Objective:To investigate the effect of β-Ecdysterone (β-Ecd), an active ingredient in cow′s knee, on hormonal femoral head necrosis in young rabbits and explore the mechanism involved.Methods:An animal study.Sixty New Zealand young rabbits were divided into control, model and intervention groups by random number table method, with 20 rabbits in each group.Prednisolone acetate (7.5 mg/kg) was injected bilaterally into the gluteal muscle of rabbits in both model and intervention groups twice a week.β-Ecd (0.5 mg/kg) was injected subcutaneously at the time of the first injection of Prednisolone acetate in the intervention group for 5 times a week.An equal amount of saline was injected into rabbits in control and model groups.Eight weeks after modelling, animals were put to death, and femoral heads were taken from both sides for gross observation.Micro computed tomography(Micro-CT) was used to analyze the microstructure of bone trabeculae and to measure bone microstructural parameters.Histological staining was used to detect changes in the morphology of bone tissues.Immunohistochemistry and Western blot were used to examine the expression of osteogenic and lipogenic factors and proteins in the femoral head tissue.Rabbit bone marrow mesenchymal stem cells (BMSCs) were divided into a blank control group, a Dexamethasone group and a β-Ecd intervention group.Cell damage was induced by Dexamethasone in Dexamethasone group and β-Ecd intervention group, and the β-Ecd intervention group was given the optimal concentration of β-Ecd.Western blot was used to detect the expression of osteogenic and lipogenic proteins in the cells of each group.Real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression of osteogenic and lipogenic marker genes.Results:After excluding 9 rabbits that died during the experimental period, 51 rabbits were finally included in the study, with 19 in the control group, 15 in the model group and 17 in the intervention group.Gross observation and Micro-CT showed that compared with that of the control group, the femoral head of the model group was obscure and greyish, with dark red necrotic areas.The bone trabeculae of the model group were sparse, thinned, disordered, and partially fractured, compared with those of the control group.The changes in the femoral head and bone trabeculae of the intervention group were between those of control and model groups.The bone mineral density, the number, thickness and relative volume of bone trabeculae significantly decreased and trabecular separation significantly increased in both model and intervention groups, compared with those in the control group ( F=12.78, 45.52, 32.74, 64.08, 8.83, all P<0.05).However, these symptoms in the intervention group were better than those in the model group.Pathological histology showed that in the control group, bone trabeculae were neatly arranged, robust and full, with a high number of osteoclasts and occasional empty bone sockets.In the model group, bone trabeculae were sparsely arranged and broken, with fewer osteoclasts, and the number of empty bone sockets increased and enlarged.In the intervention group, bone trabeculae had a more complete morphology, with fewer necrotic osteoclasts and reduced empty bone sockets, compared with the model group.Immunohistochemistry results showed that compared with the control group, the model group and intervention group had increased content of fatty acid binding proteins (FABP) and CCAAT/enhancer binding proteins α (CEBP) in the femoral head bone tissue, and decreased content of osteopontin (OPN) and Runt-related transcription factor 2 (RUNX2) .The changes in each index were greater in the model group than those in the intervention group ( F=21.07, 24.06, 17.92, 21.36, all P<0.05). Western blot detection showed that compared with the control group, the CEBP protein expression content of the femoral head in the model group and the intervention group was increased and the RUNX2 protein expression content was decreased. The changes of CEBP and RUNX2 were greater in the model group than those in the intervention group( F=73.43, 197.87, all P<0.05).Western blot detection of BMSCs showed that compared with the blank control group, the Dexamethasone group and β-Ecd intervention group had decreased expression of OPN and RUNX2 proteins and increased expression of FABP and CEBP proteins ( F=161.61, 358.01, 91.18, 69.04, all P<0.05).The changes in each index in the β-Ecd intervention group were smaller than those in the Dexamethasone group.RT-qPCR detection of BMSCs showed that in the Dexamethasone group had lower expression of OPN and RUNX2 and higher expression of CEBP and FABP than the blank control group ( F=19.71, 45.08, 61.46, 15.12, all P<0.05).The changes in each index in the β-Ecd intervention group were smaller than those in the Dexamethasone group. Conclusions:β-Ecd can attenuate the pathological changes of hormonal femoral head necrosis in young rabbits by promoting osteogenic differentiation of BMSCs, inhibiting lipogenic differentiation of BMSCs, and improving bone microstructure.

Objective:To investigate the effect of β-Ecdysterone (β-Ecd), an active ingredient in cow′s knee, on hormonal femoral head necrosis in young rabbits and explore the mechanism involved.Methods:An animal study.Sixty New Zealand young rabbits were divided into control, model and intervention groups by random number table method, with 20 rabbits in each group.Prednisolone acetate (7.5 mg/kg) was injected bilaterally into the gluteal muscle of rabbits in both model and intervention groups twice a week.β-Ecd (0.5 mg/kg) was injected subcutaneously at the time of the first injection of Prednisolone acetate in the intervention group for 5 times a week.An equal amount of saline was injected into rabbits in control and model groups.Eight weeks after modelling, animals were put to death, and femoral heads were taken from both sides for gross observation.Micro computed tomography(Micro-CT) was used to analyze the microstructure of bone trabeculae and to measure bone microstructural parameters.Histological staining was used to detect changes in the morphology of bone tissues.Immunohistochemistry and Western blot were used to examine the expression of osteogenic and lipogenic factors and proteins in the femoral head tissue.Rabbit bone marrow mesenchymal stem cells (BMSCs) were divided into a blank control group, a Dexamethasone group and a β-Ecd intervention group.Cell damage was induced by Dexamethasone in Dexamethasone group and β-Ecd intervention group, and the β-Ecd intervention group was given the optimal concentration of β-Ecd.Western blot was used to detect the expression of osteogenic and lipogenic proteins in the cells of each group.Real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression of osteogenic and lipogenic marker genes.Results:After excluding 9 rabbits that died during the experimental period, 51 rabbits were finally included in the study, with 19 in the control group, 15 in the model group and 17 in the intervention group.Gross observation and Micro-CT showed that compared with that of the control group, the femoral head of the model group was obscure and greyish, with dark red necrotic areas.The bone trabeculae of the model group were sparse, thinned, disordered, and partially fractured, compared with those of the control group.The changes in the femoral head and bone trabeculae of the intervention group were between those of control and model groups.The bone mineral density, the number, thickness and relative volume of bone trabeculae significantly decreased and trabecular separation significantly increased in both model and intervention groups, compared with those in the control group ( F=12.78, 45.52, 32.74, 64.08, 8.83, all P<0.05).However, these symptoms in the intervention group were better than those in the model group.Pathological histology showed that in the control group, bone trabeculae were neatly arranged, robust and full, with a high number of osteoclasts and occasional empty bone sockets.In the model group, bone trabeculae were sparsely arranged and broken, with fewer osteoclasts, and the number of empty bone sockets increased and enlarged.In the intervention group, bone trabeculae had a more complete morphology, with fewer necrotic osteoclasts and reduced empty bone sockets, compared with the model group.Immunohistochemistry results showed that compared with the control group, the model group and intervention group had increased content of fatty acid binding proteins (FABP) and CCAAT/enhancer binding proteins α (CEBP) in the femoral head bone tissue, and decreased content of osteopontin (OPN) and Runt-related transcription factor 2 (RUNX2) .The changes in each index were greater in the model group than those in the intervention group ( F=21.07, 24.06, 17.92, 21.36, all P<0.05). Western blot detection showed that compared with the control group, the CEBP protein expression content of the femoral head in the model group and the intervention group was increased and the RUNX2 protein expression content was decreased. The changes of CEBP and RUNX2 were greater in the model group than those in the intervention group( F=73.43, 197.87, all P<0.05).Western blot detection of BMSCs showed that compared with the blank control group, the Dexamethasone group and β-Ecd intervention group had decreased expression of OPN and RUNX2 proteins and increased expression of FABP and CEBP proteins ( F=161.61, 358.01, 91.18, 69.04, all P<0.05).The changes in each index in the β-Ecd intervention group were smaller than those in the Dexamethasone group.RT-qPCR detection of BMSCs showed that in the Dexamethasone group had lower expression of OPN and RUNX2 and higher expression of CEBP and FABP than the blank control group ( F=19.71, 45.08, 61.46, 15.12, all P<0.05).The changes in each index in the β-Ecd intervention group were smaller than those in the Dexamethasone group. Conclusions:β-Ecd can attenuate the pathological changes of hormonal femoral head necrosis in young rabbits by promoting osteogenic differentiation of BMSCs, inhibiting lipogenic differentiation of BMSCs, and improving bone microstructure.

Objective:To investigate the effects of gene silencing inducible cyclooxygenase-2 (COX-2) combined with hyperbaric oxygen (HBO) on neuronal cell edema, apoptosis, autophagy and neural functional recovery in rats with intracerebral hemorrhage.Methods:SPF-grade adult male SD rats ( n=96) were used to establish a cerebral hemorrhage model through stereotactic injection of thrombin VII into the caudate nucleus. They were randomized (random number) into 4 groups ( n=24/group): control group, hyperbaric oxygen (HBO) group, COX-2 RNAi group and combined group (COX-2 RNAi+HBO). The siRNA plasmid targeting silencing COX-2 gene expression was constructed. After group treatment, the four rats were randomly selected from each group for testing in each category. Postoperative day 1, 7, and 14 were assessed using the modified neurological severity score (mNSS) for evaluating neurofunctional deficits. On the 7th day, the water content of the brain tissue was measured using the dry/wet weight method. The blood-brain barrier permeability was assessed using the Evans method. Annexin V and TUNEL assays were employed to assess the apoptotic rate of neural cells. The mRNA expression level of COX-2 in brain tissue was determined using the RT-PCR method. The protein expression levels of Beclin-1, COX-2, aquaporin 4 (AQP-4), B cell lymphoma/lewkmia-2 (Bcl-2), caspase-3, hypoxia-inducible factor-1α (HIF-1α) and matrix metalloprotein-2/9 (MMP-2/9) were detected by Western blot (WB). SPSS software was used for data analysis. One-way ANOVA was used for inter group comparisons and LSD- t test was used for further pairwise comparison. Results:The SD rat intracerebral hemorrhage model and plasmid construction were successfully achieved. The mNSS scores were significantly decreased in COX-2 RNAi, HBO and combined groups compared with control group on the 7th day and 14th day (all P<0.01), especially in combined group ( P<0.01). The contents of Evans blue and the water content of brain tissue of all treatment groups were significantly lower than those in control group (all P<0.05), especially in combined group ( P<0.01). The apoptotic rate of neural cells decreased in all treatment groups compared with the control group (all P<0.05), and the combined group decreased the most ( P<0.01). The mRNA expression levels of COX-2 were significantly decreased in all treatment groups compared with the control group (all P<0.01), and combined group silenced COX-2 expression most obviously ( P<0.05). The results of WB showed that the protein expression levels of Beclin-1, COX-2, AQP-4, Caspase-3, HIF-1α, MMP-2/9 were significantly lower than control group (all P<0.05), while the expression of Bcl-2 was increased in all treatment groups (all P<0.01). Among them, the combined group exhibited the most pronounced trend ( P<0.01). Conclusions:Gene silencing of COX-2 in combination with hyperbaric oxygen therapy can effectively restore neurological function in rats with cerebral hemorrhage. The mechanism may be associated with reduced blood-brain barrier permeability, alleviated brain edema, and inhibition of neuronal apoptosis and autophagy.

With increasing age, more and more patients with posterior chamber intraocular lens (ICL) implantation are facing the threat of cataracts to their visual acuity.When examining the eyes of cataract patients after ICL surgery, attention should be paid to whether the density of corneal endothelial cells is greater than 2 000 cells/mm 2, the state of the anterior chamber angle, and whether there are fundus abnormalities such as retinal detachment and choroidal neovascularization.When conducting eye biometry measurement, attention should be paid to the measurement starting and ending lines of anterior chamber depth and lens thickness.If patients undergo ICL combined with corneal refractive surgery, they should be examined with two or more devices to obtain corneal refractive power according to the examination requirements after corneal laser vision correction.When selecting the type of intraocular lens, consideration should be given to the histological characteristics of high myopia.Compared to C- and L- loops, plate-haptic is relatively more stable in patients with high myopia accompanied by large capsules and larger diameters of continuous curvilinear capsulorhexis.Kane, Barrett Universal Ⅱ, Olsen, Hill-RBF formulas for calculating the refractive power of intraocular lenses are more accurate in people with long axial length.It is recommended to perform ICL removal simultaneously with phacoemulsification and intraocular lens implantation, preferably with a surgical incision greater than 2.6 mm.Femtosecond laser assisted cataract extraction surgery, although superior to traditional phacoemulsification in reducing corneal endothelial cell loss, reducing corneal edema, and high-quality capsulorhexis, can cause incomplete capsulorhexis and fragmentation due to the cavitation bubbles, manual adjustment of location, and the impact of lower vault.It is recommended to use it with caution.Ophthalmologists should fully understand and pay attention to the characteristics and difficulties of cataract surgery after ICL surgery, communicate fully with patients, and make personalized surgery to achieve better visual outcomes.

Objective:To investigate the effect of hyperbaric oxygen combined with RNA interference (RNAi) technology targeting aquaporin-4 (AQP-4) on improving cognitive function in rats with traumatic brain injury (TBI), and to explore its mechanism.Methods:Totally 112 adult male SD rats were randomly divided into four groups: control group, hyperbaric oxygen(HBO) group, AQP-4 RNAi group and combined treatment group, with 28 rats in each group.The TBI model of rat was established by hydraulic percussion and siRNA targeting aquaporin 4 was constructed. Rats were given corresponding intervention according to their groups.Then the modified neurological severity scores(mNSS)was evaluated on the 7th day and 21th day after operation. Morris water maze test was carried out from the 21st day to 25th day after operation and the percentage of target quadrant and daily escape latency were recorded.The changes of the brain permeability of blood-brain barrier and moisture in brain tissues were measured by Evans blue fluorometry and a wet-dry-weighing technique respectively. The protein expression levels of AQP-4, Caspase-3, Bcl-2, MMP-2 and MMP-9 were detected by Western blot method. The mRNA expression of AQP-4 in TBI brain tissue was measured by RT-PCR method, and the apoptosis rate of TBI brain cells was detected by TUNEL and AnnexinV methods on the 7th day after operation. SPSS 23.0 and Graphpad Prism 7.0 softwares were used for data analysis.One-way ANOVA was used for inter group comparison.Repeated measurement ANOVA was used for Morris results, and the LSD- t test was used for pairwise comparisons. Results:The results of mNSS showed that there were significant differences among the groups on the 7th day and 21st day after operation ( F=4.89, 7.59, both P<0.05). The scores of each treatment group were lower than that of the control group, and the effect of the combined treatment group was the best (7th day: t=3.98, -7.75, both P<0.05; 21st day: t=47.82, 7.94, both P<0.05). The results of Morris water maze test showed that the time and group interaction of rats in the target quadrant residence time and escape latency were not statistically significant( F=1.83, 8.42, both P>0.05). The escape latency and the percentage of stay in the target quadrant in the combined treatment group were better than those in other groups on the 24th and 25th day after operation (all P<0.05). Evans blue staining showed that the contents of Evans blue in AQP-4 RNAi group, hyperbaric oxygen group and combined treatment group were lower than that in the control group(all P<0.05), and that in the combined treatment group was the lowest( t=6.19, P<0.05). The results of dry-wet specific gravity method showed that the water content of brain tissue in the combined treatment group((68.15±1.52)%) was the lowest, and that in the AQP-4 RNAi group((76.71±1.06)%) was lower than that in the HBO group ((80.23±1.43)%)( t=4.38, P<0.05). The results of Western blot showed that the protein levels of AQP-4, Caspase-3, MMP-2 and MMP-9 in the combined treatment group were significantly lower than those in other groups(all P<0.05), while the expression of Bcl-2 was increased in the combined treatment group( P<0.05). RT-PCR results (gray value ratio) showed that AQP-4 mRNA levels in AQP-4 RNAi group(0.61±0.21), HBO group (0.83±0.12), combined treatment group(0.22±0.05) and CON group (1.31 ± 0.25) were significantly different( F=175.05, P<0.05), while the AQP-4 mRNA levels decreased in AQP-4 RNAi group which was better than that in hyperbaric oxygen group ( t=5.25, P<0.05). The decrease was the most obvious in the combined treatment group ( t=58.94, P<0.05). The results of TUNEL and AnnexinV showed that the treatment groups were more effective than the control group in inhibiting neuronal apoptosis, especially in the combined treatment group ( P<0.01). Conclusion:The combination of targeted AQP-4 RNAi and hyperbaric oxgen can effectively promote the recovery of neurological and cognitive function, and the mechanism may be related to protecting the integrity of blood-brain barrier, alleviating brain edema and inhibiting apoptosis of nerve cells after TBI.

Objective To examine the effects and mechanisms of siRNA targeting aquaporin 4 (AQP 4) in combination with hyperbaric oxygen therapy(HBO) on cerebral edema and apoptosis in the brain tissue of rats after hemorrhage.Methods Rats were randomly divided into four groups,the control group,the hyperbaric oxygen group,the AQP-4 siRNA group and the combination therapy group (24 rats).Thrombin Ⅶ was injected into the caudate nucleus to establish the hemorrhage model.Construction of siRNA targeting aquaporin 4 was conducted.The mRNA expression of AQP-4 was detected by RT-PCR at day 3.Changes in brain moisture and blood-brain barrier perme ability were measured by a wet/dry weight method and Evans blue fluorometry.The nerve cell apoptosis rate was analyzed by Annexin V andTdT-mediated dUTP-biotin nick end labeling (TUNEL).The expression of proteins including AQP-4,MMP-2,MMP-9,Bcl-2 and caspase-3 was detected by Western Blotting.All the animals were given a score for their nerve function at day 3.Results AQP-4 siRNA treatment obtained better effects than HBO in decreasing the brain edema leveland silencing AQP-4 mRNA(P＜0.05)while,the combination therapy group achieved the best results(P＜ 0.05).Compared with the control group,the percentage of apoptotic cells decreased in all the three treatment groups,with the most marked decrease observed in the combination treatment group(4.24± 0.04)％(F=13.76,P=0.001).The expression of AQP-4,MMP-2,MMP-9 and caspase-3 was lower (P＜0.05) and the expression of Bcl-2 was higher(P＜0.01)in the combination treatment group than in the other three groups.Compared with the control group,all the other three groups received better scores on nerve function defect evaluation at day 3 after hemorrhage(P＜0.05),with the combination treatment group again achieving the most favorable score (4.7 ± 1.1) (F=7.21,P =0.013).Conclusions Targeted siRNA interference combined with hyperbaric oxygen can effectively reduce cerebral edema after cerebral hemorrhage,inhibit neuronal apoptosis and promote neuron function recovery.The underlying mechanisms may be related to down-regulation of AQP-4,MMP 2,MMP-9 and caspase-3 expression and up-regulation of Bcl-2 expression.