[Antagonistic effect of modified Dahuang Zhechong Granule against the apoptosis of epididymal spermatogenic cells in varicocele rats based on Nrf2/HO-1 pathways].

OBJECTIVE: To investigate the effects of modified Dahuang Zhechong Granule (DZG) on the epididymal tissue of varicocele (VC) rats and the expressions of the nuclear factor erythroid 2 (NF-E2)-related factor (Nrf2) and heme oxygenase-1 (HO-1) protein. METHODS: Sixty SD rats were randomly divided into six groups of an equal number: sham operation, VC model control, aescuven forte (AF) and low-, medium- and high-dose DZG. The VC model was established by ligation of the left renal vein with the Turner's method, followed by intragastrical administration of normal saline to the rats in the sham operation and VC model control groups, AF Tablets at 54 mg/kg to those in the AF group, and modified DZG at 0.6, 1.2 and 2.4 g/ml to those in the low-, medium- and high-dose DZG groups respectively, all once daily for 8 weeks. Then, all the animals were sacrificed and their left epididymides harvested for examination of semen quality, observation of local ultrastructural changes, measurement of the apoptosis of spermatogenic cells by Annexin V-FITC, and determination of the expressions of Nrf2 and HO-1 in the epididymal tissue by immunohistochemistry. RESULTS: Evident pathological damage was observed in the left epididymal tissue of the VC model controls, with significantly reduced numbers of spermatogenic cells and sperm at all levels, partially destroyed cellular structure, and disappearance of some subcellular structures such as the lysosome, mitochondrion, endoplasmic reticulum, nucleus and cell membrane, which were all improved to some extent in the DZG and AF group. Sperm concentration and motility in the left epididymis were significantly higher in the medium- and high-dose DZG and AF groups than in the VC model controls (P < 0.05), even more significantly in the high-dose DZG than in the AF group (P < 0.05). The apoptosis rate of spermatogenic cells was markedly higher in the VC model control than in the sham operation group (P < 0.05), but lower in the medium- and high-dose DZG and AF groups than in the VC model controls (P < 0.05). Immunohistochemistry showed positive expressions of Nrf2 and HO-1 proteins, brown, scattered and with a low luminance of the cells, in the left epididymis tissue of the VC model control rats, but with a significantly higher cell luminance in the high-dose DZG and AF groups. CONCLUSIONS: Modified Dahuang Zhechong Granule can effectively repair pathological damage to the epididymis of varicocele rats, increase the expressions of Nrf2 and HO-1 proteins, antagonize the apoptosis of spermatogenic cells and provide a favorable condition for sperm maturation.

Simultaneous determination of 25 pesticides in Zizania latifolia by dispersive solid-phase extraction and liquid chromatography-tandem mass spectrometry.

An improved quick, easy, cheap, effective, rugged, and safe (QuEChERS) method combined with ultrapressure liquid chromatography tandem mass spectrometric method (UPLC-MS/MS) was developed to simultaneously determine 25 pesticides in Zizania latifolia. The samples were extracted with methanol(MeOH) and 0.1% formic acid (80:20, v/v) and cleaned with C18 absorbent and primary-secondary amine (PSA). LC separation was performed on a BEH C18 UPLC column under the condition of gradient elution with the mobile phase consisted of 0.5% formic acid (10 mM ammonium acetate)/MeOH. External standard calibration method with matrix-matched was used for quantification, and good linearity was obtained over a concentration range of 0.5-100 µg/l, with correlation coefficients greater than 0.9901. The limit of detection (LOD) and the limit of quantitation (LOQ) of the 25 pesticides were in the range of 0.2-1.0 µg/kg and 0.5-3.3 µg/kg, respectively. The recoveries ranged from 72% to 118%, and the relative standard deviations (RSDs) were less than 20%. Thus, the proposed method is suitable for the simultaneous determination of 25 pesticides in Z. latifolia.

[Intervention effect of Modified Dahuang Zhechong Granule on epididymal morphological changes in experimental varicocele rats].

OBJECTIVE: To explore the effect of Modified Dahuang Zhechong Granule (MDZG) on the development and maturation of epididymal sperm in experimental varicocele (VC) rats. METHODS: Sixty SD male rats were randomly divided into six groups of equal number, sham operation, VC model, Aescuven forte, and low-, medium- and high-dose MDZG. The model of left VC was made by the Turner method in all the rats except those of the sham operation group, followed by treatment with 0.9% normal saline for the animals in the sham operation and VC model groups, Aescuven forte tablets at 54 mg per kg of the body weight for those in the Aescuven forte group, and MDZG at 0.6, 1.2 and 2.4 g/ml for those in the low-, medium- and high-dose MDZG groups, all administered intragastrically qd for 8 successive weeks. Then, all the rats were sacrificed and their left epididymides harvested for examination of the quality of the epididymal sperm and the local microscopic and ultrastructural changes of the epididymal tissue. RESULTS: The VC model rats showed significant apoptosis of the epididymal sperm cells, interstitial edema, microvascular dilatation, degeneration and degeneration of the epithelial cells, degeneration of some principal cells and basal cell vacuoles, and immature spermatids in the lumen. Sperm motility was significantly increased in the Aescuven forte and low-, medium- and high-dose MDZG groups as compared with the VC models (P <0.01). Both sperm concentration and motility were markedly higher in the high-dose MDZG than in the Aescuven forte group (P <0.05). Remarkable apoptosis of epididymal sperm cells was observed in the microenvironment of sperm development in the VC models, which exhibited no statistically significant difference from that in the rats of the medium- and high-dose MDZG groups. CONCLUSIONS: Experimental varicocele induced local apoptosis of epididymal sperm cells, interstitial edema and microvascular dilatation in the rat epididymis, while Modified Dahuang Zhechong Granule could improve the stability of epididymal sperm maturation and contribute to their development.

[Antioxidating and energy metabolism improving effects of Qiangjing Decoction on oligospermia and asthenospermia: An experimental study].

OBJECTIVE: To explore the mechanisms of Qianjing Decoction in the treatment of oligoasthenospermia (OAS). METHODS: We randomly divided 100 SPF male rats into five groups of equal number: normal, model, Huangjingzanyu, levocarnitine, and Qiangjing. OAS models were established in the animals followed by intragastrical administration of normal saline, ornidazole, Huangjingzanyu Capsules (200 mg per kg body weight per day), levocarnitine (100 mg per kg body weight per day), and Qianjing Decoction (10 g per kg body weight per day), respectively, qd, for 4 successive weeks. Then, we detected the concentration and motility of the epididymal sperm, obtained the contents of superoxide dismutase (SOD), malonaldehyde (MDA), glutathione peroxidase (GSH-Px), lactate dehydrogenase (LDH), α-glucosidase, and fructose in the epididymis, and determined the mRNA expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and succinate dehydrogenase (SDH) in the epididymal tissue of the rats by real-time PCR. RESULTS: The concentration and motility of the epididymal sperm in the model, Huangjingzanyu, levocarnitine, and Qianging groups were (35.34 ± 4.22) x 10(6)/ml and (40.04 ± 7.05)%, (48.12 ± 5.56) x 10(6)/ml and (62.46 ± 7.12)%, (47.14 ± 4.87) x 10(6)/ml and (63.23 ± 6.34)%, and (50.25 ± 5.08) x 10(6)/ml and (66.34 ± 7.58)%, respectively, all significantly lower than in the normal group ([53.05 ± 4.55] x 10(6)/ml and [70.20 ± 8.54]%) (P < 0.05), but remarkably higher in the Huangjingzanyu, levocarnitine, and Qiangjing groups than in the model rats (P < 0.05). Compared with the thinned epididymal lumen walls, decreased sperm count, and disorderly and loose arrangement of the lumens in the OAS models, the rats in the Huangjingzanyu, levocarnitine, and Qiangjing groups showed evidently thicker epididymal lumen walls, with the lumens full of sperm cells and arranged regularly and compactly, similar to those of the normal rats. The levels of SOD and GSH-Px were significantly lower but that of MDA markedly higher in the model rats ([84.12 ± 23.25], [10.56 ± 3.02], and [14.04 ± 2.06] nmol/mg) than in the normal group ([110.04 ± 19.56], [17.25 ± 3.56], and [8.87 ± 1.35] nmol/mg) (P < 0.05), while the former two indexes remarkably higher and the latter one significantly lower in the animals treated with Qiangjing Decoction ([120.56 ± 23.68], [16.34 ± 3.12], and [8.45 ± 1.56] nmol/mg), Huangjingzanyu Capsules ([115.34 ± 21.35], [15.23 ± 3.67], and [8.33 ± 1.54] nmol/mg), and levocarnitine ([116.67 ± 22.67], [15.35 ± 3.45], and [8.05 ± 1.78] nmol/mg) than in the models (P < 0.05). The levels of fructose, LDH and α-glucosidase were decreased markedly in the OAS models ([100.22 ± 12.12] mg/[ ml x g], [322 ± 46.13] U/[ ml x g], and [10.48 ± 2.33] U/[ml x g]) as compared with the normal rats ([128.12 ± 13.45] mg/[ml x g], [428 ± 35.12] U/[ml x g], and [15.34 ± 3.12] U/[ ml x g]) (P < 0.05), remarkably higher in the rats treated with Qiangjing ([130.23 ± 13.67] mg/[ml x g] [455 ± 51.50] U/[ml x g], and [18.56 ± 4.67] U/[ml x g]), Huangjingzanyu ([124.16 ± 14.02] mg/[ml x g], [ 419 ± 43.14] U/[ml x g], and [17.64 ± 4.08] U/[ml x g]), and levocarnitine ([123.34 ± 14.02] mg/[ml x g], [430 ± 31.80] U/ [ml x g], and [16.85 ± 5.55] U/[ml x g]) than in the models (P < 0.05). The Nrf2 mRNA expression was significantly reduced in the models as compared with the normal rats (P < 0.05) but remarkably increased in the Huangingzanyu, Qiangjing and levocarnitine groups as compared with the model and normal animals (P < 0.05). The SDH mRNA expression was significantly lower in the model than in the normal rats (P < 0.05) but markedly elevated in the Huangjingzanyu, Qiangjing and levocarnitine groups as compared with the model and normal animals (P < 0.05), remarkably higher in the Qiangjing than in the Huangjingzanyu group (P < 0.05). CONCLUSION: Ornidazole induces OAS in rats, which is closely associated with excessive oxidation and energy metabolism dysfunction. Qiangjing Decoction can improve and even reverse ornidazole-induced OAS in rats as well as improve the ultrastructure of their testicular and epididymal tissues. Antioxidation and improvement of energy metabolism are probably the action mechanisms of Qiangjing Decoction in the treatment of OAS.

[Effects of mammalian-target-of-rapamycin pathway on lapatinib resistance in breast cancer MDA-MB-231 cells].

OBJECTIVE: To establish a lapatinib resistance cell line for elucidating the mechanisms of drug resistance of lapatinib in human breast cancer cells. METHODS: The human breast cancer MDA-MB-231 cells were exposed in an incremental dose of lapatinib to establish a lapatinib resistance rMDA-MB-231 cell line. The assay of methyl thiazolyl tetrazolium (MTT) was used to detect the cytotoxic activity of lapatinib against MDA-MB-231 and rMDA-MB-231 cells. The protein expression was detected by Western blot. Small interfering RNA was used to specifically knock down mammalian-target-of-rapamycin (mTOR) in rMDA-MB-231 cells. Apoptosis was determined by fluorescein isothiocyanate (FITC)-annexin V/PI staining and flow cytometry. RESULTS: The human breast cancer lapatinib resistance cell line rMDA-MB-231 was induced by lapatinib. The half maximal inhibitory concentration (IC50) values of lapatinib against MDA-MB-231 and rMDA-MB-231 cells were (6.1 ± 0.6) and (34.9 ± 2.7) µmol/L respectively (P < 0.01). Compared with MDA-MB-231 cells, the protein expression of mTOR in rMDA-MB-231 cells was significantly up-regulated. The protein expression of mTOR was significantly down-regulated by specific siRNA duplexes in rMDA-MB-231 cells. After siRNA interference, 20 µmol/L lapatinib was added into control, negative siRNA control and mTOR-targeted siRNA groups respectively. The percents of cell apoptosis in control, negative control and targeted siRNA groups were 13.4% ± 2.5%, 14.2% ± 2.8% and 34.6% ± 5.8% respectively, there was no significance between the first two groups (P > 0.05) , and there was significant difference between the control and targeted siRNA group (P < 0.01) . CONCLUSIONS: The up-regulation of mTOR plays an important role in the lapatinib-resistant phenotype of human breast cancer rMDA-MB-231 cells. And the down-regulation of mTOR increases the apoptotic death of lapatinib against rMDA-MB-231 cells.

[TRUS-guided transperineal biopsy with 9 + X method for the diagnosis of prostate carcinoma: retrospective analysis of 420 cases].

OBJECTIVE: To explore the clinical value and safety of TRUS-guided transperineal biopsy with the 9 + X method in the diagnosis of prostate carcinoma. METHODS: A total of 420 men underwent TRUS-guided transperineal biopsy with the 9 + X method for suspected prostate carcinoma. Their clinical data were retrospectively analyzed. RESULTS: Prostate carcinoma was detected in 160 (38.1%) of the 420 cases, accounting for 7.4%, 17.8% and 65.4% in those with PSA < 4.0 microg/L, 4 -10 microg/L and > 10 microg/L respectively, 25.0% in those with abnormal findings on digital rectal examination (DRE), and 22.2% in those with abnormal echoes on TRUS or abdominal ultrasound examination. Complications after prostatic biopsy included gross hematuria in 79 cases (18.8%), acute urinary retention in 13 (3.1%) and fever in 9 (2.1%), but no other serious complications were observed. CONCLUSION: TRUS-guided transperineal biopsy with the 9 + X method, with high accuracy and fewer complications, is an ideal approach to the diagnosis of prostate carcinoma.

[Diagnosis and treatment of primary epididymal tumor: a report of 35 cases].

OBJECTIVE: To explore the diagnosis and treatment of primary epididymal tumor. METHODS: We retrospectively analyzed the clinical data of 35 cases of pathologically confirmed primary epididymal tumor. Of the total number of patients, 10 underwent tumor excision, 23 received epididymectomy, 1 was treated by simple orchidoepididymectomy, and by radical orchidoepididymectomy with second-stage retroperitoneal lymph node dissection. RESULTS: Postoperative pathology confirmed 33 cases of benign tumor (including 21 adenomatoid tumor, 7 leiomyoma, 4 fibroma, and 1 papillary cystadenoma), and 2 cases of malignancy (1 malignant fibrous histiocytoma and 1 adenocarcinoma). The follow-up lasted 10 months to 6 years, which revealed no recurrence, metastasis and death. CONCLUSION: Primary epididymal tumor is difficult to be definitely diagnosed preoperatively. Surgical exploration is the first choice for those highly suspected of the disease. Tumor excision or epididymectomy can be considered for benign cases, while radical orchidoepididymectomy with retroperitoneal lymph node dissection is recommended in case of malignancy.

[The advantages for Snail expression to promote cell migration and induce actin reorganization and to protect against the serum-deprivation-triggered apoptosis of bone marrow stem cells].

The Snail transcription factor has been described as a strong repressor of E-cadherin and its stable expression induces epithelial-mesenchymal transitions responsible for the acquisition of motile and invasive properties during tumor progression. A fascinating analogy that has been raised is the seemingly similar and shared characteristics of stem cells and tumorigenic cells, which prompted us to investigate whether the mechanisms of the acquisition of invasiveness during tumor progression are also involved in bone marrow stem cells (MSCs). In this study, we examined whether Snail gene expression acts in the mobility, cytoskeleton and anti-apoptosis of MSCs. Cell Transmigration Assay and Western Blotting were performed to evaluate the cell migratory capability and the related Signaling pathways in MSCs transfected with the Snail expression vector of pCAGGSneo-SnailHA (MSCs-Sna), compared with MSCs(MSCs-neo) transducted with the control vector(pCAGGSneo). Actin cytoskeleton by Immunofluorescence and Sub-G1 detection by a FACScan flow cytometer were performed to analyze the cytoskeleton and antiapoptotic capability of MSCs-Sna. Compared with MSCs-neo, MSCs-Sna show significantly more migration in the transwell migration system (P < 0.05). And suppression of PI-3K activation by the specific PI-3K inhibitor, Wortmannin, brought on a reduction in Snail-mediated MSCs migration. In addition, we provide evidences that high expression of Snail inhibited the serum-deprivation triggered apoptosis and cytoskeleton changement of MSCs. These data suggest the possibility of facilitating MSCs migration to injured tissue and subsequent survival and maintenance in the local microenvironment after their transplantation, by investigating and increasing the advantage factors such as Snail high expression in MSCs.

[Inverse correlation between Snail and E-cadherin expression in carcinoma cell lines and invasive ability in vitro].

OBJECTIVE: Malignant transformation of epithelial cell frequently coincides with loss of E-cadherin. Here we study the expression of Snail and E-cadherin and correlate their expression with cell differentiation and in vitro invasion. METHODS: The expression and localization of Snail and E-cadherin were studied by Northern blot and laser confocal microscopy in two normal cell lines (MDCK, NIH 3T3) and six carcinoma cell lines (A431, MCF-7, MDA-MB-453, HepG2, MDA-MB-435s, MDA-MB-231). Boyden chamber assay was done to detect the invasive ability of cells in vitro. RESULTS: Snail mRNA and protein were detected in fibroblasts NIH 3T3 and poorly differentiated carcinoma cell lines HepG2, MDA-MB-435s and MDA-MB-231. On the contrary, E-cadherin mRNA and protein were detected in normal epithelial cell line MDCK and well differentiated carcinoma cell lines A431 and MDA-MB-453. In MCF-7 cells, Snail and E-cadherin expressions were revealed both at mRNA and protein levels. The cells with higher expression of Snail had stronger ability of invasion than those with lower expression of Snail. CONCLUSION: There is an inverse correlation between Snail and E-cadherin expressions and their expressions are correlated with cell differentiation and tumor invasiveness.

[High glucose stimulates synthesis of fibronectin in human mesangial cells via serum and glucocorticoid induced protein kinase-1 signaling pathway].

OBJECTIVE: To investigate the role of serum and glucocorticoid induced kinase-1 (SGK(1)) pathways in fibronectin (FN) synthesis in human mesangial cell (HMC) under high glucose condition and the mechanism by which SGK(1) contributes to glomerulosclerosis in diabetic nephropathy (DN). METHODS: HMCs were cultured and transfected with (P)IRES2-EGFP-(S422D) SGK(1) mutant (SD), plasmid containing SGK(1) dominant activation mutant, or blank plasmid. Non-transfected HMCs were used as control group. Then the HMCs were divided into 6 groups: transfected with SD + high glucose (SD-HG, 25 mmol/L D-glucose), transfected with FP + high glues (FP + HG), non-transfected + high glucose (NT-HG), transfeted with SD + normal glucose (SD-NG, 5.5 mmol/L D-glucose), transfected with FP + normal glues (FP + NG), and non-transfected + normal glucose (NT-NG). Eight hours after the glucose stimulation, RT-PCR was used to examine the SGK(1) mRNA expression and fibronectin (FN). Western blotting was used to detect the fibronectin (FN) protein expression. RESULTS: The SGK(1) mRNA expression of the SD + HG group was 0.709, significantly higher than those of the FP + HG and NT + HP groups (0.497 and 0.491, both P < 0.01). The SGK(1) protein expression of the SD + HG group was 1,178,497, significantly higher than those of the FP + HG and NT + HP groups (193,875 and 195,597 respectively, both P < 0.01). The FN mRNA expression of the SD + HG group was 0.749, significantly higher than those of the FP + HG and NT + HP groups (0.463 and 0.475 respectively, both P < 0.01). The FN protein expression of the SD + HG group was 659,550, significantly higher than those of the FP + HG and NT + HG groups (342,354 and 340,428 respectively, both P < 0.01). There were not significant differences in the expressions of FN mRNA and protein among different NG groups. CONCLUSION: SGK(1) may be involved in the signal transduction leading to the increase of fibronectin production in DN and therefore may play an active part in glomerulosclerosis in DN.

[Effect of transcriptional factor snail on epithelial-mesenchymal transition and tumor metastasis].

BACKGROUND & OBJECTIVE: Transcription factor Snail mediates epithelial-mesenchymal transition (EMT), and is associated with tumor metastasis. This study was designed to observe the enhancive effect of Snail and the reverse effect of antisense-Snail on EMT of tumor cells, and explore the role of Snail in tumor metastasis. METHODS: Snail cDNA was transfected into canine renal epithelial cell line MDCK; antisense-Snail was transfected into human breast cancer cell line MDA-MB231. The expression of epithelial markers E-cadherin, beta-catenin and Cytokeratin 18, mesenchymal marker Fibronectin, metastasis-related marker matrix metalloproteinase-2 (MMP-2), and RhoA were detected by Western blot. The metastatic potential of tumor cells was examined by in vitro cell wound model and Boyden chamber invasion assay. RESULTS: The invasion potential of MDCK cells was enhanced after transfection of Snail. The expression of E-cadherin, beta-catenin, and Cytokeratin 18 was significantly lower in Snail-transfected MDCK cells than in control cells (P < 0.05); the expression of Fibronectin, MMP-2, and RhoA was significantly higher in Snail-transfected cells than in control cells (P < 0.05). Inhibiting the expression of Snail with antisense-Snail in MDA-MB231 cells led to opposite results. CONCLUSION: Snail promotes EMT in normal epithelial cells, and inhibiting the expression of Snail may reverse EMT and suppress tumor metastasis.