ATP2B1 gene Silencing Increases Insulin Sensitivity through Facilitating Akt Activation via the Ca2+/calmodulin Signaling Pathway and Ca2+-associated eNOS Activation in Endothelial Cells.

Endothelial cell insulin resistance may be partially responsible for the higher risk of atherosclerosis and cardiovascular disease in populations with insulin resistance and type 2 diabetes mellitus (T2DM). A genome-wide association study revealed a significant association between the ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) gene and T2DM in two community-based cohorts from the Korea Association Resource Project. However, little is known about the implication of the ATP2B1 gene on T2DM. In the present study, we investigated the role of the ATP2B1 gene in endothelial cell insulin sensitivity. ATP2B1 gene silencing resulted in enhanced intracellular calcium concentrations and increased insulin-induced Akt activation compared to that in the negative siRNA-transfected HUVECs (Human Umbilical Vein Endothelial Cells). The elevated insulin sensitivity mediated by ATP2B1 gene silencing was Ca2+/calmodulin-dependent, as verified by administration of the calcium chelator BAPTA-AM or the calmodulin-specific antagonist W7. Moreover, higher levels of phosphorylation of eNOS (Ser1177) were observed in ATP2B1-silenced HUVECs. In addition to BAPTA-AM and W7, L-NAME, an eNOS antagonist, abolished insulin-induced Akt phosphorylation at Ser473 in both si-Neg and si-ATP2B1-transfected endothelial cells. These results indicate that the enhanced insulin sensitivity in ATP2B1-silenced endothelial cells is alternatively dependent on an increase in intracellular Ca2+ and the subsequent activation of the Ca2+/calmodulin/eNOS/Akt signaling pathway. In summary, ATP2B1 gene silencing increased insulin sensitivity in endothelial cells by directly modulating the Ca2+/calmodulin signaling pathway and via the Ca2+/calmodulin/eNOS/Akt signaling pathway alternatively.

[Lentiviral vectors carrying siRNA inhibit S1PR3 gene expression in the corpus cavernosum smooth muscle cells of rats with spontaneous hypertension].

OBJECTIVE: To screen lentiviral vectors carrying siRNA which can specifically down-regulate the gene expression of the sphingosine-1-phosphate receptor 3 (S1PR3) in the corpus cavernosum smooth muscle (CCSM) cells of rats with spontaneous hypertension (SHT) and investigate the influence of the vectors on the signaling pathways of ROCK1, ROCK2 and eNOS in the CCSM cells of SHT rats. METHODS: Using the S1PR3 mRNA sequence of the rat as an interfering target, we designed and synthesized three pairs of siRNA sequences (siRNA1, 2 and 3) targeting S1PR3 and one pair of negative control, and then constructed and packaged them into lentiviral vectors. We cultured the CCSM cells of SHT and Wistar-Kyoto (WKY) rats in vitro and randomly divided them into groups A (SHT untransfected control), B (SHT transfected and carrying negative control virus), C (SHT transfected and carrying siRNA1 targeting S1PR3), D (SHT transfected and carrying siRNA2 targeting S1PR3), E (SHT transfected and carrying siRNA3 targeting S1PR3), and F (WKY untransfected control). With the multiplicity of infection (MOI) = 60, we transfected the CCSM cells of the SHT rats with the lentiviral vector and then determined the expression of the green fluorescent protein (GFP) as well as the mRNA and protein expressions of S1PR3, ROCK1, ROCK2 and eNOS in the CCSM cells of the SHT and WKY rats by RT-PCR and Western blot. RESULTS: Gene sequencing proved the successful construction of the lentiviral vector. The transfection efficiency of the CCSM cells of the rats was >80% in groups B, C, D and E. Compared with group A, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 exhibited no significant difference in group B but were remarkably decreased in groups C, D, E and F (P< 0.05), most significantly in group E, with the inhibition rates of the mRNA and protein expressions of S1PR3 of (34.2±2.9) and (77.7±4.7)%, those of ROCK1 of (33.3±1.4) and (51.1±7.3)%, and those of ROCK2 of (30.8±3.6) and (58.32±5.5)%, respectively. The mRNA and protein expressions of eNOS in group A showed no significant difference from those in groups B, C, D and E (P>0.05) but remarkably lower than those in group F (P< 0.05). Compared with group F, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 were not significantly different from those in group E (P>0.05) but markedly increased in groups A, B, C and D (P< 0.05), while those of eNOS remarkably decreased in groups A, B, C, D and E (P< 0.05). CONCLUSIONS: The three constructed lentiviral vectors carrying siRNA targeting different loci of the S1PR3 gene could significantly inhibit the expression of S1P3 as well as RhoA/Rho kinase signaling pathways in the CCSM cells of SHT rats, and the vector carrying siRNA3 exhibited the highest inhibitory effect.

[Molecular mechanisms of androgens regulating the eNOS expression in rat corpus cavernosum].

OBJECTIVE: To investigate whether androgens can regulate the expression of eNOS in rat corpus cavernosum through AKT3, PIK3CA, CALM, and CAV1 and influence erectile function. METHODS: Thirty-six 8-week-old male SD rats were randomly divided into groups A (4-week control), B (6-week control), C (4-week castration), D (6-week castration), E (4-week castration + testosterone replacement), and F (6-week castration + testosterone replacement). Both the testis and epididymis were removed from the rats in groups C, D, E and F, and on the second day after surgery, the animals of groups E and F were subcutaneously injected with testosterone propionate at 3 mg per kg of the body weight qd alt while all the others with isodose oil instead. At 4 weeks (for groups A, C and E) and 6 weeks (for groups B, D and F) after treatment, we detected the maximum intracavernous pressure (ICPmax), the mean carotid arterial pressure (MAP) and their ratio (ICPmax/MAP), measured the level of serum testosterone (T), and determined the expressions of eNOS, P-eNOS, AKT3, PIK3CA, CALM and CAV1 in the corpus cavernosum by Western blot and immunohistochemistry. RESULTS: No statistically significant differences were observed in the body weight and MAP among different groups. The serum T level and ICPmax/MAP were remarkably lower in groups C and D than in the other four groups (P<0.01) as well as in groups E and F than in A and B (P<0.05) but exhibited no significant differences either between E and F or between A and B. Immunohistochemistry showed that eNOS and P-eNOS were mainly expressed in the vascular endothelial cell membrane and cavernous vascular lumen, while AKT3, PIK3CA, CALM and CAV1 chiefly in the vascular endothelial cell cytoplasm and membrane, with a few in the smooth muscle cells. Western blot analysis manifested that the expressions of eNOS, P-eNOS, AKT3, PIK3CA, CALM and CAV1 were markedly lower in groups C and D than in A, B, E and F (P<0.01) as well as in D than in C (P<0.05) but those in groups E and F did not showed any significant difference from those in A and B, nor E from F or A from B. CONCLUSIONS: Androgens can improve erectile function by upregulating the expressions of AKT3, PIK3CA, CALM and CAV1 protein molecules and activating eNOS after its phosphorylation, though the exact molecular mechanisms are yet to be further studied.

[Expressions of S1P1-3 in the corpus cavernosum of castrated male rats].

OBJECTIVE: To investigate the expressions of sphingosine-1-phosphate receptors 1-3 (S1P1- 3) in the corpus cavernosum of castrated male rats and its relationship with the NOS/NO/cGMP and RhoA/Rho kinase signaling pathways. METHODS: We equally randomized 18 eight-week-old healthy male SD rats into a sham-operation control, a castration, and a testosterone replacement (TR) group and harvested the bilateral testes and epididymides from the rats in the latter two groups, followed by 4 weeks of subcutaneous injection of testosterone propionate at 3 mg per kilogram of the body weight per day for those in the TR group and that of plant oil for those in the control and castration groups. At the age of 12 weeks, we measured the serum testosterone (T) level and maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP) of the animals and determined the expressions of SlP1-3, eNOS, P-eNOS, ROCK1, and ROCK2 in the corpus cavernosum by Western blot and immunohistochemistry. RESULTS: The serum T level was significantly decreased in the rats of the castration group as compared with those of the control and TR groups ([0.41 ± 0.04] vs [16.01 ± 1.02] and [15.84 ± 1.32] nmol/L, P < 0.01), with no statistically significant difference between the latter two groups. The ICPmax/MAP at 0 V, 3 V, and 5 V electric stimulation was remarkably lower in the rats of the castration group (0.088 ± 0.014, 0.323 ± 0.014, and 0.432 ± 0.012) than in those of the control group (0.155 ± 0.011, 0.711 ± 0. 010, and 0.819 ± 0.024) and TR group (0.153 ± 0.012, 0.696 ± 0.017, and 0.763 ± 0.027) (P < 0.01), with no significant difference between the latter two groups. With GAPDH as internal control, the animals of the castration group showed markedly reduced expressions of S1P1 ([49.99 ± 3.39]%), eNOS ([46.82 ± 3.81]%) , and P-eNOS ([45.42 ± 4.35]%) in comparison with those in the control group ([72.57 ± 3.06], [89.76 ± 3.98], and [82.53 ± 8.92] and TR group ([71.77 ± 4.43], [87.19 ± 4.23], and [79.82 ± 7.38]%) (P < 0.01) , while the expressions of S1P2, S1P3, ROCK1, and ROCK2 were significantly upregulated in the castration group ([82.35 ± 4.13], [61.03 ± 5.14], [74.50 ± 4.02], and [69.83 ± 5.75]%) as compared with those in the control group ([41.67 ± 1.68], [31.66 ± 2.67], [35.69 ± 5.56], and [39.85 ± 7.17]%) and TR group ([42.80 ± 3.87], [32.25 ± 4.22], 38.06 ± 5.21], and [42.36 ± 4.44]%) (P < 0.01). CONCLUSION: Androgen deficiency induces significant reduction of ICPmax/ MAP in male rats, which is possibly associated with the decline of S1P1 in the corpus cavernosum, inhibition of the eNOS/NO/cGMP signaling pathway, increased expressions of S1P2 and S1P3, and activation of the RhoA/Rho kinase signaling pathway.

Retinoic acid aliphatic amide inhibits the AMPK-HIF-1α pathway in human ovarian cancer.

Ovarian carcinoma the commonly observed gynecological cancers has a high mortality rate. In the present study effect of retinoic acid aliphatic amide (RACA) in ovarian cancer cells was investigated using proliferation, migration and invasion assays. Western blot was used to examine the Bcl-2, cleaved caspase 3, p-ERK, MMP-2, p-FAK, P-P38, p-AMPKα and HIF-1α protein expression. CoCl2 was used to induce HIF-1α expression in SKOV3ip. 1 and HEY-A8 cells. The results revealed that RACA treatment prompted cell proliferation, invasion and migration but inhibited apoptosis of SKOV3ip. 1 and HEY-A8 cells. RACA treatment also induced upregulation of Bcl-2 and MMP-2, activation of p-P38, p-ERK and p-FAK, inhibition of cleaved caspase 3. RACA treatment also caused upregulatation of HIF-1α in ovarian cells with the activation of p-AMPKα. Upregulation of HIF-1α expression in CoCl2-treated cancer cells resulted in decrease in SDHB. Thus RACA plays a key role in cell proliferation, invasion, migration and apoptosis of human ovarian carcinoma through AMPK-HIF-1α pathway.

[Expressions of aquaporins decrease in the prostate and seminal vesicles of castrated rats].

OBJECTIVE: To investigate the levels of secretions from the prostate and seminal vesicles and their association with the expressions of aquaporins (AQP) in the prostatic tissue and seminal vesicles of castrated rats. METHODS: We randomly divided 18 eight-week-old male SD rats into a control, a castration, and a testosterone (T) replacement group. Four weeks after surgical castration, we detected the plasma T level and measured the volumes of the secretions and the expressions of AQPs 3, 7, and 10 - 12 in the prostate and seminal vesicles of the rats. RESULTS: The plasma T level was significantly lower in the castrated models ([30. 98 ± 28. 84] ng/dl) than in the rats of the control ([700.78 ± 123.8] ng/dl) and T replacement groups ([688.08 ± 132. 47] ng/dl) (P <0. 05). The castration group, in comparison with the control and T replacement groups, showed remarkably reduced ratios of prostatic secretion volume / prostate weight ([11.1 ± 0.30] vs [2.32 ± 0.61] and [2.13 ± 0.56] %, P <0. 05) and seminal vesicle secretion volume / seminal vesicle weight ( [4. 78 ± 1. 97 ] vs [57. 36 ± 11. 86] and [55. 74 ± 7. 21] %, P < 0. 05). Immunohistochemistry revealed the expressions of AQPs 3 and 7 in the epithelial envelop and cytoplasm and that of AQP 11 the in endothelial envelop and cytoplasm of the prostate and seminal vesicles. Western blot exhibited significantly lower expressions of AQPs 3, 7, and 10 - 12 in the prostate and seminal vesicles of the castrated rats than in the animals of the control and T replacement groups (P <0. 05). CONCLUSION: Significant decreases of the secretions from the prostate and seminal vesicles may be related to the reduced expressions of AQPs 3, 7, and 10 - 12 in the prostatic tissue and seminal vesicles in castrated rats.

[Expressions of CSE and CBS in the penile corpus cavernosum of hyperglycemia rats and their implications].

OBJECTIVE: To investigate the impact of hyperglycemia on the hydrogen sulfide (H2S) signaling pathway in rat penile tissue and its relationship with erectile function. METHODS: Twenty healthy male Sprague Dawley (SD) rats aged 8 weeks were randomly divided into groups A (4-week healthy control), B (4-week diabetes mellitus model), C (6-week healthy control) and D (6-week diabetes mellitus model). The rats in groups B and D were injected intraperitoneally with streptozotocin at 50 mg/kg to induce diabetes mellitus, while those in groups A and C with the same volume of normal saline. The animals were killed at 4 (groups A and B) and 6 weeks (groups C and D) after treatment for measurement of the maximal intracavernous pressure/mean arterial blood pressure (ICP(max)/MAP) by electrostimulation, determination of the H2S concentration in the plasma and penile tissue, and detection of the expressions of cystathionine-beta-synthetase (CBS) and cystathionine-gamma-lyase (CSE) in the penile corpus cavernosum by immunohisto- chemistry and Western blot. RESULTS: With electrostimulation of the pelvic ganglia at 5V and 7 V, ICP(max)/MAP was significantly reduced in groups B (0.19 +/- 0.03 and 0.29 +/- 0.04) and D (0.14 +/- 0.04 and 0.25 +/- 0.04) as compared with A (0.46 +/- 0.07 and 0.68 +/- 0.09) and C (0.43 +/- 0.07 and 0.65 +/- 0.16) (P < 0.05). No statistically significant differences were found in the level of serum testosterone either between groups A and B ([469.19 +/- 126.46] ng/dl vs [359.08 +/- 60.06] ng/dl, P > 0.05) or between C and D ([470.44 +/- 209.28] ng/dl vs [297.01 +/- 96.58] ng/dl, P > 0.05). Groups B and D showed remarkable reduction in the H2S concentration (P < 0.05) and the expressions of CBS and CSE (P < 0.05) in comparison with A and C, and the CBS and CSE expressions were even more significantly decreased in D than in B (P < 0.05). CONCLUSION: The reduced concentration of H2S and decreased expressions of CBS and CSE in the penile corpus cavernosum of the diabetic rats suggested that the H2S signaling pathway might be involved in hyperglycemia-induced erectile dysfunction.

Left ventricular strain examination of different aged adults with 3D speckle tracking echocardiography.

The purpose of this study was to identify traits of the left ventricular (LV) global longitudinal strain (GLS), global radial strain (GRS), global circular strain (GCS), and global area tracking (GAT) with three-dimensional speckle tracking echocardiography (3DSTE), and to determine the relationship between strain and age in healthy adults of different ages. A total of 153 volunteers were divided into young adult, middle-aged, and elderly groups, and examined with echocardiography to obtain general data and live two-dimensional (2D) images of the apical four-chamber view, which were assembled to obtain the full volume view of the LV. The images were then analyzed with 3DSTE software. Compared with the young adult and middle-aged groups, elderly adults demonstrated lower GLS, GRS, GCS, and GAT. Significant differences were not noted in GLS, GRS, and GCS between the young adult and middle-aged groups; however, the GAT of the middle-aged group was lower than that of the young adult group. The longitudinal strain (LS), radial strain (RS), and area tracking (AT) of 16 LV segments of the young adult group decreased gradually in level from the mitral valve to the apex, and increased in circular strain (CS). The LS, RS, CS, and AT of the middle-aged group also decreased gradually. The LS, RS, CS, and AT of the elderly people were highest from the mitral valve to the apex level and lowest at the papillary muscle. The results of this study demonstrated that LV GLS, GRS, GCS, and GAT decrease with age.

[Effects of Ling Qi Juan Gan capsule drug-containing serum on PDGF-induced proliferation and JAK/STAT signaling of HSC-T6 cells].

OBJECTIVE: To analyze the effects of Ling Qi Juan Gan capsule drug-containing serum at different concentrations on the platelet-derived growth factor (PDGF)-induced proliferative capabilities of and JAK2 and p-STAT3 protein expression in hepatic stellate cells (HSC) using an in vitro system. METHODS: Twenty-five Sprague-Dawley rats were randomly divided into five equal groups for intragastric administration of physiological saline (10 ml/kg; group A), Fufang Biejia Ruangan tablet solution (1.5 g/kg; group B), or Ling Qi Juan Gan capsule solution at low dose (2.125 g/kg; group C1), mid dose (4.25 g/kg; group C2), or high dose (8.5 g/kg; group C3). The post-administration serum isolated from each rat (200 ml/L) was used to treat the HSC-T6 cell line following induction by PDGF (10 ng/ml). At 24, 48 and 72 h post-exposure, the cells' proliferation was measured using the Cell Counting Kit-8 (CCK-8) colorimetric assay. In addition, at 24 h post-exposure the expression of JAK2 and p-STAT3 was measured by western blotting (expressed as grey scale intensity). Multiple group comparison of repeated measures data was made by one-way ANOVA with Student-Newman-Keuls post hoc test. RESULTS: Compared to group A, groups C2 and C3 had significantly higher inhibited proliferation at all post-exposure time points examined (24 h: A = 1.550 +/- 0.065, C2 = 1.335 +/- 0.106, C3 = 1.241 +/- 0.205; 48 h: A = 1.311 +/- 0.650, C2 = 1.090 +/- 0.106, C3 = 0.909 +/- 0.191; 72 h: A = 1.039 +/- 0.103, C2 = 0.719 +/- 0.116, C3 = 0.641 +/- 0.110, F = 36.292, all P less than 0.05); in contrast, compared to group A, group C1 showed no inhibition of proliferation at 24 h (1.522 +/- 0.128, P = 0.717) but showed significantly higher inhibition of proliferation at 48 h and 72 h (1.153 +/- 0.183 and 0.753 +/- 0.210, respectively, F = 36.292, P less than 0.05). Compared with group A, all Ling Qi Juan Gan capsule-containing serum-treated groups showed significantly lower expression of both JAK2 (A = 1.605 +/- 0.024 vs. C1 = 1.170 +/- 0.042, C2 = 0.842 +/- 0.036, C3 = 0.555 +/- 0.036, F = 43.091) and p-STAT3 (A = 1.401 +/- 0.030 vs. C1 = 1.229 +/- 0.025, C2 = 0.668 +/- 0.034, C3 = 0.630 +/- 0.026, F = 78.426, all P less than 0.01). CONCLUSION: Ling Qi Juan Gan capsule drug-containing serum can inhibit the proliferation of HSC-T6 cells in a dose-dependent manner and cause an overall decrease in the expression of JAK2 and p-STAT3 in activated HSC, thereby leading to a suppression of the JAK/STAT signal transduction pathway.

[Influence of angiotensin-(1-7) on cell activation in rat renal interstitial fibroblasts induced by aldosterone].

AIM: To explore the influence of angiotensin-(1-7) [Ang-(1-7)] on cell activation and extracellular matrix secretion in rat renal interstitial fibroblasts (NRK-49F) induced by aldosterone (ALD). METHODS: The NRK-49F cells were cultured in vitro, and then were divided into control group, ALD group, Ang-(1-7) group, and ALD+Ang-(1-7) group. When the cells were cultured for 48 h, the expression of α-smooth muscle actin (α-SMA) (a sign of cell activation) was detected by immunocytochemistry; the level of collagen type I (Col I ) in the cultured supernatant was measured by enzyme-linked immunosorbent assay (ELISA). When the cells were cultured for 30 min, the expressions of phosphorylated and total ERK1/2 (pERK1/2, tERK1/2) in the cell lysate were detected by Western blotting. RESULTS: Compared with control group, the expressions of α-SMA, Col I and the Phos/Total ERK1/2 ratio in ALD group and ALD+Ang-(1-7) group increased significantly (P<0.05). Compared with ALD group, the expressions of α-SMA, Col I and the Phos/Total ERK1/2 ratio in ALD+Ang-(1-7) group decreased significantly (P<0.05). CONCLUSION: Ang-(1-7) can inhibit ALD-induced cell activation and decrease the secretion of Col I in rat renal interstitial fibroblasts. Inhibition of ERK1/2 pathway may play an important role in this process.