial respiratory function and enzyme activity in the rats with ischemia reperfusion, and to observe the changes of ATP content in the myocardial cells. METHODS: (1) The healthy male Sprague-Dawley rats were randomly divided into 4 groups:normal group (Nor group), ischemia reperfusion group (IR group), Ucn I preconditioning group (Ucn I group), 5-hydroxy acid (5-HD)+Ucn I group. Langendorff perfusion was used to establish the in vitro model of cardiac ischemia reperfusion. At the end of the balance (T₁), before ischemia (T₂) and at the end of the reperfusion (T₃) respectively, the myocardial mitochondria was extracted, the mitochondrial respiratory function and respiratory enzyme activity in each group were determined. (2) The method of MPA isolated heart perfusion was used to isolate myocardial cells of the adult rats. After cultured for 24 h, myocardial cells were divided into 4 groups:Nor group, hypoxia/reoxygenation group (I/R group), Ucn I group, 5-HD+Ucn I group. Hypoxia/reoxygenation model of myocardial cells was established. At the end of reoxygenation, the changes of myocardial ATP content were measured by high performance liquid chromatography.RESULTS: (1) Compared with T₁, T₂ time points, the respiratory function (state 3 respiratory rate, respiratory control rate) and NADH oxidase, succinate oxidase and cytochrome C oxidase activities at T₃ time point were significantly decreased (P<0.05) in all groups except Nor group. At T₃ time point, the myocardial mitochondrial respiratory function and respiratory enzyme activity in Ucn I group were superior to 5-HD+Ucn I group and IR group (P<0.05), but was inferior to Nor group (P<0.05). At T₃ time point, the respiratory function of myocardial mitochondria and respiratory enzyme activities (NADH oxidase, succinate oxidase) in 5-HD+Ucn I group were better than those in IR group (P<0.05), but no statistical difference of the cytochrome C oxidase activity between the 2 groups was observed. The respiratory function and 3 kinds of respiratory enzyme activities at T₁, T₂ time points had no statistical change. (2) At the end of the reoxygenation, the myocardial ATP content in Nor group was higher than that in other groups (P<0.01). The myocardial ATP contents in I/R group and 5-HD+Ucn I group were lower than that in Ucn I group (P<0.05). In additon, 5-HD+Ucn I group was higher ATP content compared with I/R group (P<0.05). CONCLUSION: Ucn I preconditioning attenuates the ischemia/reperfusion induced damages of myocardial mitochondrial respiratory function and respiratory enzyme activity, thus ensuring the myocardial ATP contents under the condition of hypoxia/reoxygenation.     

Effects of different lighting on reproductive system in depressive female rats

AIM: To investigate the effects of different lighting on the reproductive system in depressive female rats. METHODS: Healthy adult female rats were randomly chosen as control group, and the depressive adult female rats in SPF grade were randomly divided into 5 groups(7 rats each):depressive model group, sulfur lamp group, heat radiation lamp group, fluorescent lamp group and LED lamp group. After 45 d of continuous illumination, the estrous cycle was observed by the vaginal exfoliated cells, and the organ indexes of ovary and uterus were calculated. The concentrations of estiadrol(E₂), prolactin(PRL), progesterone(PROG) and testosterone(T) in the serum were detected by ELISA, and the histopathological lesion of ovary was observed under microscope with HE staining. RESULTS: The estrous cycle exhibited serious disorder, the ovaries exhibited serious congestion, and the organ indexes of ovary and uterus and the concentrations of E₂, PRL, PROG and T decreased significantly in the rats in depressive model group compared with control group(P<0.05). The estrous cycle and histopathological damage of ovary were obviously improved, and the concentrations of E₂, PRL, PROG and T were significantly increased after the sulfur lamp lighting in the depressive female rats compared with depressive model group. No obvious change and improvement of the reproductive functions in the heat radiation lamp group, fluorescent lamp group and LED lamp group was observed. CONCLUSION: The reproductive functions of depressive female rats are improved by sulfur lamp lighting.     

Effect of Zhengtian pills on P2X3 receptor expression in trigeminal gan-glion of migraine rats

AIM: To explore the effect of Zhengtian pills on P2X₃ receptor expression in trigeminal ganglion (TG) of migraine rat. METHODS: Sprague-Dawley (SD) rats were randomly divided into control group, migraine group, Zhengtian pills (ZTP) group and A-317491 group. After given corresponding drugs for 7 d, migraine rat model was established by subcutaneously injection of nitroglycerin (10 mg/kg), while the control rats were injected with saline. The beha-vioral manifestations of the rats were observed. The expression of P2X₃ receptor in rat TG was detected by the methods of immunofluorescence, Western blot and rea-time PCR. RESULTS: About 5 min after subcutaneousl injection, the behavioral manifestations such as scratching head and climbing cage were observed. The behavioral manifestations were observed within the first 30 min in control group, but the erythroid ears did not appear. After 2 h of molding, the behavioral manifestations disappeared in ZTP group and A-317491 group, while those in migraine group lasted for 3 h. Immunofluorescence results showed that the expression of P2X₃ receptor in TG was positive in each group. The expression level in migraine group was significantly higher than that in other groups. The P2X₃ receptor protein and mRNA levels in the TG of migraine group were higher than those in control group (P<0.01), while those in ZTP group were lower than those in migraine group (P<0.01). No difference of the P2X₃ receptor expression between ZTP group and A-317491 group was observed. CONCLUSION: Zhengtian pills may effectively alleviate migraine by inhibiting the expression of P2X₃ receptor in TG.     

Experimental study on setting up hypercapnic model and its pathophysiological changes in rats

AIM:To reproduce hypercapnic models and approach some pathophysiological changes in rats. METHODS:The mixed gases of high concentrated carbon dioxide (8% CO₂, 21% O₂, 71% N₂) were given to wistar rats 7 hours a day for 28 days. The various indexes were compared between control group (group A) and hypercapnic group (group B). RESULTS:The PaCO₂ and the lipid peroxides (LPO) contents in plasma, lung tissue and right ventricle were significantly higher in group B than those in group A (P＜0.05 ); while artery pH values (7.205±0.037) and superoxide dismutase (SOD) activities in erythrocytes, lung tissue and right ventricle were significantly decreased in group B compared with group A (P＜0.05). The weight of right ventricle was significantly heavier than that of group A (P＜0.05). There were no significant differences were found in mean right artial pressure (Rap), mean right ventricle pressure (Rvp) and mean pulmonary artery pressure (Pap) between both groups.CONCLUSION:Hypercapnic model can be reproduced with 8% CO₂ mixed gases in rats. High concentration of CO₂ can cause a series of positively toxic injuries to the body. Care should be seriously taken when using permissive hypercapnic ventilation in the clinic.     

Comparison of the levels of serum prolactin and the concentrations of anti-infective factors of human colostrum in normal labor and cesarean section

AIM: To compare the difference in serum prolactin levels and the concentrations of anti-infective factors of human colostrums between normal labor and cesarean section. METHODS: The serum prolactin levels were measured at 48 h, 96 h after parturition in 100 cases of normal labor and cesarean using radioimmunoassay. Furthermore, the levels of SIgA, IgG, IgM, C₃, C₄ were tested in 100 cases at the same time using the technique of immuno-turbidimetry. RESULTS: The serum prolactin levels in normal labor tested at 48 h after parturition were significantly different with that of the cesarean section group. But there was no significant difference in serum prolatin levels 96 h after parturition between the two groups. The levels of SIgA,IgG,IgM,C₃ and C₄ of the colostrum in the cesarean group were higher significantly than those of the normal group at 48 h after parturition. (P＜0.05 or P＜0.01). At 96 h after parturition, there was no significant difference in SIgA, IgG, IgM and C₃ between the two groups except C₄. CONCLUSION:At 48 h after paturition, the serum prolactin levels of normal labor group are higher than that of the cesarean section group. The concentrations of anti-infective factors (SIgA, IgG, IgM, C₃ and C₄) of human colostrums at 48 h after paturition in cesarean section group are higher than those of normal labor group.     

Effects of rapamycin on hydrogen peroxide-induced vascular endothelial cell senescence

AIM:To investigate the effects of rapamycin (Rapa) on hydrogen peroxide (H₂O₂)-induced vascular endothelial cell senescence and to explore the underlying mechanisms. METHODS:The human umbilical vascular endothelial cells (HUVECs) were divided into 4 groups:control group, senescence group, Rapa+H₂O₂ group and 3-methyladenine (3-MA)+H₂O₂ group. MTT assay was performed to assess the cell viability. Senescence-associated β-ga-lactosidase (SA-β-Gal) staining was performed to measure the senescent cells in each group. The subcellular structures were observed under transmission electron microscope (TEM). The protein levels of phosphorylated Rb (p-Rb), Rb, p21, LC3-Ⅱ and beclin-1 were determined by Western blot. RESULTS:Compared with control group, the cell viability in H₂O₂ group was significantly decreased accompanied with higher rate of SA-β-Gal staining positive cells (P<0.05) and markedly damaged structure. Additionally, the protein levels of p-Rb and p21 in senescence group were increased markedly compared with control group (P<0.05). However, the cells pre-treated with Rapa prior to stimulation with H₂O₂ showed increased viability, decreased number of senescent cells and decreased protein levels of p-Rb and p21 as compared with the cells stimulated with H₂O₂ alone (P<0.05). Moreover, the TEM observation showed that the structure of the cells in Rapa+H₂O₂ group was roughly normal and the autophagosome was captured, and the expression levels of beclin-1 and LC3-Ⅱ were increased (P<0.05). Conversely, pre-treatment with autophagy inhibitor 3-MA resulted in opposite results. The cell viability was decreased significantly, more senescent cells were stained blue, higher protein levels of p-Rb and p21 were detected (P<0.05), poor subcellular structures were captured, and no beclin-1 and LC3-Ⅱ was detected. CONCLUSION:Rapa may retard the senescence of HUVECs induced by H₂O₂, and promoting autophagy may be the underlying mechanism.     

Role of central PGE2 on sympathetic excitation in chronic heart failure

AIM: To observe the effect of central prostaglandin E₂ (PGE₂) on sympathetic activation in chronic heart failure (CHF) and to explore the underlying mechanism. METHODS: Male SD rats were subjected to coronary artery ligation to induce heart failure (HF), and the intracerebroventricular infusion was performed by osmotic pump continuously. The rats in sham group and HF group were given artificial cerebrospinal fluid (0.25 μL/h). The rats in HF plus treatment group was given celecoxib (CLB; 20 mg/h). After 4 weeks, the levels of PGE₂ in cerebrospinal fluid (CSF), the sympathetic nerve excitability and cardiac function were measured, and the changes of corticotropin-hormone releasing hormone (CRH)-containing neurons activation and neurotransmitter contents in the hypothalamic paraventricular nucleus (PVN) were also determined. RESULTS: Compared with the sham-operated rats, the HF rats had raised level of PGE₂ in CSF, up-regulated renal sympathetic nerve activity and plasma norepinephrine, increased left ventricular end diastolic pressure, lung-to-body weight and right ventricular-to-body weight ratios, and decreased maximal increase and decreased rate of left ventricular pressure (P<0.05). In addition, the number of CRH positive neurons in PVN and the level of plasma adrenocorticotropic hormone were higher in HF rats than those in sham-operated rats (P<0.05). After administration of CLB into the lateral ventricle of HF rats, the contents of PGE₂ in CSF were significantly reduced, the number of activation CRH neurons in PVN was decreased, the excitability of sympathetic nerves was down-regulated and cardiac function was improved (P<0.05). Compared with the sham-operated rats, the content of glutamic acid in PVN of HF rats was increased, the content of γ-aminobutyric acid and the number of glutamate decarboxylase 67-positive neurons were decreased (P<0.05). After the CLB was given, the above indexes were reversed (P<0.05). CONCLUSION: These findings indicate that in CHF, the increased central PGE₂ may activate CRH-containing PVN neurons and contribute to the augmented sympathetic drive possibly by modulating the neurotransmitters within the PVN.     

Effect of chelerythrine on TGF-β/Smads signaling pathway in mouse livers with hepatic fibrosis

AIM:To investigate the anti-hepatic fibrosis effect of chelerythrine on mice and the regulation of transforming growth factor-β (TGF-β)/Smads signaling pathway. METHODS:C57BL/6N mice (n=50) were randomly divided into control group, model group and chelerythrine groups (10 mg·kg^-1·d^-1, 20 mg·kg^-1·d^-1 and 40 mg·kg^-1·d^-1, ig). The mouse model of hepatic fibrosis was established by intraperitoneal injection of carbon tetrachloride (CCl₄) in combination with the olive oil for 8 weeks. At the 5th week, different doses of chelerythrine was used to treat hepatic fibrosis in the mice. At the 14th week, hepatic index was detected. Histopathological changes and the degree of hepatic fibrosis were observed by hematoxylin-eosin staining and Van Gieson staining. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA), and hepatic hydroxyproline (Hyp) content were assayed by spectrophotometry and ELISA. The mRNA expression of TGF-β₁, Smad3, Smad4 and Smad7 in the liver was detected by RT-qPCR, and the protein expression of TGF-β₁, Smad4 and Smad7 was determined by Western blot. RESULTS:The degree of hepatic fibrosis changed markedly in model group compared with control group. The hepatic index, the serum levels of ALT and AST, and the contents of HA and Hyp were significantly increased (P<0.05). The mRNA expression of TGF-β₁, Smad3 and Smad4 was significantly up-regulated, while the mRNA expression of Smad7 was significantly down-regulated (P<0.05). The protein expression of TGF-β₁ and Smad4 was significantly up-regulated, while the protein expression of Smad7 was significantly down-regulated (P<0.05). Compared with model group, the changes of the above indexes in chelerythrine groups were inhibited. CONCLUSION:Chelerythrine protects the mouse liver from CCl₄-induced fibrogenesis injury by regulating TGF-β/Smads signaling pathway.     

Blockade of L-type Ca2+ channels suppresses activation of calcineurin and development of right ventricle cardiac hypertrophy induced by chronic hypoxia

AIM: To study the role of calcineurin in the progression of right ventricle cardiac hypertrophy in the chronic hypoxia rats and examine the effect of Ca²⁺ channel blockers on the activation of calcineurin. METHODS: Sixty rats were divided into three groups: treatment group with amlodipine besylate ablets, chronic hypoxia group, normal control group with normal oxygen. The rats in treatment group and chronic hypoxia group were exposed to normobaric chronic hypoxia(10±0.5)% O₂ for 21 days. All hearts were removed immediately after dissection for further investigation. RESULTS: (1)The RV/(LV+S),RV/BW were significantly higher in hypoxia group than that of control group and treatment group(P<0.01,respectively); (2) Right ventricular cardiomyocytes [Ca²⁺]_i in treatment group were significantly higher than that of control group and lower that that of hypoxia group(P<0.01,respectively); (3) The activity of calcineurin of the heart in hypoxia group were significantly increased when compared with control group. Amlodipine besylate ablets apparently suppressed the activity of calcineurin(P<0.01). CONCLUSION: Calcineurin possibly plays a role in the progression of right ventricle cardiac hypertrophy in the chronic hypoxia rats;Blockade of L-type Ca²⁺ channels with amlodipine besylate ablets effectively prevents its development possibly by inhibition of calcium inflow and suppression of the calcineurin activity.     

Roles of gap junction in TGF-β1-induced proliferation of vascular smooth muscle cells from spontaneous hypertensive rats

AIM: To investigate whether gap junction participates in transforming growth factor β₁(TGF-β₁)-induced proliferation of spontaneous hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). METHODS: The thoracic aorta of the rats were sampled. The primary SHR VSMCs were isolated and cultured in vitro. The cells were divided into 4 groups: control group, TGF-β₁ group,18α-glycyrrhetinic acid(18α-GA) group and TGF-β₁+18α-GA group. The proliferation of SHR VSMCs was observed by the methods of MTT and flow cytometry. The protein expression and co-localization of connexin(Cx)43 and Cx40 in SHR VSMCs were detected by immunofluorescence staining. The protein levels of Cx43 and Cx40 in the cells were also measured by Western blotting. The method of molecular dye transfer (scrape dye transfer method) was applied to detect the function of gap junction in SHR VSMCs. RESULTS: The protein expression of Cx43 and Cx40 in SHR VSMCs was positive and co-localized in the cytoplasm. Compared with control group, the percentage of S-phase detected by cell cycle and A value detected by MTT in TGF-β₁ group were obviously increased (P<0.05), indicating that the proliferation of the cells was enhanced. However, the proliferation of the cells decreased in 18α-GA group (P<0.05). Compared with TGF-β₁ group, the percentage of S-phase and A value in TGF-β₁+18α-GA group were both significantly decreased (P<0.05), indicating that the proliferation of the cells decreased. Compared with control group, the protein expression of Cx43 in TGF-β₁ group was increased (P<0.05), whereas the protein expression of Cx40 was not changed (P>0.05), and the protein expression of Cx43 and Cx40 in 18α-GA group were decreased (P<0.05). Compared with TGF-β₁ group, the expression of Cx43 in TGF-β₁+18α-GA group was significantly decreased (P<0.05),but no difference of the Cx40 protein levels between the two groups was observed. Compared with control group, the function of gap junction detected by scrape dye transfer method in TGF-β₁ group was enhanced (P<0.05), and weakened in 18α-GA group (P<0.05). Compared with the TGF-β₁ group, the function of gap junction in TGF-β₁+18α-GA group was significantly attenuated (P<0.05). CONCLUSION: TGF-β₁ enhances the function of gap junction to stimulate the proliferation of SHR VSMCs through the expression of Cx43 protein. The expression of Cx40 protein may not play a major role in this process.