Objective:To explore the effects and mechanism of B-cell lymphoma-2/E1B 19 000 interacting protein 3 (BNIP3) on the migration of human dermal microvascular endothelial cells (HDMECs) under hypoxia.Methods:The experimental research method was applied. (1) HDMECs were divided into normoxic group received routine culture and hypoxic 6, 12, 24 h groups treated with hypoxia under oxygen volume fraction of 2% for corresponding time. Western blotting was used to detect the protein expressions of BNIP3 and microtubule-associated protein 1 light chain 3Ⅱ (LC3Ⅱ) in HDMECs. (2) HDMECs were divided into normoxia+unloaded group, normoxia+BNIP3 knockdown group, hypoxia+unloaded group, and hypoxia+BNIP3 knockdown group which were transfected with unloaded virus or BNIP3 knockdown virus and were subjected normoxic or hypoxic treatment. The BNIP3 protein expression was detected by Western blotting and immunofluorescence staining. The scratch area at 24 h post scratching was detected by scratch test, and the wound healing rate was calculated. The curve distance of cell movement was measured with the living cell workstation, and the speed of movement was calculated within 3 hours. (3) HDMECs were grouped and treated as experiment (2). Western blotting and immunofluorescence staining were performed to detect the protein expression of LC3Ⅱ. The samples were 3 in the above-mentioned experiments. Data were statistically analyzed with one-way analysis of variance and LSD test.Results:(1) Compared with normoxic group, the protein expressions of BNIP3 and LC3Ⅱ of cells in hypoxic 6, 12, 24 h groups were significantly increased (P<0.01). (2) After 6 hours of culture, compared with hypoxia+unloaded group, the BNIP3 expression of cells in hypoxia+BNIP3 knockdown group was significantly decreased (P<0.05). The red fluorescence of BNIP3 expression of cells in normoxia+unloaded group and normoxia+BNIP3 knockdown group was weak, the red fluorescence of cells in hypoxia+unloaded group was strong, and the red fluorescence of cells in hypoxia+BNIP3 knockdown was significantly decreased compared with that in hypoxia+unloaded group. After scratching for 24 hours, the scratch of cells in hypoxia+unloaded group basically healed, while the remaining scratch area in the other three groups were large. The wound healing rates in normoxia+unloaded group, normoxia+BNIP3 knockdown group, hypoxia+unloaded group, and hypoxia+BNIP3 knockdown group were (61±4)%, (58±4)%, (88±4)% and (57±4)%, respectively. There was significant difference in general comparison among these groups (F=14.57, P<0.01). The wound healing rate in hypoxia+unloaded group was significantly higher than that in normoxia+unloaded group (P<0.01) and hypoxia+BNIP3 knockdown group (P<0.05). Within 3 hours of observation, the range of cell movement in hypoxia+unloaded group was significantly larger than that in normoxia+unloaded group, and the range of cell movement in hypoxia+BNIP3 knockdown group was significantly smaller than that in hypoxia+unloaded group. Within 3 hours of observation, the curve movement velocity of cells in hypoxia+unloaded group was significantly higher than that in normoxia+unloaded group and hypoxia+BNIP3 knocdown group (P<0.01). (3) After 6 hours of culture, compared with hypoxia+unloaded group, the BNIP3 protein expression of cells in hypoxia+BNIP3 knockdown group decreased significantly (P<0.05). After 6 hours of culture, the red fluorescence of LC3 expression of cells was weak in normoxia+unloaded group and normoxia+BNIP3 knockdown group, the red fluorescence of LC3 expression of cells was significantly enhanced in hypoxia+unloaded group, and the red fluorescence of LC3 expression of cells was significantly inhibited in hypoxia+BNIP3 knockdown group.Conclusions:BNIP3 can promote the migration and motility of HDMECs under hypoxia, and autophagy may be involved in the regulation migration and motility of HDMECs by BNIP3.

Oxidative stress is one of the main causes leading to the occurrence and development of oste-oporosis,and the most of the current nursing interventions on osteoporosis are related to reducing the level of oxidative stress,but they are still limited to the stage after disease occurrence,and less attention is paid to the population in the sub-disease period,moreover the dietary intervention in the related interventional measures still focuses on calcium and vitamin D supplementation,and lacks the guidance of anti-oxidative stress diet.This article summarizes the relationship and mechanism between the occurrence and development of osteopo-rosis and oxidative stress,by combining with the current progress of osteoporosis related nursing interven-tion,the consideration of age-stepped bone mineral density examination combined with ultra-early nursing in-tervention is proposed,in order to improve the level of bone mineral density in the population,and provide a theoretical basis for the development of new nursing intervention measures for the prevention and treatment of osteoporosis.

Objective:To explore the effects of B-cell lymphoma-2/adenovirus E1B 19 000 interacting protein 3 (BNIP3) on the migration and motility of human dermal microvascular endothelial cells (HDMECs) under hypoxia and the mechanism.Methods:The experimental research method was applied. (1) HDMECs were divided into normoxia group received routine culture and hypoxia 6, 12, 24 h groups treated under hypoxia with oxygen volume fraction of 2% for corresponding time according to the random number table (the same grouping method below). Western blotting was used to detect the protein expressions of BNIP3 and microtubule-associated protein 1 light chain 3Ⅱ (LC3Ⅱ) in HDMECs. (2) HDMECs were divided into normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group which were transfected with unloaded virus or BNIP3 knockdown virus and were subjected to normoxic or hypoxic treatment. The BNIP3 protein expression was detected by Western blotting and immunofluorescence staining. The scratch area at 24 h post scratching was detected by scratch test, and the healing rate of scratch was calculated. The curve distance of cell movement was measured with the living cell workstation, and the speed of movement was calculated within 3 hours. (3) HDMECs were grouped and treated as experiment (2). Western blotting and immunofluorescence staining were performed to detect the protein expression of LC3Ⅱ. The number of sample was 3 in the above-mentioned experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test.Results:(1) Compared with those of normoxia group, the protein expressions of BNIP3 and LC3Ⅱ of cells in hypoxia 6, 12, 24 h groups were significantly increased ( P<0.01). (2) After 6 hours of culture, compared with that of hypoxia+ unloaded group, the BNIP3 protein expressions of cells in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were significantly decreased ( P<0.05 or P<0.01). The red fluorescence denoting BNIP3 protein expression of cells in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group was weak, the red fluorescence of cells in hypoxia+ unloaded group was strong, and the red fluorescence of cells in hypoxia+ BNIP3 knockdown group was significantly decreased compared with that in hypoxia+ unloaded group. After scratching for 24 hours, the scratch of cells in hypoxia+ unloaded group basically healed, while the remaining scratch area in the other three groups were large. The healing rates of scratch of cells in normoxia+ unloaded group, normoxia+ BNIP3 knockdown group, hypoxia+ unloaded group, and hypoxia+ BNIP3 knockdown group were (61±4)%, (58±4)%, (88±4)%, and (57±4)%, respectively. The healing rate of scratch of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group ( P<0.01) and hypoxia+ BNIP3 knockdown group ( P<0.05). Within 3 hours of observation, the range of cell movement in hypoxia+ unloaded group was significantly larger than that in normoxia+ unloaded group, the range of cell movement in hypoxia+ BNIP3 knockdown group was significantly smaller than that in hypoxia+ unloaded group, and the curve movement velocity of cells in hypoxia+ unloaded group was significantly higher than that in normoxia+ unloaded group and hypoxia+ BNIP3 knockdown group ( P<0.01). (3) After 6 hours of culture, compared with hypoxia+ unloaded group, the LC3Ⅱ protein expressions of cells in hypoxia+ unloaded group and hypoxia+ BNIP3 knockdown group were decreased significantly ( P<0.05 or P<0.01). After 6 hours of culture, the red fluorescence denoting LC3 protein expressions of cells was weak in normoxia+ unloaded group and normoxia+ BNIP3 knockdown group, the red fluorescence of cells was significantly enhanced in hypoxia+ unloaded group, and the red fluorescence of cells was significantly inhibited in hypoxia+ BNIP3 knockdown group. Conclusions:BNIP3 can promote the migration and motility of HDMECs under hypoxia, and autophagy may be involved in the regulation migration of HDMECs by BNIP3.

Objective: To investigate the effect of P62 on the migration and motility of human epidermal cell line HaCaT in high glucose microenvironment and its possible molecular mechanism, so as to explore the mechanism of refractory diabetic foot wound healing. Methods: The method of experimental research was used. HaCaT cells in logarithmic growth phase was taken for experiment. The cells were collected and divided into normal control group (culture solution containing glucose with final molarity of 5.5 mmol/L) and high glucose (culture solution containing glucose with final molarity of 30.0 mmol/L) 24 h group, high glucose 48 h group, and high glucose 72 h group according to the random number table (the same grouping method below). The cells in normal control group were routinely cultured for 72 h, cells in high glucose 72 h group were cultured with high glucose for 72 h, cells in high glucose 48 h group were routinely cultured for 24 h then cultured with high glucose for 48 h, cells in high glucose 24 h group were routinely cultured for 48 h then cultured with high glucose for 24 h. Then the protein expression of P62 was detected by Western blotting. The cells were collected and divided into normal control group and high glucose group. After being correspondingly cultured for 48 h as before, the protein expression of P62 was detected by immunofluorescence method (indicated as green fluorescence). The cells were collected and divided into negative control small interfering RNA (siRNA) group, P62-siRNA-1 group, P62-siRNA-2 group, and P62-siRNA-3 group, and transfected with the corresponding reagents. At post transfection hour (PTH) 72, the protein expression of P62 was detected by Western blotting. The cells were collected and divided into normal glucose+negative control siRNA group, normal glucose+P62-siRNA group, high glucose+negative control siRNA group, and high glucose+P62-siRNA group. After the corresponding treatment, the protein expression of P62 was detected by Western blotting at PTH 72 h, the cell migration rate was detected and calculated at 24 h after scratching by scratch test, with the number of samples being 9; and the range of cell movement was observed and the trajectory velocity was calculated within 3 h under the living cell workstation, with the number of samples being 76, 75, 80, and 79 in normal glucose+negative control siRNA group, normal glucose+P62-siRNA group, high glucose+negative control siRNA group, and high glucose+P62-siRNA group, respectively. The cells were collected and divided into normal glucose+phosphate buffered solution (PBS) group, high glucose+PBS group, and high glucose+N-acetylcysteine (NAC) group. After the corresponding treatment, the protein expression of P62 at 48 h of culture was detected by Western blotting and immunofluorescence method, respectively. Except for scratch test and cell motility experiment, the number of samples was all 3 in the rest experiments. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results: Compared with the protein expression in normal control group, the protein expressions of P62 of cells in high glucose 24 h group, high glucose 48 h group, and high glucose 72 h group were significantly increased (P<0.01). At 48 h of culture, the green fluorescence of P62 of cells in high glucose group was stronger than that in normal control group. At PTH 72, compared with the protein expression in negative control siRNA group, the protein expressions of P62 of cells in P62-siRNA-1 group, P62-siRNA-2 group, and P62-siRNA-3 group were significantly decreased (P<0.01). At PTH 72, compared with the protein expression in normal glucose+negative control siRNA group, the protein expression of P62 of cells in normal glucose+P62-siRNA group was significantly decreased (P<0.01), while the protein expression of P62 of cells in high glucose+negative control siRNA group was significantly increased (P<0.01); compared with the protein expression in high glucose+negative control siRNA group, the protein expression of P62 of cells in high glucose+P62-siRNA group was significantly decreased (P<0.01). At 24 h after scratching, compared with (55±7)% in normal glucose+negative control siRNA group, the cell migration rate in normal glucose+P62-siRNA group was significantly increased ((72±14)%, P<0.01), while the cell migration rate in high glucose+negative control siRNA group was significantly decreased ((37±7)%, P<0.01); compared with that in high glucose+negative control siRNA group, the cell migration rate in high glucose+P62-siRNA group was significantly increased ((54±10)%, P<0.01). Within 3 h of observation, the cell movement range in high glucose+negative control siRNA group was smaller than that in normal glucose+negative control siRNA group, while the cell movement range in normal glucose+P62-siRNA group was larger than that in normal glucose+negative control siRNA group, and the cell movement range in high glucose+P62-siRNA group was larger than that in high glucose+negative control siRNA group. Compared with that in normal glucose+negative control siRNA group, the cell trajectory speed in normal glucose+P62-siRNA group was significantly increased (P<0.01), while the cell trajectory speed in high glucose+negative control siRNA group was significantly decreased (P<0.01); compared with that in high glucose+negative control siRNA group, the cell trajectory speed in high glucose+P62-siRNA group was significantly increased (P<0.01). At 48 h of culture, compared with that in normal glucose+PBS group, the protein expression of P62 of cells in high glucose+PBS group was significantly increased (P<0.01); compared with that in high glucose+PBS group, the protein expression of P62 of cells in high glucose+NAC group was significantly decreased (P<0.01). At 48 h of culture, the green fluorescence of P62 of cells in high glucose+PBS group was stronger than that in normal glucose+PBS group, while the green fluorescence of P62 of cells in high glucose+NAC group was weaker than that in high glucose+PBS group. Conclusions: In HaCaT cells, high glucose microenvironment can promote the protein expression of P62; knockdown of P62 protein can promote the migration and increase the mobility of HaCaT cells; and the increase of reactive oxygen species in high glucose microenvironment may be the underlying mechanism for the increase of P62 expression.
Humans ; RNA, Small Interfering/genetics* ; Cell Line ; Epidermis ; Glucose/pharmacology* ; Epidermal Cells