Effects of nitric oxide on steroidogenesis and apoptosis in goat luteinized granulosa cells.

The aim of this study was to investigate effects of nitric oxide (NO) on steroidogenesis and apoptosis in goat luteinized granulosa cells (LGCs). We cultured goat LGCs from healthy follicles in culture medium supplemented with the NO donor sodium nitroprusside (SNP) or the NO synthase inhibitor Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), then examined steroid synthesis, oxidative stress and apoptosis in vitro. The results showed that SNP treatment significantly increased the cGMP concentration in the LGCs (P < 0.05), whereas the l-NAME treatment significantly decreased cGMP concentration (P < 0.05). Then Inhibition of NO production significantly inhibited the expression of CYP19A1, a key gene that is involved in sex steroid hormones synthesis and is responsible for the decrease of E2. Inhibition of NO production resulted in an increased percentage of apoptosis, which was accompanied by upregulating expression levels of apoptosis-related markers BAX, CASP3 and CASP9. These data indicate that NO is required for goat LGCs steroidogenesis and cell survival. Furthermore, Inhibition of NO production decreased the expression of mitochondrial biogenesis related genes and proteins (PPARGC1A, NRF-1 and TFAM) and the mtDNA copy number. Simultaneously, inhibition of NO production suppressed the transcription and translation of SOD, GPX1, and CAT, and decreased the glutathione level and increased the 8-OHdG level. However, SNP treatment increased the expression of genes involved in mitochondrial function and biogenesis, and elevated the anti-oxidant stress system and steroid synthesis. Together, our results indicate that NO may up-regulate the expression of PPARGC1A and its downstream factors through the cGMP pathway, thereby decreasing granulosa cell apoptosis, and may participate in the regulation of granulocyte steroid production through the mitochondrial-dependent pathway.

MicroRNA-34a promoting apoptosis of human lens epithelial cells through down-regulation of B-cell lymphoma-2 and silent information regulator.

AIM: To investigate the role of microRNA-34a (miR-34a) in the induction of apoptosis of human lens epithelial (HLE-B3) cells. METHODS: The apoptosis of HLE-B3 cells was detected by Annexin V-PE apoptosis detection kit after the treatment with 200 µmol/L H2O2 for 24h and lentiviral miR-34a vector transfection. The expression of miR-34a in the cells was quantified by quantitative real time polymerase chain reaction (qRT-PCR) in response to H2O2 exposure and the vector transfection. The effects of overexpression of miR-34a on the expression of B-cell lymphoma-2 (Bcl-2) and silent information regulator 1 (SIRT1) was determined by qRT-PCR and Western blot. RESULTS: The expression of miR-34a was up-regulated by the treatment of H2O2 in HLE-B3 cells. The increased expression of miR-34a is accompanied with the cell apoptosis. Consistence with the H2O2 exposure, ectopic overexpression of miR-34a in HLE-B3 cells promoted cells apoptosis. Importantly the anti-apoptosis factors Bcl-2 and SIRT1 were reduced significantly by up-regulation of miR-34a in HLE-B3 cells. CONCLUSION: MiR-34a promotes the apoptosis of HLE-B3 cells by down-regulating Bcl-2 and SIRT1, suggesting that miR-34a may involve in the pathogenesis of cataract formation and targeting miR-34a may be a potentially therapeutic approach for treatment of cataract.

[Effects of extract of Buddleja officinalis eye drops on androgen receptors of lacrimal gland cells of castrated rats with dry eye].

OBJECTIVE: To observe the effects of the extract of Buddleja officinalis eye drops (EBOED) on basic tears secretory volume, tear film stability, and expressions of androgen receptors (AR) in castrated rats with dry eye, and to investigate the mechanism of EBOED on dry eye caused by decreased anti-androgen levels. METHODS: Forty-five male Wistar rats were randomly divided into the blank group, the model group, and the treatment group (treated by EBOED), respectively. Rats in each group were further divided into three sub-groups (fed for one month, two months, and three months, respectively). There were totally nine groups, with five in each. The dry eye model was established with orchiectomy of rats in the model group and the treatment group. EBOED was given to rats in the treatment group for one successive month. Schirmer I test (SIT) and breakup time of tear film (BUT) were determined in all experimental rats. Expressions of AR was analyzed by flow cytometer. RESULTS: Ths SIT value, BUT, and AR positive rate in the model group at the 1st, 2nd, 3rd month were lower than those in the blank group of the same time points (P < 0.01). There was statistical difference in SIT value, BUT, and AR positive rate between the model group and the treatment group at the three time points (P < 0.01). Take the three-month subgroup as an example, the SIT value in the treatment group was (12.667 +/- 5.221) mm, obviously higher than that in the model group (2.676 +/- 1.987) mm. The BUT in the treatment group was (11.758 +/- 4.415) s, obviously longer than that of the model group (4.667 +/- 2.108) s. The AR positive rate in the treatment group was 49.33% +/- 3.44%, obviously higher than that of the model group (33.32% +/- 7.12%, all P < 0.01). CONCLUSIONS: The main components of EBOED was the flavonoids which could significantly inhibit the occurrence of dry eye in rats with decreased androgen levels. Its mechanism might possibly be similar to androgen.

Effects of eye drops of Buddleja officinalis Maxim. extract on lacrimal gland cell apoptosis in castrated rats with dry eye.

OBJECTIVE: To explore the possible mechanism of eye drops of Buddleja officinalis extract in treating dry eye of castrated rats by analyzing the expressions of Bax and Bcl-2 proteins. METHODS: Forty-five Wistar male rats were randomly divided into sham-operated group, untreated group and eye drops of Buddleja officinalis Maxim. extract (treatment) group. The dry eye model was established with orchiectomy in the untreated group and treatment group. Rats in the treatment group were treated with eye drops of Buddleja officinalis Maxim. extract, one drop once, three times daily. Eyes of rats in the sham-operated group and untreated group were instilled with normal saline. After one-, two-, or three-month treatment, five rats in each group were scarified respectively. Then samples were taken to detect related indices. Expressions of Bax and Bcl-2 of lacrimal gland were checked by immunohistochemical method and quantity of apoptotic cells was counted. RESULTS: After one-, two- or three-month treatment, the quantities of expressions of Bax in acinar epithelial cells and glandular tube cells were significantly lower, and those of Bcl-2 were significantly higher in the treatment group than in the untreated group, and the quantities of apoptotic cells of the treatment group were significantly lower than those of the untreated group (P<0.01). CONCLUSION: The main components of extract of Buddleja officinalis Maxim. are flavonoids, which can significantly inhibit cell apoptosis in lacrimal gland.

Effect of Buddleia flavonoids drug-containing plasma on the expression of STAT1 phosphoprotein in lacrimal gland epithelial cells in vitro.

AIM: To explore the effect of Buddleia flavonoids drug-containing plasma and androgen receptor (AR) blocker on the expression of STAT1 phosphoprotein. METHODS: In vitro lacrimal gland epithelial cells were cultivated with H(2)O(2) to establish the dry eye apoptosis state. Blank plasma group, Buddleia officinalis plasma total flavonoids interfere with drug-containing group, and the intervention group of testosterone propionate were set. The expressions of STAT1 phosphoprotein of each group were observed by Western blot. AR blocker flutamide was used to explore the intended androgen effect of Buddleia flavonoids. RESULTS: After the intervention of drug-containing plasma, the expression of STAT1 phosphoprotein in Buddleja officinalis drug-containing plasma intervention group (0.353±0.494) and testosterone propionate intervention group (0.502±0.036) were enhanced and the differences between the two groups were significant (P<0.01). After using AR blocker, the expression of STAT1 phosphoprotein in each group (0.268±0.061, 0.283±0.106, 0.213±0.071) had no difference. CONCLUSION: Buddleja officinalis drug-containing plasma total flavonoids can promote the expression of STAT1 phosphorylation.

Effects of extract of Buddleja officinalis eye drops on androgen receptors of lacrimal gland cells of castrated rats with dry eye.

AIM: To evaluate the effects of the extract of Buddleja officinalis eye drops in basic tears secretory volume, tear film stability, expression of androgen receptors (AR) in castrated rats with dry eye, and to investigate the therapeutic effects of the extract of Buddleja officinalis on dry eye caused by gonadal hormones level imbalance. METHODS: Forty-five Wistar masculinity rats were divided at random into nine groups, including normal groups (A1, A2 and A3); model groups (B1, B2 and B3); therapy groups with extract of Buddleja officinalis eye drops (C1, C2 and C3). The "1" stood for being fed for 1 month, and "2" for 2 months, and "3" for 3 months. The dry eye model was established with orchiectomy on groups B and C. Group C was treated with Buddleja officinalis extract eye drops for one month. All rats were checked with Schirmer I test (SIT) and tear film break-up time (BUT). Expression of AR was analyzed by flow cytometer (FCM). RESULTS: The SIT value of group C was significantly higher than that of group B (P<0.01) and the BUT value of group C was significantly longer than that of group B (P<0.01), which indicated the eye drop could significantly keep basic tears secretory volume and tear film stability. And the expression of AR of group C was much higher than that of group B, which showed that available composition of the eye drops maybe display androgen-like activity. CONCLUSION: The main components of the extract of Buddleja officinalis is the flavonoids that can significantly inhibit happening of dry eye of rat after androgen level lowered. Its mechanism is like androgen's and it can display androgen-like activity to keep basic tears secretory volume and tear film stability.

Effects of extract of Buddleja officinalis on partial inflammation of lacrimal gland in castrated rabbits with dry eye.

AIM: To assess the effects of extract of Buddleja officinalis on tear secretion volume, tear film stability, expressions of TGF-ß1, IL-1ß, TNF-α in lacrimal gland of castrated rabbits with dry eye. METHODS: A total of 30 victory rabbits were divided averagely into normal group(A), model group(B), therapy group with low dose extract of Buddleja officinalis (C), therapy group with high dose extract of Buddleja officinalis (D) and therapy group with genistein (E). The dry eye model was established with orchiectomy on Group B, C, D, E. Group C, D, E were administered intragastrically with corresponding dose extract of Buddleja officinalis or genistein for 30 days. All rabbits were detected with SIT. TGF-ß1, IL-1ß, TNF-α were detected with immunohistochemistry and the ultrastructure of lacrimal gland was observed under transmission electron microscope. RESULTS: The SIT value of group C, D, E were respectively 13.167±4.957, 14.667±5.279, 8.667±0.516, obviously higher than that of group B 5.667±2.338 (P<0.01). The positive expression of IL-1ß in acinar cell and glandular tube cell of group C, D were 0.470±0.048, 0.510±0.088, obviously lower than that of group B 0.770±0.118 (P<0.01). The positive expression of TNF-α of group C, D were 0.498±0.156, 0.435±0.069, obviously lower than that of group B 0.769±0.095 too (P<0.01). The positive expression of TGF-ß1 of group C, D were 0.406±0.171, 0.497±0.147, obviously higher than that of group B 0.222±0.113(P<0.01). Any result of group C, D was positive compared with that of group E (P <0.05). Ultrastructure of the lacrimal gland of group C, D, E was well preserved, especially in D group it was remarkable. CONCLUSION: The extract of Buddleja officinalis can adjust lacrimal gland partial inflammation of dry eye.

[Effects of Huoxue Tongmai Lishui method on fundus fluorescein angiography of non-ischemic retinal vein occlusion: a randomized controlled trial].

BACKGROUND: Huoxue Tongmai Lishui method, a traditional Chinese medicine treatment for eliminating water, activating and promoting blood circulation, could inhibit fundus hemorrhage on experimental retinal vein occlusion (RVO) with high obvious effective rate, and improve symptoms in traditional Chinese medicine. The action mechanism may be related to reducing plasma viscosity and non-perfusion area, and the formation of collateral circulation. OBJECTIVE: To explore the therapeutic effects of Huoxue Tongmai Lishui method (Sanxue Mingmu Tablet) on fundus fluorescent angiograph of non-ischemic retinal vein occlusion (RVO). DESIGN, SETTING, PARTICIPANTS AND INTERVENTIONS: Thirty-four patients with non-ischemic RVO in Department of Ophthalmology, the First Affiliated Hospital, Hunan University of Traditional Chinese Medicine from April 2005 to April 2009 were included. All the patients were diagnosed as qi stagnation and blood stasis syndrome or hyperactivity of liver yang syndrome, and they were randomly divided into two groups, with 17 eyes of 17 patients in treatment group treated by Sanxue Mingmu Tablet combined with conventional treatment, and 18 eyes of 17 patients in control group treated by Xueshuantong Tablet combined with conventional treatment. The patients were treated for two months. MAIN OUTCOME MEASURES: Fundus colour photography, and fundus fluorescent angiograph were detected in two groups before and after the treatment. RESULTS: The curative effect of Sanxue Mingmu Tablet was better than that of Xueshuantong Tablet. Huoxue Tongmai Lishui method could significantly shorten the retinal circulation time, reduce the non-perfusion area, decrease the formation of angiogenesis and promote the formation of collateral circulation. CONCLUSION: Huoxue Tongmai Lishui method is an effective traditional Chinese medicine treatment with high obvious effective rate in reducing non-perfusion area and avoiding venous occlusion and formation of collateral circulation.

[Effects of extract of Buddleja officinalis on prevention of dry eye in castrated rabbits].

OBJECTIVE: To assess the preventive effects of extract of Buddleja officinalis on dry eye in castrated rabbits and to discuss the mechanism of these effects. METHODS: It was a experimental study. Thirty male rabbits were divided equally into normal group (A), disease group (B) and treatment group (C, D, and E). The dry eye model was established with orchiectomy (ORX) in Group B, C, D and E. Group C, D and E were gastrically perfused with single-dose or double-does of Buddleja officinalis extract or genistein for 30 days. All rabbits were examined with Schirmer I test (SIT). TGF-beta1, IL-1beta, TNF-alpha, Fas, FasL, Bax and bcl-2 were detected by immunohistochemistry. Morphological and ultrastructure changes were observed by electron microscopy. RESULTS: The SIT value of group C, D, E was significantly greater than that of group B (P < 0.01). The expression of IL-1beta, TNF-alpha, Fas, FasL and Bax in acinar cells and glandular tube cells of group C, D, E were significantly lower than those of group B (P < 0.01) and the expression of TGF-beta1 and bcl-2 in acinar cells and glandular tube cells of group C, D, E were significantly higher than those of group B (P < 0.01). Furthermore, ultrastructure of lacrimal gland in group C, D, E was much healthier than that of group B. The results obtained from all of these studies showed that the lacrimal glands status in group C, D was significantly better than that of group E (P < 0.05). CONCLUSION: Extract of Buddleja officinalis has a significant effect on the prevention of experimental dry eye in castrated male rabbits. The main components of extract of Buddleja officinalis are the flavonoids. The flavonoids display androgen-like activity. Therefore, it can adjust gonadal hormone level in vivo. As a result, it can inhibit local inflammation in lacrimal gland and reduce apoptosis of lacrimal gland cells.