[Inhibitory effects of doxycycline on argyrophilic nucleolar organizing regions and a-smooth muscle actin expression in proliferative bovine corneal myofibroblasts in vitro].

OBJECTIVE: To investigate the effect of doxycycline on the nucleolar organizing regions and a-smooth muscle actin expression in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repair mechanisms. METHODS: Cell culture and identification: bovine corneal fibroblasts were cultured after the stroma was incubated in 1.0 and 2.0 g/L type I collagenase in two stages.Isolated cells were plated at mantaryay culture flask in 10% of BSA RPMI-1640. Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry. The cells staining positive for Vimentin and α-SMA indicated the presence of corneal myofibroblasts. Bovine corneal myofibroblasts were treated with different concentrations of doxycycline (10, 20, 40, 60, 80 mg/L) , a bland control group and the dexamethasone group (120 mg/L) were set up, each group had 30 cases. The argyrophilic nucleolar organizing regions (AgNOR) staining and the immunohistochemistry for α-SMA were performed when the cells were treated for 24 hours and 48 hours. The AgNOR count (Ag-c), AgNOR area (Ag-a) and the expression of α-SMA in the bovine corneal myofibroblasts among each experiment group and control group were compared using one-way ANOVA, further pairwise comparisons using Independent-Samples t test. RESULTS: Cell culture techniques were successfully used to establish a method for the isolation and culture of bovine corneal myofibroblasts. Microscopic examination and immunohistochemical staining confirmed that the cells cultured were bovine corneal myofibroblasts. The Ag-c and Ag-a of bovine corneal myofibroblasts progressively decreased as the concentrations of doxycycline was increase. 24 h:bland control group Ag-c was 6.40 ± 0.6, 60 mg/L doxycycline group Ag-c was 2.23 ± 0.43;bland control group Ag-a was (34.80 ± 2.36) µm(2), 60 mg/L doxycycline hormone group Ag-a was (19.91 ± 2.15) µm(2). 48 h: bland control group Ag-c was 7.27 ± 0.6,60 mg/L doxycycline hormone group Ag-c was 2.80 ± 0.76, bland control group Ag-a was (36.27 ± 1.99) µm(2), 60 mg/L doxycycline group Ag-a was (13.75 ± 2.09) µm(2). The differences were statistically significant: in the same time intervention (FAg-c 24 h = 252.55, FAg-a 24 h = 202.16, P < 0.05, FAg-c 48 h = 169.38, FAg-a 48 h = 853.23, P < 0.05), in the same concentrations intervention (tAg-c = 6.98, tAg-a = 11.62, P < 0.05). And 60 mg/L of doxycycline had an obviously inhibitory action as 120 mg/L dexamethasone in the same treated hours (dexamethasone group Ag-a 24 h = 30.56 ± 3.66, dexamethasone group Ag-a 48 h = 28.35 ± 1.23 ),the differences were not statistically significant (tAg-a 24h = 1.182, P = 0.242,tAg-a 48 h = 0.21, P = 0.832). As the concentrations investigated, doxycycline can inhibit the expression of α-SMA in the bovine corneal myofibroblasts (189.90 ± 7.48, 140.20 ± 7.79, 113.20 ± 8.98, 98.00 ± 3.50, 85.50 ± 4.99), the difference was statistically significant (F = 761.79, P = 0.00). While dexamethasone had no significant role in the expression of α-SMA (bland control group was 225.10 ± 6.74, the dexamethasone group was 228.50 ± 7.12), and the statistically difference was not obvious (t = 1.096, P = 0.287). CONCLUSIONS: As the concentrations of doxycycline was increased from 10 mg/L to 80 mg/L, the AgNOR count and AgNOR area of bovine corneal myofibroblasts can be significantly reduced in vitro. Compared with dexamethasone, doxycycline significantly suppressed the expression of α-SMA in bovine corneal myofibroblasts in a dose-dependent positive trend.

[Relationship between Chinese medicine syndrome type and psychological factor in patients with functional dyspepsia].

OBJECTIVE: To explore the relationship between Chinese medicine syndrome type and psychological factor in patients with functional dyspepsia (FD). METHODS: With an epidemiologic method adopted, 297 FD patients received psychologic mensuration and their Chinese medicine syndrome type was differentiated. The distribution of Chinese medicine syndrome type in FD patients was studied and the differences among various types were analyzed using self-rating depression scale (SDS) and self-rating anxiety scale (SAS). RESULTS: (1) Patients' Chinese medicine syndrome could be differentiated into 5 types: the Pi-deficiency qi-stagnancy type (I), the Gan-qi accumulation type (II), the Gan-qi invading Wei type (III), the dampness-heat stagnating in Wei type (IV) and the Pi-Wei qi-deficiency type (V). Patients of type I (96 cases, 32.3%) held the dominant share. (2) Depressive and anxiety states presented in patients with various syndrome types, among them, patients of type II held the highest percentage of depressive status (30 cases, 62.5%), type III held the highest percentage of anxiety state (19 cases, 35.8%), while type IV possessed the lowest percentages of both. (3) Analysis between symptoms and syndrome types showed that post-prandial fullness presented in most patients of types V and I; early satiation presented more prominently in patients of type V; upper abdominal pain presented frequently in patients of types II and V, and upper abdominal burning sensation presented more evidently in patients of type IV. (4) Comparisons of SDS and SAS scores in patients with different syndrome types showed that the highest SDS score presented in type II, highest SAS score presented in type III; and the lowest scores of SDS and SAS all presented in type IV. CONCLUSIONS: Psychological states are different in FD patients with various syndrome types. The Chinese medicine pathogenetic mechanisms of FD is complex in deficiency/excessive nature, and the condition of disease is closely related with organs Gan and Pi.

[Effect of depside salt from salvia miltiorrhizae in repairing advanced glycation end products-induced late endothelial progenitor cell dysfunction and its molecular mechanism].

OBJECTIVE: To investigate the effects of depside salt from salvia miltiorrhizae (DSSM) in repairing advanced glycation end products (AGE)-induced late endothelial progenitor cell (EPC) dysfunction, and its possible molecular mechanism. METHODS: Mononuclear cells (MNCs) were separated using density gradient centrifugation from human umbilical cord blood, and cultured with EGM-2-MV culture fluid to late EPCs. Then the EPCs were divided into 5 groups: Group A incubated with 200 microg/mL AGE-modified bovine serum albumin (AGE-albumin) alone (A), Groups B, C and D with equal dosage of AGE-albumin plus DSSM at different dosages (0.1 microg/mL, 1 microg/mL, and 10 microg/mL), Group E with 200 microg/mL of unmodified-AGE. The late EPCs apoptosis was detected by Annexin V+/PI double-stain, angiogenic capacity was detected by ECMatrix-gel, mRNA expressions of the receptor for AGE (RAGE) and endothelial nitric oxide synthase (eNOS) were measured by reverse-transcriptase polymerase chain reaction (RT-PCR), and the protein expressions of RAGE, eNOS and protein kinase (Akt) were measured by Western blot. RESULTS: Compared with Group E, in Group A, the Annexin V+/PI- ratio and expression of RAGE in EPCs increased, the angiogenic capacity, mRNA and protein expressions of eNOS, and protein expression of Akt decreased significantly. These abnormal changes in Groups C and D were significantly smaller than those in Group A (P < 0.05 or P < 0.01). And all the indices in Group D were adjacent to those in Group E, showing insignificant difference between the two groups (P > 0.05). CONCLUSIONS: AGE could injure the function of EPCs, revealing increase of cell apoptosis and migration, deprivation of angiogenic capacity in vitro. DSSM could repair the EPCs dysfunction induced by AGE-albumin. Up-regulation of eNOS and Akt in these cells may be involved in the mechanism.