| Objective:（1）By collecting femoral head tissue from patients with alcoholic femoral head necrosis and femoral neck fracture,the expression of typical autophagy,LAP related marker proteins,apoptosis related proteins,and osteoclast marker proteins was detected;Simultaneously,immunohistochemistry was used to detect the expression of autophagy,apoptosis,and osteoclast marker proteins in bone tissue;It is speculated that the occurrence and development of alcoholic femoral head necrosis are related to typical autophagy,LAP,apoptosis,and high expression of osteoclasts.（2）Establish an alcohol intervention mouse RAW264.7 cell model for osteoclast precursor cells,based on the regulatory relationship between typical autophagy and LAP,as well as the relationship between autophagy and apoptosis,with a focus on discussing the promoting effect of alcohol on osteoclast differentiation and the therapeutic targets of icariin.Exploring the dynamic balance of icariin regulation of autophagy and apoptosis,maintaining cell homeostasis,and resisting the promoting effect of alcohol on osteoclast differentiation.（3）Explore whether there is a competitive relationship between typical autophagy and LAP autophagy pathways,whether alcohol promotes osteoclast differentiation by mediating the relationship between typical autophagy and LAP autophagy pathways,and whether icariin inhibits osteoclast differentiation by mediating these two pathways.Methods:（1）Part 1:This study selected a total of 10 patients who underwent total hip arthroplasty due to alcoholic femoral head necrosis and femoral neck fracture from June 2023 to January 2024,including 5 patients with alcoholic femoral head necrosis and 5 patients with femoral neck fracture.General basic information on the patient’s age,gender,and weight was collected,and relevant clinical data during hospitalization were collected,including preoperative and postoperative X-ray examinations,preoperative CT scans,biochemical indicators,blood routine,etc.Obtain the patient’s bone tissue for Western blot and immunohistochemistry after surgery,WB detection of typical autophagy in femoral head tissue LAP、Protein expression of apoptosis and osteoclast related markers,immunohistochemical detection of autophagy,apoptosis,and osteoclast marker proteins in bone tissue.（2）Part 2:Select mouse osteoclast precursor cell line（RAW264.7）for the experiment.The CCK-8 method was used to detect the effects of different concentrations of icariin(10-4M,10-5M,10-6M,10-7M,10-8M,10-9M,10-10M)and alcohol(1.25 x 10-1M,2.5 x 10-1m M,3.75 x 10-1m M,5 x 10-1m M,6.25 x10-1m M,7.5 x 10-1m M,8.25 x 10-1m M)on cell proliferation over 1-3 days.Three days were selected as the time node for subsequent experiments,and alcohol concentrations of 1.25 x 10-1M and icariin concentrations of 10-7M and10-8M were selected for cell intervention.According to different intervention measures,cells were divided into 6 groups:（1）Control group:adding complete culture medium+RANKL inducer;（2）Alcohol group:Add complete culture medium containing 1.25m x 10-1M alcohol+RANKL;（3）Epimedium 10-7M group:Add complete culture medium containing Epimedium 10-7M+RANKL;（4）Epimedium 10-8M group:Add complete culture medium containing Epimedium 10-8M+RANKL;（5）Epimedium 10-7M+alcohol group:Add complete culture medium containing 1.25×10-1M alcohol+RANKL+Epimedium10-7M;（6）Epimedium 10-8M+alcohol group:Add complete culture medium containing 1.25×10-1M alcohol+RANKL+Epimedium 10-8M.TRAc P detection of osteoclast formation and reactive oxygen species（ROS）detection of intracellular ROS levels in the above groups;TUNEL apoptosis detection method to detect cell apoptosis;Autophagy detection using m RFP-GFP-LC3double labeled adenovirus,reverse transcription real-time fluorescence quantification polymerase chain reaction（qRT PCR）detection of CAMKII The relative expression levels of Rubicon,Cathespin K,P62,LC3B,and Caspase-3m RNA;Western blot detection of Caspase-3 Expression of p62,LC3II/LC3I,Rubicon,Cam KII,and Cathespin K proteins.（3）Part 3:Select RAW264.7 for the experiment.Detect the effect of KN93on cell proliferation using CCK-8 method.According to the detection results of CCK-8,10-3M concentration was selected to intervene in the cells,and they were divided into 7 groups according to the intervention method:（1）Normal group:added with RANKL complete culture medium;（2）KN93 group:Add complete culture medium containing 10-3MKN93+RANKL;（3）KN93 alcohol group:Add complete culture medium containing 10-3MKN93+1.25×10-1M alcohol+RANKL;（4）KN93 icariin 10-7M group:Add complete culture medium containing 10-3MKN93+icariin 10-7M+RANKL;（5）KN93 icariin 10-8M group:Add complete culture medium containing 10-3MKN93+icariin 10-8M+RANKL;（6）KN93 icariin 10-7M+alcohol group:Add complete culture medium containing 10-3MKN93+1.25×10-1M alcohol+RANKL+icariin 10-7M;（7）KN93icariin 10-8M+alcohol group:Add complete culture medium containing10-3MKN93+1.25×10-1M alcohol+RANKL+icariin 10-8M.The above groups,TRAc P detection of osteoclast formation and reactive oxygen species（ROS）detection of intracellular reactive oxygen species levels in each group;TUNEL apoptosis detection method to detect cell apoptosis;Autophagy was detected using m RFP-GFP-LC3,and the relative expression levels of CAMKII,Rubicon,Cathespin K,P62,LC3B,and Caspase-3 m RNA were detected using reverse transcription real-time fluorescence quantification polymerase chain reaction（qRT PCR）;Western blot was used to detect the expression of Caspase-3,p62,LC3II/LC3I,Rubicon,Cam KII,and Cathespin K proteins.Results:（1）Part 1:Immunohistochemical results of bone tissue showed that compared with normal tissue,the expression levels of apoptosis related proteins Caspase-3,Benlin-1,autophagy related protein LC3B,and osteoclast related protein Cathespin K in alcoholic femoral head necrosis bone tissue were significantly increased（P<0.01）.WB results showed that compared with the normal group,the expression levels of Caspase-3,p62,LC3II/LC3I,Rubicon,Cam KII,and Cathespin K proteins were significantly higher than those in the normal group（P<0.01）.（2）Part 2:CCK-8 results showed that compared with Control,when the alcohol concentration was maintained at 3.75×10-1M-8.75×10-1M,alcohol had a significant（P<0.01）inhibitory effect on the proliferation of osteoclast precursor cells in all five groups on DAY1,DAY2,and DAY3 days.As the alcohol concentration decreased,it showed an upward trend,while during DAY1-DAY4,it showed a downward trend.Among them,the inhibitory effect of DAY1 had a relatively small impact on other days.When the alcohol concentration was 2.5×10-1M,compared with the normal group,DAY1,DAY2,and DAY3 still showed a downward trend,but there was no significant difference（P>0.01）.When the alcohol concentration was maintained at 1.25×10-1M,there was an upward trend compared to the normal group,and this upward trend was most obvious on the third day,with P<0.01.Therefore,we chose to culture osteoclast precursor cells at an alcohol concentration of 1.25×10-1M for 3 days,and icariin was selected at concentrations of 10-7M and 10-8M based on previous experiments conducted by the research group.The TRAP staining results showed that compared with the normal induction group of osteoclasts,the number of osteoclasts was highest under alcohol intervention.However,under the intervention of different concentrations of icariin,the number of osteoclasts formed decreased,and there was no significant difference in the number of osteoclasts formed between the alcohol+icariin group and the normal induction group.The ROS results showed that compared with the normal group,the level of reactive oxygen species in the alcohol group decreased significantly（P<0.01）,and the expression of reactive oxygen species in each concentration group of icariin increased significantly（P<0.01）.The alcohol+icariin concentration groups showed a downward trend compared to the normal group,but there was no significant difference（P>0.01）;The alcohol+icariin group showed an upward trend compared to the alcohol group,with a significant difference（P<0.01）.The TUNEL apoptosis detection results showed that compared with the normal group,the apoptosis level of the alcohol group showed a significant decrease（P<0.01）,and the apoptosis level of cells in each concentration group of icariin also showed a downward trend,but there was no significant difference（P>0.01）.The apoptosis level of cells in each concentration group of alcohol+icariin showed a downward trend,and there was no significant difference（P>0.01）;Compared with the alcohol group,the apoptosis of cells in each concentration group of icariin showed an upward trend,and the alcohol+icariin group also showed a certain upward trend compared to the alcohol group.The qRT PCR results showed that in the m RNA detection of CAMKII,compared with the normal group,the m RNA content of CAMKII in the AL group significantly increased（P<0.01）,while the content of ICA-7 and ICA-8groups significantly decreased（P<0.01）.The content of AL+ICA-7 and AL+ICA-8 groups slightly decreased.Compared with the AL group,the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.In the m RNA detection of Caspase-3,it was found that compared with the normal group,the m RNA content of Caspase-3 in the AL,ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.Compared with the AL group,the content of ICA-7,ICA-8,and AL+ICA-8 groups significantly increased（P<0.01）,while the content of AL+ICA-7 group slightly increased.In the m RNA detection of Cathepsin K,it was found that compared with the normal group,the m RNA content of Cathepsin K in the AL group increased significantly（P<0.01）,while the content of ICA-7 and ICA-8 groups decreased significantly（P<0.01）.The content of AL+ICA-7 and AL+ICA-8groups slightly decreased.Compared with the AL group,the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.In the m RNA detection of LC3B,it was found that compared with the normal group,the m RNA content of LC3B in the AL group significantly increased（P<0.01）,while the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.Compared with the AL group,the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.In the m RNA detection of P62,it was found that compared with the normal group,the m RNA content of P62 in the AL group increased significantly（P<0.01）,while the content of ICA-7 and ICA-8 groups decreased significantly（P<0.01）.The content of AL+ICA-7 and AL+ICA-8 groups slightly decreased.Compared with the AL group,the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.In the m RNA detection of Rubicon,it was found that compared with the normal group,the m RNA content of Rubicon in the AL group significantly increased（P<0.01）,while the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.Compared with the AL group,the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.Western blot results showed that in CAMKII protein detection,compared with the normal group,the protein content of CAMKII in the AL group significantly increased（P<0.01）,while the content of ICA-7 and ICA-8 groups significantly decreased（P<0.01）.The content of AL+ICA-7 and AL+ICA-8groups slightly decreased.Compared with the AL group,the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.In the protein detection of Caspase-3,it was found that compared with the normal group,the protein content of Caspase-3 in the AL,ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups was significantly reduced（P<0.01）.Compared with the AL group,the content of ICA-7,ICA-8,and AL+ICA-8 groups significantly increased（P<0.01）,while the content of AL+ICA-7 group slightly increased.In the protein detection of Cathepsin K,it was found that compared with the normal group,the protein content of Cathepsin K in the AL group increased significantly（P<0.01）,while the content of ICA-7 and ICA-8 groups decreased significantly（P<0.01）.The content of AL+ICA-7 and AL+ICA-8 groups slightly decreased.Compared with the AL group,the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.In the protein detection of LC3B,it was found that compared with the normal group,the protein content of LC3B in the AL group increased significantly（P<0.01）,while the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups decreased significantly（P<0.01）.Compared with the AL group,the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.In the protein detection of P62,it was found that compared with the normal group,the protein content of P62 in the AL group increased significantly（P<0.01）,while the content of ICA-7 and ICA-8 groups decreased significantly（P<0.01）.The content of AL+ICA-7 and AL+ICA-8groups slightly decreased.Compared with the AL group,the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.In the protein detection of Rubicon,it was found that compared with the normal group,the protein content of Rubicon in the AL group significantly increased（P<0.01）,while the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.Compared with the AL group,the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P<0.01）.The results of autophagy double labeled adenovirus detection showed that compared with the normal group,the alcohol group had an increase in green,red,and yellow spots,indicating a higher formation of autophagosomes,autophagosomes,and lysosomes（P<0.01）.The formation of autophagosomes and lysosomes in ICA-7 and ICA-8 was less（P<0.01）,and there was no significant difference in the number of autophagosomes between the alcohol+ICA-7 and alcohol+ICA-8 groups compared to the normal group（P>0.01）（3）Part 3:CCK-8 results showed that compared with Control,when the concentration of KN-93 was maintained at 10-2M-10-1M,KN-93 had a significant inhibitory effect on the proliferation of osteoclast precursor cells in both groups on Day 3,with P<0.01.Among them,the inhibitory effect of 10-1M on osteoclast precursor cells was more significant,with P<0.001.When the concentration of KN-93 was between 1×10-3-1×10-8M,there was no significant difference between the six groups and the normal group,with P>0.01;The TRAP staining results showed that compared with the normal induction group of osteoclasts,there was no significant difference in the number of osteoclast differentiation in the KN-93 group.The highest number of osteoclasts was observed under the intervention of KN-93+alcohol,while the number of osteoclasts formed decreased under the intervention of different concentrations of icariin+KN-93.Moreover,there was no significant difference in the number of osteoclasts formed between the KN-93+alcohol+icariin group and the normal induction group;The ROS results showed that compared with the normal group,the level of reactive oxygen species in the KN-93 group showed a certain upward trend,but there was no significant difference;Compared with the KN-93 group,the level of reactive oxygen species in the KN-93+alcohol group showed a significant decrease（P<0.01）,while the expression of reactive oxygen species in each concentration group intervened with KN-93+icariin increased significantly（P<0.01）.The concentration groups of KN-93+alcohol+icariin showed a downward trend compared to the KN-93 group,but there was no significant difference（P>0.01）;The KN-93+alcohol+icariin group showed an upward trend compared to the KN-93+alcohol group,but there was no significant difference（P>0.01）;The TUNEL apoptosis detection results showed that compared with the normal group,the apoptosis of KN-93 showed a downward trend,but the difference was not significant（P>0.01）.Compared with the KN-93 group,the apoptosis level of the KN-93+alcohol group showed a significant decrease（P<0.01）,and the apoptosis level of cells in each concentration group of KN-93+icariin also showed a downward trend,but there was no significant difference（P>0.01）.The apoptosis level of cells in each concentration group of KN-93+alcohol+icariin showed a downward trend,There was no significant difference（P>0.01）;Compared with the KN-93+alcohol group,the apoptosis of cells in each concentration group of KN-93+icariin showed an upward trend,and the KN-93+alcohol+icariin group also showed a certain upward trend compared to the KN-93+alcohol group;The qRT PCR results showed that in the m RNA detection of Caspase-3,compared with the normal group,the m RNA content of Caspase-3 in the normal+inhibitory group significantly increased（P>0.01）,while the content of AL and ICA-7 groups significantly decreased（P>0.01）.The content of ICA-8,AL+ICA-7,and AL+ICA-8 groups slightly decreased.Compared with the normal+inhibitory group,the levels of AL,ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P>0.01）.In the m RNA detection of Cathepsin K,it was found that compared with the normal group,the m RNA content of Cathepsin K in the normal+inhibitory group slightly increased,the AL group significantly increased（P>0.01）,the ICA-7 group significantly decreased（P>0.01）,and the ICA-8,AL+ICA-7,and AL+ICA-8 groups slightly decreased.Compared with the normal+inhibitory group,the AL group showed a significant increase in content（P>0.01）,while the ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups showed a significant decrease in content（P>0.01）.In the m RNA detection of LC3B,it was found that compared with the normal group,the m RNA content of LC3B in the normal+inhibitory group slightly increased,the AL group significantly increased（P>0.01）,the ICA-7 and ICA-8 groups significantly decreased（P>0.01）,and the AL+ICA-7 and AL+ICA-8 groups slightly decreased.Compared with the normal+inhibitory group,the content of AL group increased significantly（P>0.01）,while the content of ICA-7 and ICA-8 groups decreased significantly（P>0.01）.The content of AL+ICA-7 and AL+ICA-8 groups slightly decreased.In the m RNA detection of P62,it was found that compared with the normal group,the m RNA content of P62 in the normal+inhibitory and AL groups increased significantly（P>0.01）,while the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups decreased significantly（P>0.01）.Compared with the normal+inhibitory group,the AL group showed a significant increase in content（P>0.01）,while the ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups showed a significant decrease in content（P>0.01）.In the m RNA detection of Rubicon,it was found that compared with the normal group,the m RNA content of Rubicon in the normal+inhibitory and AL groups significantly increased（P>0.01）,while the content of ICA-7 and ICA-8 groups slightly decreased,and the content of AL+ICA-7 and AL+ICA-8groups slightly increased.Compared with the normal+inhibitory group,the AL group showed a slight increase in content,while the ICA-7,ICA-8,and AL+ICA-7 groups showed a significant decrease（P>0.01）,while the AL+ICA-8group showed a slight decrease in content.In the m RNA detection of CAMKII,it was found that compared with the normal group,the m RNA content of CAMKII in the normal+inhibitory,AL,ICA-7,ICA-8,AL+ICA-7,AL+ICA-8groups was significantly reduced（P>0.01）.Compared with the normal+inhibitory group,the content of AL group increased significantly（P>0.01）,while the content of ICA-7 group decreased significantly（P>0.01）.The content of ICA-8,AL+ICA-7,and AL+ICA-8 groups slightly decreased;Western blot results showed that in Caspase-3 protein detection,compared with the normal group,the protein content of Caspase-3 in the normal+inhibitory group was significantly increased（P>0.01）,while the content of AL and ICA-7 groups was significantly decreased（P>0.01）.The content of ICA-8,AL+ICA-7,and AL+ICA-8 groups slightly decreased.Compared with the normal+inhibitory group,the levels of AL,ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups significantly decreased（P>0.01）.In the protein detection of Cathepsin K,it was found that compared with the normal group,the protein content of Cathepsin K in the normal+inhibitory group slightly increased,the AL group significantly increased（P>0.01）,the ICA-7 group significantly decreased（P>0.01）,and the ICA-8,AL+ICA-7,and AL+ICA-8 groups slightly decreased.Compared with the normal+inhibitory group,the AL group showed a significant increase in content（P>0.01）,while the ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups showed a significant decrease in content（P>0.01）.In the protein detection of LC3B,it was found that compared with the normal group,the protein content of LC3B in the normal+inhibitory group slightly increased,the content of AL group significantly increased（P>0.01）,the content of ICA-7and ICA-8 groups significantly decreased（P>0.01）,and the content of AL+ICA-7 and AL+ICA-8 groups slightly decreased.Compared with the normal+inhibitory group,the content of AL group increased significantly（P>0.01）,while the content of ICA-7 and ICA-8 groups decreased significantly（P>0.01）.The content of AL+ICA-7 and AL+ICA-8 groups slightly decreased（P>0.01）.In the protein detection of P62,it was found that compared with the normal group,the protein content of P62 in the normal+inhibitory and AL groups increased significantly（P>0.01）,while the content of ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups decreased significantly（P>0.01）.Compared with the normal+inhibitory group,the AL group showed a significant increase in content（P>0.01）,while the ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups showed a significant decrease in content（P>0.01）.In the protein detection of Rubicon,it was found that compared with the normal group,the protein content of Rubicon in the normal+inhibitory and AL groups significantly increased（P>0.01）,while the content of ICA-7 and ICA-8 groups slightly decreased,and the content of AL+ICA-7 and AL+ICA-8 groups slightly increased.Compared with the normal+inhibitory group,the AL group showed a slight increase in content,while the ICA-7,ICA-8,and AL+ICA-7 groups showed a significant decrease（P>0.01）,while the AL+ICA-8 group showed a slight decrease in content.In the protein testing of CAMKII,it was found that compared with the normal group,the protein content of CAMKII in the normal+inhibitory,AL,ICA-7,ICA-8,AL+ICA-7,and AL+ICA-8 groups was significantly reduced（P>0.01）.Compared with the normal+inhibitory group,the AL group showed a significant increase in content（P>0.01）,while the ICA-7 group showed a significant decrease（P>0.01）.The content of ICA-8,AL+ICA-7,and AL+ICA-8groups slightly decreased;The results of autophagy double labeled adenovirus detection showed that compared with the normal group,there was no significant difference in the number of autophagosomes between the KN-93 group,alcohol+ICA-7,and alcohol+ICA-8 groups（P>0.01）.The number of green,red,and yellow spots in the alcohol group increased,indicating that there was more formation of autophagosomes,autophagosomes,and lysosomes（P<0.01）.ICA-7 and ICA-8have less formation of autophagosomes and autophagosomes（P<0.01）,Conclusion:（1）The occurrence and development of alcoholic femoral head necrosis are related to cellular autophagy and apoptosis,which may be related to p62 The expression levels of LC3,Rubicon,Ca MKII,and Caspace-3 proteins are correlated.（2）High concentration alcohol(3.75×10-1M-8.75×10-1M)has a killing effect on RAW264.7 cells,while low concentration alcohol(1.25×10-1M)has a proliferative effect on the cells.Low concentration alcohol can promote cell autophagy,inhibit intracellular oxidative stress levels,reduce apoptosis of osteoclast precursor cells,promote osteoclast differentiation,and icariin can increase intracellular oxidative stress levels and inhibit osteoclast differentiation by inhibiting cell autophagy;（3）Alcohol can jointly promote both typical autophagy and LAP atypical autophagy pathways,thereby increasing the autophagy level of osteoclast precursor cells,and icariin has inhibitory effects on both pathways.（4）High concentration of KN-93((10-2M---10-1M))inhibits the proliferation of osteoclast precursor cells.When the concentration is controlled at 10-3M,it has no effect on cell proliferation.When the protein level of Ca MK II is inhibited,The RUBICON protein will be enhanced,and there may be a competitive relationship between the two.When typical autophagy is inhibited,the atypical autophagy LAP pathway will be enhanced,thereby maintaining the balance and stability of cellular autophagy. |
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