The Impact Of Traditional Chinese Medicine On The Femoral Head Necrosis On Histology And L-type Calcium Channel

Glucocorticoid-induced Avasadar Necrosis of the Femoral Head, (GANFH) is induced by using lots of glucocorticoid. The bone marrow cells, osteoblasts and osteocyts cause degeneration and necrosis; The lipocyte increase; Bubble or hardening band occur in bone trabeeula. By now, the mechanism is still not completely known. With the glucocorticoid used more and more, the incidence rises gradually, especially after SARS. Because of the refractory arthrosis disease, lots of patients become handicapped, and cannot live independently. So the research of GANFH goes deeply further and further.Objective: Through the research on which Chinese Herb prevent and cure GANFH on the changes in ion channel and bone tectology, we analyze the correlation of the changes, and discuss the possibility of using Ca²⁺ channel currents in osteopathy.Methods : In the histology study, we use 30 SD rats, each one of which weighs (250±10) g, (Number: males=females).They are randomly divided into 3 groups: Control, Model and Chinese Herb groups. Both LPS and mPSL are used to copy GANFH successfully. Procedures: disinfect the rats as usual, give them LPS 15ug/kg intravenous injection, 24 hours later, the rats will get intramuscular injection of mPSL 30mg/kg/d, the following 3 days, Chinese Herb group are taken with chengzai pill 1．5g/kg/d orally. They are killed 6 weeks later. On the 13^th, 3^rd day before they are killed, they are injected with tetracycline with fluorescence twice. We use 0．3% pentobarbital sodium to anesthetize the rats, shear the hair, fix the limbs, and disinfect the rats as usual. Open the abdomen to expose the abdominal aorta, the right and left femoral artery. Ligate the abdominal aorta and the right femoral artery, then inject the Indian Ink towards the left toe untill all of the left toes become black at once. We carve the rats at iliac crest, take both femoral heads. All the right femoral heads should be put in the 10% formaldehyde fixative for HE dyeing. All the left should be put on the 4% paraformalclehyde fixative for decalcification bone section and decoration. We use LEICA photograph analysis system for bone trabecula area(BTA,μm²) , bone mineralization apposition rate ( MAR,μm/10d) , osteo-lacuna rate(OLR,%) and capillary vessel area (CVA,μm²) . The MC3T3-E1 cells are divided into control, model, low-dose and high-dose groups. We measure ten cells in each group. The concentration of the high-dose group (serum containing Chinese Herb) is 10%, and the lower one is 5%. The making methods of the serum containing Chinese Herb are as follows: Add 200 ml pure water to Chengzai pill 60g(provided by Beijing Emperor Special Hospital of Femur Head Necrosis) ;One SD rat is taken 2．5 ml/d orally,8 rats totally. Draw blood at the fourth day 2 hours after the rats taking Chinese Herb, without food before they are killed. We separate the serum from the drawn blood , put 56℃water into it for 30 mins, get rid of the bacteria with iltration membrane, place it to-18℃fridge finally. Except that the rats should be taken 2．5 ml normal saline, we will get the final serum without Chinese Herb, while other procedures are the same. With the density of 10^-5 mol/L, mPSL should be added to MC3T3-E1 cells, which is the model group. We use the whole cell patch clamp technique to measure L-type voltage-sensitive calcium channels current (L-VSCCsC) .The experimental data is expressed by ( (?)±s) .The statistic software is SPSS 11．5．P<0．05 means statistic diversity. The correlative analysis is linear correlation.Results:1．The results of photograph analysis:groupsnBTAMAROLRCVA(μm²) (μm/10d)(%)(μm²) Control1066．26±6．327．713±0．49112．08±0．9536．48±6．90Model1049．56±4．704．818±0．493723．19±9．2919．22±9．03Chengzai1075．63±8．94●9．39±0．3460●4．14±1．02●36．56±7．68pillcompared with control P<0．01,●compared with model P <0．01From the results, we can observe that there are significant differences between model and control (P<0．01) . Compared with model, Chinese Herb group is of great differences (P<0．01) . The results show us: 1．The methods that we combine LPS with mPSL to make the model of GANFH is successful. In model group, histological observation shows that trabecular bones are mussily distributed, loose and even broken. The number of osteo-lacuna increases, while me number of capillary vessel decreases, mean while bone mineralization apposition rate is suppressed. 2．The Chinese Herb group shows the opposite changes which can prevent and cure GANFH.2．The results of current:groupnpeak current (nA)control100．2284±0．0209model100．1839±0．0179low dose100．2526±0．0093●high dose100．2671±0．0120●▼compared with control P0．01,●compared with model P<0．01,â–¼compared with low dose P<0．05The results shows: mPSL can inhibit the L-VSCCsC of osteoblast significantly. However, Chinese Herb can recover the inhibited currents and the high dose is more effective.3．The results of correlation analysis:The L-VSCCsC has positive correlation with MAR, BTA and CVA(r is 0．89, 0．78 and 0．64) , but it has negative correlation with OLR (r is-0．66) .Theresults show that L-VSCCsC is intimate with bone metabolism. So L-VSCCsC is one of the ways to research osteopathy.Conclusion:1．We can manage to make the model of GANFH with the association with LPSand mPSL.2．Chinese Herb can prevent and cure GANFH.3．L-VSCCsC can be inhibited by mPSL, and it has higher correlation with MAR, BTA, CVA and OLR, so it can be used as one of the ways to research osteopathy.