The Rabbit Serum With Huoluo Gukang Pills Inhibits Differentiation Of Marrow Stromal Cells Into Adipocytes Induced By Alcohol And Promotes The Osteoblast Growth

Objective: Huoluo Gukang Pills was given to rabbits by the method of gavage administration through the mouth for preparation of containing serum,then applied it to marrow stromal cells(MSCs)which was extracted by method of whole bone marrow culture.By observing the influences of the rabbit on differentiation of MSCs into adipocytes induced by alcohol and osteoblast growth,then to discuss the mechanism of action of the rabbit serum with Huoluo Gukang Pills that can inhibit MSCs from becoming adipocytes and promote the generation of osteoblast.Confirming the feasibility that Huoluo Gukang Pills could prevent the development of alcohol-induced osteonecrosis of femoral head(ONFH).The result is that the way provides the oretical support for the clinical application of Huoluo Gukang Pills.Methods: The total numbers of New Zealand rabbits were 10,and the dosage of Huoluo Gukang Pills was calculated by basing on body surface area and converted to animal equivalent dose.After that the Pills was mixed with water according to the 2ml/Kg ratio.The mixture of liquid was fed to New Zealand rabbits.Then 7 days later,the rabbits were drew blood from abdominal aorta for preparation of serum that containing Huoluo Gukang Pills.The next step was to acquire MSCs by way of the whole bone marrow adherent cultivation in vitro.After that these MSCs were inoculatingd within 6 holes culture plate,and each plate was divided into three groups(2 holes per row)precedeing intervention experiment.Group A: MSCs were cultured in L-DMEM supplemented with 10% normal rabbit serum;Group B: MSCs were cultured in L-DMEM supplemented with 10% normal rabbit serum and 0.09 mol/L alcohol;Group C: MSCs were cultured in L-DMEM supplemented with 10% rabbit serum containing Huoluo Gukang Pills and 0.09 mol/L alcohol.After interfered culturing,it can be observe and measure adipocytes index(the number of adipocytes that were stained with Sudan ? and observed though the light microscope,and the quantity of Triglyceride from the disruptive cells by means of repeated freezing and thawing)and osteoblast index [the activity of endocellular alkaline phosphatase(ALP)and the radio-immunity of bone gla protein(BGP)in the media].12 days later,partial cells were collected by cytology brush and cracked firstly,then the liquid supernatant from centrifugation was tested the ALP activity in the automatic biochemical analyzer;14 days later,the content of BGP was measured in cells liquid media by radio immunoassay;After 21 days culture,the cover slips were took out from 6 holes culture plate,and the cells on cover slips were stained with Sudan ?,so we could observe the color of lipid drops with invert microscope and count the number of adipocytes;21 days later,partial cells were collected by cytology brush and cracked firstly,then the TG content was collected and measured by the triglyceride reagent.In final,the raw data was analyzed by SPSS 22.0 statistical software for statistical analysis.Results:(1)12~14 days,the lipid drops began to appear in cells cytoplasm,they were tiny,granular and cycloidal.At the same time,we could observe that vacuoles and black refractional rings encircled intracellular in Group B and Group C by invert microscope,among that the quantity of Group B was the fewer and Group A the fewest.Then continue to train and observe,the characteristics of adipocyte differentiation were further strengthening while the MSCs were stimulating by alcohol persistently.The number of adipocytes increased gradually as well as the volume of lipid drops incorporated to larger fat globule together gradually.The cells appeared polygon or quasi-circular.After cultured for 21 days,lipid drops were stained orange and cell nucleus was azury through the dyeing experiment.(2)The number of adipocytes and the content of TG in group B were significantly higher than that of the group C and group A,the difference was statistically significant(P<0.05),the difference of group C and group A has no statistical significance(P>0.05).(3)The activity of ALP and the content of BGP in group B significantly lower than that of the group A and group C,the difference was statistically significant(P<0.05),but the difference of group C and group A was not statistical significance(P>0.05).Conclusion: The toxicity of alcohol enabled the quality of adipocytes increase and the volume become larger within the femoral head.Meanwhile,the deposition and metabolism disorder of lipid led the bone cells to degeneration and necrosis,and the osteoblasts became to inactivate at the same time.The result later,it could cause ONFH.The rabbit serum with Huoluo Gukang Pills could inhibit differentiation of MSCs into adipocytes induced by alcohol;It also can encourage and maintain the normal procedure of osteogenic differentiation.It can improve local micro circulation and reduce pressure in the femoral head bone against the toxicity of alcohol.Furthermore,the dead bone can get repaired and new bone formation.So it proved that the Huoluo Gukang Pills is able to counter the toxic effect of alcohol.