Experimental Study On The Preventive Effect Of Baicalein On Glucocorticoid-Induced Osteonecrosis Of The Femoral Head

Research background and purpose:Glucocorticoid-induced osteonecrosis of the femoral head is one of the serious adverse reactions after treatment with this kind of drug.A large number of existing research results have shown that the pathogenesis of steroid-induced osteonecrosis of the femoral head involves many factors and is the result of the combined action of multiple factors.Patients with steroid-induced osteonecrosis of the femoral head often become disabled in the late stage of this disease,and they would have to undergo surgery of artificial total hip replacement.The hazards of this disease to patients’s health cannot be ignored.Early intervention is of great significance,but there are no certain drugs can be recommended for preventive treatment.Most of the therapeutic regimen for intervention that have been proposed are aimed at a certain aspect of the pathogenesis,and the effect is not very satisfactory.In some existing in vitro studies and animal model experiments,baicalein has been reported to have a variety of regulatory effects that can all theoretically be applied to the intervention of the pathogenetic process of the disease,and it is possible to intervene the pathogenetic process of the disease through effects on various aspects,which leads to a protective effect and suggesting that it may be a potential interventional drug with pleiotropic effect.This study intends to apply baicalein to the SD rat model of steroid-induced osteonecrosis of the femoral head in early stage,and to explore its actual protective effect on the disease from level of phenotype,and to provide experimental basis for discovering new drugs used for preventative treatment of GIOFH.Materials and Methods:Thirty-six healthy male SD rats at 10 weeks of age were selected,and after 1 week of adaptive feeding,the rats were distributed into 3 groups of 12 rats each with method of random number table,3 groups are blank control group(CG),model group(MG),and baicalein intervention group(IG)respectively.Model group(MG)treatment:rats receive intraperitoneal injection of lipopolysaccharide(LPS)20μg/kg twice at 24h interval firstly;24h after the second LPS injection rats receive intramuscular injection of methylprednisolone sodium succinate(MPSS)40mg/kg at 24h interval for 3 days(24h interval between injections);initial injection of methylprednisolone sodium succinate accompanied by saline instead of baicalein gavage once daily for 6 weeks.Baicalein intervention group(IG):Giving the modeling drugs with the same method used in MG group;baicalein(300mg/kg)that confected into turbid liquid with saline is applied to rats after first injection of steroid by gavage,once daily for 6 weeks.Blank control group(CG):the drugs for injection/gavage used in other experimental groups were replaced with saline given at the same operation time period.Rats were allowed free access to purified water and feed during the rearing period.Following conditions were observed and detected:1.General conditions and changes of body weight of ratsDuring the experiment,the food and water intake,activity,hair condition,and mental state of the rats in each group were observed and recorded daily;The body weight of the rats in each group was measured weekly.2.Serum indices①Level of malondialdehyde(MDA):Two weeks after the beginning of experiment,2ml of blood was collected from the tail vein from rats and was centrifuged to extract the upper serum.The level of MDA was detected by TBA method.②Level of blood lipids:Veterinary automatic biochemical analyzer to detect blood lipids(total cholesterol for TC,triglyceride for TG).③Level of bone metabolism markers:Bone metabolism markers(BALP、PINP、β-CTX)were detected with ELISA methodAt the end of 6th week during the experiment,rats in each group were anesthetized with pentobarbital sodium 40mg/kg by intraperitoneal injection,and about 6ml of blood was collected from the lower abdomen through the abdominal aorta for detection of indices in②and ③.3.Detection of femoral head3.1 General appearance of femoral head specimensAfter collecting blood sample,the rats were euthanized by injecting pentobarbital sodium 200mg/kg into the inferior vena cava,and then the femoral heads of both hind limbs were completely taken out,and the general appearance of the femoral heads was observed and photographed.The femoral heads were fixed with 4%paraformaldehyde and stored.3.2Micro-CT scanning of femoral headMicro-CT scanning was performed on one of the femoral heads of the rat,and the reconstruction program was used to perform three-dimensional reconstruction of the bone image;the subchondral cancellous area of the femoral head was selected as the region of interest(ROI),and the five bone tissue parameters:bone volume fraction(BV/TV),trabecular thickness(Tb.Th),trabecular separation(Tb.Sp),trabecular number(Tb.N),bone mineral density(BMD)of ROI were measured and recorded.3.3 Observation and analysis of femoral head specimen section①HE staining:The tissue morphology was observed,and the empty lacuna rate of bone was calculated.According to the pathological diagnosis standard of osteonecrosis,the osteonecrosis rate of femoral head was calculated.②TRAP staining:Counting of osteoclast were performed.③TUNEL staining:Apoptosis rate of cell was calculated4.Statistical analysis:SPSS 26.0 and GraphPad Prism 8.0.2 were used to analyze the data,the quantitative data for each group were first tested for normality as well as chisquare,and the data whose distribution passed the normality test were expressed using the mean ±standard deviation(x±S).Differences between groups were first examined by one-way ANOVA,if the variance is homogeneous then pairwise comparison of differences among the groups will be performed using LSD test,if the variance is not homogeneous Welch’s correction will be performed and pairwise comparison would be using Dunnett’s T3 test.Qualitative data was expressed by frequency,and Fisher exact test is performed for the test.P<0.05 was considered statistically significant.Result:1.General condition and changes of body weight of ratsGeneral situation:The general condition of rats in CG group was normal,the body weight continued to increase steadily.Rats in MG group took less water and food,were depressed and less responsive to external stimuli,and had dry hair and lost hair,these conditions lasted for about 2 weeks and then gradually returned to normal,and were basically the same as the CG group by the 4th week;the body weight decreased significantly during the modeling week(within the first week),followed by slow increase in the next 2 weeks,and began to increase significantly after the 3rd week.General condition of rats in IG group was similar to that in MG group during the first week after modeling,but the extent was relatively mild,and it returned to normal from the 2nd week,and it was no different from the CG group in the 3rd week,the change of body weight was similar to that in MG group within modeling week,then continued to increase steadily.There was no significant difference in the body weight of the rats between the groups at the baseline level.During the experimental period,the body weight of the rats in the IG group was greater than that of the MG group and less than that of the CG group(the differences were all statistically significant).2.Serum indices①Level of malondialdehyde(MDA):At 2nd week,MG group>CG group(*P<0.05);IG group<MG group(&P<0.05),and it has no significant difference from that of CG group(P>0.05)②Level of blood lipidsTC:No statistically significant difference between groups.TG:It was higher in both the MG and IG group than that in the CG group,with statistically significant differences(all P value<0.05);there was no significant difference between the MG and IG group(&P>0.05).③Level of bone metabolism markersBALP:MG group<CG group(*P<0.05);there is no significant difference between CG group and IG group(#P>0.05);there is no significant difference between IG group and MG group(&P>0.05).PINP:MG group<CG group(*P<0.05);IG group<CG group(#P<0.05);there is no significant difference between MG group and IG group(&P>0.05)β-CTX:MG group>CG group(*P<0.05);there is no significant difference between IG group and CG group(#P>0.05);there is no significant difference between IG group and MG group(&P>0.05)3.Detection of femoral head3.1 General appearance of femoral head specimensCG group:the femoral head was structurally intact,without obvious collapse and deformation,with smooth and moist surface cartilage showing milky white color.MG group:compared with the CG group,the femoral head did not show significant collapse and flattening,and the structure was intact,but the cartilage area was rougher and slightly darker,and some specimens showed dark red lesions in the perichondrium area.IG group:structures were intact without obvious collapse and deformation,no significant difference in cartilage texture and color compared to CG group,some specimens showed dark red lesions in the perichondrium area but to a smaller extent and lesser degree compared to MG group.3.2 Bone tissue parameters of the femoral headCG group vs MG group:As for bone volume fraction(BV/TV),bone trabecular thickness(Tb.Th),bone trabecular number(Tb.N),and bone mineral density(BMD),the CG group>MG group(*P<0.05);MG group>CG group in trabecular separation(Tb.Sp)(*P<0.05).CG group vs IG group:There were no significant differences in bone volume fraction(BV/TV),bone trabecular number(Tb.N),and bone mineral density(BMD);CG group>IG group in bone trabecular thickness(Tb.Th)(#P<0.05);CG group<IG group in bone trabecular separation(Tb.Sp)(#P<0.05).MG group vs IG group:In terms of bone volume fraction(BV/TV)and trabecular thickness(Tb.Th),IG group>MG group(&P<0.05);in terms of trabecular separation(Tb.Sp),IG group<MG group(&P<0.05);in terms of trabecular number(Tb.N)and bone mineral density(BMD),there was no significant difference between IG and MG group.3.3 Observation and analysis of femoral head specimen section①HE stainingObservation of pathological changes in the slice:CG group:the tissue structure was intact,with thick bone trabeculae and a small number of fat cells in the surrounding bone marrow cavity and a large number of hematopoietic cells,with no obvious abnormalities in the structural continuity of the bone trabeculae,and arrangement is regular,in which occasional bone empty lacuna existing.MG group:bone trabeculae were significantly thinner and more sparsely arranged than in the CG group,with more interruptions in continuity,significant increase in adipocytes in the bone marrow cavity,and significant increase in bone empty lacuna in the bone trabeculae.IG group:Bone trabeculae thickness was thinner than CG group but thicker than MG group,with sparse arrangement and interrupted continuity,increased adipocytes in the bone marrow cavity,and bone empty lacuna existing in some areas but less than MG group.Rate of bone empty lacuna:MG group>IG group>CG group in terms of rate of bone empty lacuna,all of which were statistically significant.Osteonecrosis rate of femoral head:The comparison of rate of osteonecrosis of the femoral head between MG group and IG group was tested by Fisher’s exact test,and the exact significance(bilateral and unilateral)results were<0.05.Statistically,baicalein intervention treatment was effective in reducing the occurrence of modeling drugsinduced osteonecrosis of the femoral head.②TRAP stainingResults of osteoclast counting:MG group>CG group(*P<0.05);IG group<MG group(&P<0.05);there was no significant statistical difference between IG group and CG group.③TUNEL staining:Rate of apoptotic cell:MG group>IG group>CG group,the differences between groups were all statistically significant.Conclusion:The intervention of baicalein failed to significantly improve the dyslipidemia and promote the osteogenic differentiation of BMMSCs and bone formation in rats,but it effectively reduced the formation of bone empty lacunae in bone tissue and the occurrence of apoptosis;and reduced the number of osteoclasts,inhibited the loss of bone tissue quality and improved bone tissue parameters partly,and effectively reduced the rise of MDA levels in the early stage.In this experiment,baicalein significantly reduced the occurrence of osteonecrosis of femoral head induced by GC,suggesting it is an effective drug which could be used in the early preventative treatment of GIOFH,possible mechanisms could be inhibition of differentiation and formation of osteoclast,and/or induction in apoptosis of mature osteoclast,resulting in decrease of number of osteoclast and its bone resorption,and against excessive GC-induced early oxidative damage by which reducing the occurrence of osteoclast apoptosis,through these effects baicalein could partly protect femoral head from damages induced by overdose of GC.