[Effects of 4'-O-methylochnaflavone on endothelial dysfunction induced by palmitic acid in rat cavernous endothelial cells].

OBJECTIVE: To investigate the effect of biflavonoid 4'-O-methylochnaflavone (MF) on palmitic acid-induced endothelial dysfunction in rat cavernous endothelial cells (RCECs). METHODS: The isolated RCECs were commercially available and randomly divided into four groups: normal+BSA group (NC group), palmitic acid (PA) group, MF group, and icariside Ⅱ (ICA Ⅱ) group. The protein expression levels of protein kinase B (PKB/AKT) and endothelial nitric oxide synthase (eNOS) in each group were evaluated via Western blotting. The differences in the intracellular nitric oxide of RCECs treated by MF or ICA Ⅱ were detected by DAF-FM DA that served as a nitric oxide fluorescent probe. Effects of MF and ICA Ⅱ on cell proliferation of PA-stimulated RCECs were determined via CCK-8 assay. RESULTS: The content of nitric oxide in RCECs was significantly increased after the treatment of MF and ICA Ⅱ in comparison with the NC group (P < 0.05). Moreover, compared with ICA Ⅱ group, MF demonstrated a more obvious effect in promoting nitric oxide production (P < 0.05). Compared with the NC group, the expression levels of eNOS and AKT in the PA group were significantly decreased, indicating that a model for simulating the high-fat environment in vitro was successfully constructed (P < 0.05). Meanwhile, the intervention of MF and ICA Ⅱ could effectively increase the expression of eNOS and AKT, suggesting that MF and ICA Ⅱ could promote the recovery of endothelial dysfunction caused by high levels of free fatty acids (P < 0.05). The results of CCK-8 assays showed that PA could significantly reduce the proli-feration ability of RCECs (P < 0.05). Furthermore, the decreased cell viability induced by PA was significantly elevated by treatment with ICA Ⅱ and MF (P < 0.05). CONCLUSION: In RCECs, MF and ICA Ⅱ could effectively increase the content of nitric oxide. The down-regulation of the expression of proteins associated with the AKT/eNOS pathway after PA treatment revealed that this pathway was involved in the development of endothelial dysfunction, which could be effectively reversed by MF and ICA Ⅱ. In addition, the cell proliferation ability was significantly decreased following PA treatment, but MF and ICA Ⅱ could restore the above changes. Overall, biflavonoid MF has an obvious repairing effect on PA-stimulated endothelial dysfunction.

[Testosterone levels in patients with varicocele and azoospermia].

OBJECTIVE: Androgen deficiency is common in aging males and may have unfavourable health consequences. Large-scale studies suggested low testosterone level might increse mortality and morbidity in ageing males. However, young men with low testosterone level might be neglected. Recent studies reported young men with infertility may have reduced testosterone level. To investigate the incidence of androgen deficiency in males with infertility and possible factors affecting the low testosterone level. METHODS: Between January 2011 and December 2012, 407 men with infertility caused by varicocele (VC), obstructive azoospermia (OA) and nonobstructive azoospermia (NOA) in our center were included. The number of men in each group of OA, NOA and VC was 141, 97 and 169, respectively. All the eligible patients underwent a serum testosterone assessment by a single morning blood draw (between 8:00 to noon) to test for concentration of the total testosterone. All serum samples were determined by radioimmunoassay in our andrology laboratory. Androgen deficiency was defined as having a total testosterone level less than 300 ng/dL. RESULTS: The mean age was (30.4±5.8) years. The mean testosterone level was (4.18±1.64) ng/dL (range 0.30 to 11.32 ng/dL). The overall incidence of androgen deficiency was 26.5% (108/407). The incidences of androgen deficiency in NOA, OA and VC groups were 40.2% (39/97), 19.1% (27/141) and 24.9% (42/169), respectively, which were significantly higher in the NOA than in the VC and OA groups (P < 0.001). The incidences had no difference between the VC and OA groups (P=0.229). Univariate analysis revealed the cause of infertility, FSH and the mean testis volume as possible affecting factors for androgen deficiency. However, on multivariate analysis the only cause of infertility was an independent predictor. The incidence of androgen deficiency was the highest in the NOA group [OR 0.492 (95% confidence interval 0.288-0.840)]. CONCLUSION: NOA and varicocele might be risk factors of androgen deficiency. Young men with NOA may have a higher possibility of low testosterone level. Testosterone level should be followed up after NOA and varicocele treatment. Androgen deficiency should be assessed in males with infertility in clinical practice.

[Effect of bone marrow stimulation on arthroscopic repair of anterior talofibular ligament].

Objective: To investigate the effect of simultaneous bone marrow stimulation in the treatment of osteochondral lesions of the talus (OLTs) on arthroscopic repair of anterior talofibular ligament. Methods: The clinical data of 33 patients with ankle instability treated by operation in Sun Yat-Sen Memorial Hospital from January 2018 to December 2019 were retrospectively analyzed. There were 20 males and 13 females with a mean age of (31±9) years. According to the type of surgery, the patients were divided into two groups: ligament repair group (arthroscopic anterior talofibular ligament repair) and bone marrow stimulation group (arthroscopic anterior talofibular ligament repair+talus microfracture). Visual analogue scale (VAS), American Orthopedic Foot And Ankle Society (AOFAS) score, time to return to work and time to return to sport were compared between the two groups. Results: In the ligament repair group, the preoperative VAS score and the AOFAS score was 5.0±1.9 and 72.4±9.0, respectively, and it was 1.1±1.6 and 95.5±5.2 at the last follow-up, respectively. In the bone marrow stimulation group, the preoperative VAS score and the AOFAS score was 5.8±1.9 and 64.8±12.6, respectively, and it was 1.8±1.5 and 93.1±5.6 at the last follow-up, respectively. The scores of VAS and AOFAS were significantly improved in both groups after the operation, but there was no significant difference between the two groups (both P>0.05). There was no significant difference in time to return to work and return to sport between the two groups (both P>0.05). Conclusion: When repairing the anterior talofibular ligament under arthroscopy, simultaneous bone marrow in the treatment of osteochondral lesions of the talus does not affect the postoperative functional recovery and the time to return to work/sport, and satisfactory clinical results also can be obtained.

[Appllication of human acellular dermal matrix in surgical treatment of genitourinary disease].

Humanacellular dermal matrix (HADM) is widely used in the field of burn wound repair and tissue engineering plastic surgery. HADM is manufactored by physical and chemical decellular process to remove the antigenic components that might cause immune rejection in dermis.The extracellular matrix of three-dimensional cell scaffold structure with collagen fibers had been used for wound repair and tissue regeneration, while HADM characterized with low absorption rate after implantation and strong ability to induce angiogenesis in host tissue. Studies reported that after the HADM was implanted into the patient, the host cells, such as fibroblasts and myofibroblasts, as well as lymphocytes, macrophages, granulocytes and mast cells, rapidly infiltrated the graft. The connective tissue and neovascularization were then formed within the HADM three-dimensional cell scaffold, the lymphatic system also appears after vascular reconstruction. Traditional urethral reconstruction using autologous skin flaps has some defects, such as complexity of the technology, risk of necrosis of the skin flaps after transplantation, and failure to achieve functional repair of the urethral epithelium. It has been reported that using HADM to reconstruct the urethra in patients with urethral stricture, hypospadias and bladder-vaginal fistula, showed promising results. Others have reported the experience of using HADM to repair and reconstruct congenital classic bladder exstrophy. HADM has also been used for tissue repair in patients with penile skin defect caused by Fonier's gangrene and hidradenitis suppurativa, and implanted under Bucks' fascia to enlarge the penis. The report of HADM implantation for treating premature ejaculation also deserves attention. Researchers found that HADM implantation can form a tissue barrier between the skin and corpus cavernosum, which can effectively reduce penile sensitivity and treat premature ejaculation. The safety and effectiveness of HADM implantation in the treatment of premature ejaculation need to be further standardized by data from multi-center, large-sample clinical studies. In summary, HADM is the extracellular matrix and three-dimensional cell scaffold of human dermis. As a new type of tissue repair material, new blood vessels are formed actively after implantation, which shows good histocompatibility. HADM has shown increasingly broad application prospects in treatment of genitourinary diseases including penis, urethra and bladder diseases. HADM has also been used in the treatment of premature ejaculation in recent clinical studies, and its long-term safety and efficacy need to be further investigated.

[Chloroquine inhibits viability of renal carcinoma cells and enhances sunitinib-induced caspase-dependent apoptosis].

OBJECTIVE: To determine whether chloroquine (CQ), an often used inhibitor of late autophagy and autophagosome/lyosome fusion, can inhibit proliferation of renal carcinoma cells and investigate its effect on sunitinib (ST)-induced apoptosis. METHODS: Renal carcinoma cell line 786 O and ACHN had been used as cellular model and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay was carried out to detect the cell viability in response to CQ or ST treatment. Both transmission electron microscope and immunoblotting had been employed to observe apoptotic and autophagic process. To examine the involvement of autophagy in ST-dependent apoptosis, autophagy had been inhibited either chemically or genetically via utilizing autophagy inhibitor or specific small interference RNA (siRNA) targeted to either Ulk1 (unc-51-like kinase 1) or LC3 (microtubule associated protein 1 light chain 3 fusion protein), two essential autophagic proteins. RESULTS: Both ST and CQ induced cell viability loss, indicating that either of them could inhibit renal cancer cell proliferation. Clone formation experiments confirmed the aforementioned results. Furthermore, the combined ST with CQ synergistically promoted the loss of cell viability. By transmission electron microscopy and immunoblotting, we found that the ST induced both autophagy and caspase-dependent apoptosis. While 3-MA, an early autophagy inhibitor, reduced the ST-induced cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), a substrate of caspase 3/7 and often used marker of caspase-dependent apoptosis, CQ promoted the ST-dependent PARP-1 cleavage, indicating that the early and late autophagy functioned differentially on the ST-activated apoptotic process. Moreover, the knock down of either Ulk1 or LC3 decreased the ST-caused apoptosis.Interestingly, we observed that rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin) and an inducer of autophagy, also showed to inhibit cell viability and increased the cleavage of PARP-1 in the ST-treated cells, suggesting that autophagy was likely to play a dual role in the regulation of the ST-induced apoptosis. CONCLUSION: ST activates both apoptotic and autophagic process in renal carcinoma cells. Although autophagy precedes the ST-induced apoptosis, however, early and late autophagy functions differentially on the apoptotic process induced by this compound. Additionally, ST can coordinate with the inducer of autophagy to inhibit the cell proliferation. Further research in this direction will let us illuminate to utilize CQ as a potential drug in the treatment of renal carcinoma.

[Variation of neurotrophic factors expression in spinal cord and muscle after root avulsion of brachial plexus].

OBJECTIVE: To investigate the variation of neurotrophic factors expression in spinal cord and muscle after root avulsion of brachial plexus. METHODS: Forty-eight Wistar rats were involved in this study and according to the observing time in 1st day, 1st week, 4th week, 8th week, and 12th week after avulsion, and the control, were divided into 6 groups. By immunohistochemical and hybridization in situ assays, the expression of nerve growth factor (NGF) on muscle, basic fibroblast growth factor(bFGF) and its mRNA on the neurons of corresponding spinal cord was detected. Computer image analysis system was used to calculate the result. RESULTS: After the root avulsion of brachial plexus occurred, expression of NGF increased and reached to the peak at the 1st day. It subsided subsequently but was still higher than normal control until the 12th week. While expression of bFGF and its mRNA increased in the neurons of spinal cord and reached to the peak at the 1st week. Then it dropped down and at the 12th week it turned lower than normal control. CONCLUSION: After root avulsion of brachial plexus, neurotrophic factors expression increase on target muscle and neurons of corresponding spinal cord. It maybe the autoregulation and may protect neuron and improve nerve regeneration.

[Relationship between lipid peroxidase and superoxide dismutase in bronchoalveolar lavage fluid and pulmonary qi deficiency syndrome].

Investigating the levels of lipid peroxidase (LPO) and superoxide dismutase (SOD) in serum, bronchoalveolar lavage fluid (BALF) and alveolar macrophage (AM) were determined in 40 chronic bronchitis patients with Pulmonary Qi Deficiency (PQD) Syndrome and 36 normal subjects. Results showed: (1) No significant differences were found between PQD syndrome and normal subjects on serum SOD or LPO levels. (2) Patients with PQD Syndrome. LPO level in BALF was significantly higher, and SOD significantly lower, when compared with normal subjects. (3) Through correlation analysis, it was found in BALF that SOD level was markedly correlated with AM, while LPO was significantly correlated with neutrophil. In short, SOD and LPO in BALF play an important role in the development of the PQD Syndrome, and are good indications in evaluating the PQD Syndrome.

Fibronectin and hyaluronan in bronchoalveolar lavage fluid from young patients with chronic obstructive pulmonary diseases.

Fibronectin (FN) and hyaluronan (HA) in bronchoalveolar lavage fluid (BALF) and FN released by alveolar macrophages (AM) were examined in 7 nonsmoking healthy control subjects, and 20 smoking patients with chronic obstructive pulmonary diseases (COPD). All patients and subjects were no more than 40 years old. According to the 95% confidence limits of HA and FN in BALF from nonsmoking healthy control subjects, the smoking patients were divided into two groups, those who had HA and FN levels within the limits for nonsmoking controls were classified as the first group (group 1) and those with levels above the control limits were classified as the second group (group 2). Our data showed that the concentrations of HA and FN in BALF and FN released by AM were significantly higher in group 2 than in group 1 patients. There were also significant differences between the two groups in pulmonary function measurements (DLco, FEV1, and FEV1/FVC) which were lower in group 2. HA levels in group 2 patients correlated directly with counts of inflammatory cells in BALF (BALF cells/ml, and numbers/ml of total cells, macrophages, neutrophils and lymphocytes), and the concentration of FN released by AM, and showed an inverse correlation with pulmonary function (DLco and FEV1/FVC). Our results suggest that the inflammatory repair response and fibrosis may play a role in the development of emphysema in young patients with COPD.

[Relationship between local effect of cortisol used by chronic bronchitis patients and syndrome differentiation in TCM].

Serum cortisol levels and bronchoalveolar lavage fluid (BALF) from 105 patients with chronic obstructive pulmonary disease (COPD) and 36 normal subjects were examined and the relationship between cortisol inhibiting the TXB2 secreted by alveolar macrophage (AM) and the theory of Syndrome Differentiation in TCM was explored. Results showed: (1) No significant differences were found between chronic bronchitis and normal subjects on cortisol levels in serum, but in BALF, cortisol levels was significantly lower in Lung Qi Deficiency when compared with that in normal subjects. (2) the levels of cortisol inhibiting the TXB2 secreted by AM which were significantly lower in chronic bronchitis when compared with that in control. In short, the amount and function of cortisol in BALF were significantly different in various syndromes in TCM of chronic bronchitis.