| Objective:To study the effect of a blocker of integrin from Gloydius Brevicaudus venom(ItgB-Gbv)on experimental osteoporosis animals and investigate its mechanism.Methods:1.Isolated,purified,and identified a blocker of integrin from the Gloydius Brevicaudus venom:a blocker of integrin containing arginine,glycine and aspartic acid(RGD)was isolated and purified from the Gloydius Brevicaudus venom(Gbv)by Superdex 75 gel filtration and HiTrapTM DEAE FF ion exchange according to the protocol of our lab.Born method was used to determine its antiplatelet activity which is considered to be an indicator of antagonistic integrin activity.The purity of the ItgB-Gbv was identified by Tris-Tricine-SDS-PAGE.2.Determination of affinity of ItgB-Gbv to integrin α2bβ3 and αvβ3:OpenSPR technology was used to analyze the binding of ItgB-Gbv to the integrin receptorsα2bβ3 and αvβ3 related to the RGD sequence and determine their affinity.3.Effect of ItgB-Gbv on osteoclasts in vitro:The osteoclast differentiation factor RANKL was used to induce RAW264.7 cell differentiate into osteoclasts,and the effects of ItgB-Gbv on osteoclast differentiation were observed.The changes of cells were observed under inverted phase contrast microscope,and the differentiation and maturation of OCS were observed by tartrate-resistant acid phosphatase(TRAP)staining.4.Preparation of animal model of osteoporosis and anti-osteoporosis effect of ItgB-Gbv in vivo1)The effects of ItgB-Gbv on osteoporosis mice induced by retinoic acid:Sixty three-month-old Kunming mice of clean grade,weighing(30±5)g,half male and half female,were randomly divided into six groups,namely control group(control),model group(model),ItgB-Gbv low,medium and high dose groups and positive drug(VitD3)groups.Except the normal control group,the other groups were given retinoic acid(RA)90mg.kg-1.d-1 by intragastric administration every morning for 14 days.At the same time,the positive control group was given intraperitoneal injection of VitD 33 mg.kg-1,and the ItgB-Gbv low,medium and high dose groups were administration of ItgB-Gbv 1.25mg.kg-1,2.5 mg.kg-1 and 3.75 mg.kg-1 by intraperitoneal injection,respectively,once a day for 14 days.The body weight of mice were monitored during the experiment.After two weeks,the eyeballs were removed for blood collection and serum was separated.Theconcentration of calcium(Ca),phosphorus(P),osteocalcin(BGP),estradiol(E2),alkaline phosphatase(ALP),and tartrate-resistant acid phosphatase(TRAP)in serum were measured.2)Effect of ItgB-Gbv on ovariectomy-induced osteoporosis in rats:Sixty 8-weeks SD rats were randomly apportioned to receive a sham-operation(n=10),or ovariectomy(n=50).The latter group was equally divided as the model(OVX control)or to receive Estradiol Valerate,or ItgB-Gbv 1.25、2.5和3.75mg.kg-1.One month after surgery,after the model was successfully prepared,the corresponding dose of drugs was continuously given for two months.The positive control group rats received weekly gastric irrigation Estradiol Valerate 4.Omg.kg-1;aline was daily administration by intraperitoneal injection of in sham operation group rats and model group rats for 8 weeks;ItgB-Gbv groups were received different dose of ItgB-Gbv by intraperitoneal injection once a day for 8 weeks also.After 8 weeks of treatments,the uterus of the rats was extracted and weighed under the anesthesia condition.Blood samples were collected from the heart and serum were prepared to measure the concentration of calcium(Ca),phosphorus(P),estradiol(E2)and alkaline phosphatase(ALP)in serum and the concentration of calcium(Ca)and phosphorus(P)in urine were measured too.The bone mineral density of lumbar vertebrae L1-5 and left femur were measured by GE Lunar dienergy X-ray absorptiometry(DEXA).The femur was examined by HE staining.Results:1.Physicochemical Properties and Biological Activities of ItgB-Gbv:According to the protocol of isolation and purification of ItgB-Gbv in our research group,eight fractionswere obtained by Superdex 75 gel filtration.The result of the anti-platelet aggregation experiment using the Born method showed that IV peak,which was purified by the HiTrapTM DEAE FF ion exchange chromatographyon AKTA avant protein purification process development system,had strong anti-platelet aggregation activity.Five factions were obtained from the above mentioned ion exchange procress.The first fraction was identified as having anti-platelet aggregation activity.Tris-Triscine-SDS-PAGE was identified as electrophoresis purity.According to its electrophoretic distance,the molecular weight was about 7.4kDa,which was similar to previous reports,denoted as ItgB-Gbv.2.Evaluation the affinity of ItgB-Gbv binding to the integrin α2bβ3 and αvβ3:ItgB-Gbv specifically binds to integrin α2bβ3,a glycoprotein receptor present in platelet membrane,and integrin αvβ3,mainly present in osteoclasts and vascular endothelial cells,with affinity of 1.23 uM and 650 nM,respectively.3.Effect of ItgB-Gbv on osteoclasts differentiationin in vitro:ItgB-Gbv inhibited RANKL-induced differentiation of RAW264.7 cells into mature OCs.No TRAP-positive osteoclast-like cells were observed in the negative control group after 6 days of culture.The TRAP staining color of the positive control group(50 ng/mL RANKL)was the darkest.The number of TRAP staining positive cells in the positive control group,low,medium and high dose groups was 109.00±19.98,83.33±24.91,59.67±2.08 and 37.33±5.51,respectively.It was found that ItgB-Gbv inhibited osteoclastogenesis in a dose-dependent manner(P<0.05 or P<0.01).4.The preventive and therapeutic effects of ItgB-Gbv on osteoporosis model induced by retinoic acid in mice.1)Effects of ItgB-Gbv on general state and body weight of mice:During the intragastric administration of retinoic acid,mice showed varying degrees of abnormal performance,such as reduced food intake,uneasy movement,prone to lying,depressed spirit,arched back,erect hair,daily weight loss and so on,while the normal control group did not appear the above phenomenon,which indicates that the successful modeling and modeling dose of retinoic acid have more serious adverse reactions.ItgB-Gbv 3.75mg.kg-1 had an antagonistic effect on retinoic acid-induced weight loss in mice,ItgB-Gbv 1.25 mg.kg-1 and 2.5mg.kg-1 and vitamin D3 had no obvious effect.2)Effect of ItgB-Gbv on blood biochemical indexes of osteoporosis mice induced by Retinoic Acid:The serum calcium(s-Ca)in the high-conversion osteoporosis model mice induced by retinoic acid decreased from 2.6±0.03 mmol/L to 2.4±0.05 mmol/L,which was significantly different from that in the normal control group(P<0.05).The s-Ca levels in the ItgB-Gbv low,medium,high dose groups and vitamin D3 groups were 2.5±0.06 mmol/L,2.5±0.05 mmol/L,2.6±0.03 mmol/L and 2.7±0.08 mmol/L,respectively.The level was significantly higher than the model group(P<0.05 or P<0.01).The administration of retinoic acid in mice increased s-P and s-ALP,which were significantly different from those in the control group(P<0.01).3)Effect of ItgB-Gbv on bone metabolism related hormone levels in mice:Tartrate-resistant acid phosphatase(TRAP),osteocalcin(BGP)and estradiol(E2)are hormones closely related to the occurrence and development of osteoporosis.The serum s-BGP level of osteoporosis mice induced by retinoic acid was significantly higher than that of the control group(P<0.01).Compared with the model group,VitD3 and different doses of ItgB-Gbv could significantly antagonize the increase of s-BGP level induced by retinoic acid(P<0.01),and ItgB-Gbv decreased the s-BGP level in a dose-dependent manner.Two-way analysis of variance was used to analyze the E2 level in mice,and the results confirmed that gender had no effect on the change of E2.Compared with the control group,the s-E2 level in the model group was significantly decreased(P<0.01).Different doses of ItgB-Gbv made the s-E2 level in mice positively correlated with the dose,especially in the high-dose group,which was even higher than that in the normal control group(P<0.01).Compared with the normal control group,the s-TRAP level in the model group was significantly increased(P<0.01).Compared with the model group,the levels of s-TRAP in VitD3 group and ItgB-Gbv retinoic acid-induced osteoporosis mice were significantly different(P<0.01 or P<0.05).5.Therapeutic effect of ItgB-Gbv on osteoporosis in ovariectomized rats1)Effects of ItgB-Gbv on general status and body weight of OVX rats:After bilateral ovariectomy,rats ’food intake and water intake were reduced,their hairs were erected,their reaction was slow and their movements were less.About one week after operation,the living conditions of rats,such as activity,drinking water,diet,and response to the surrounding environment,gradually returned to normal,the incision healed completely,and there was no infection such as redness,swelling and abdominal distension.There was no significant difference in the body weight of rats in each group before operation(p>0.05).At the end of the experiment(the 90th day),compared with the sham operation group,the body weight of rats in the model group and ItgB-Gbv groups was significantly increased(P<0.01),indicating that the low estrogen level caused weight gain and the modeling was successful.Estradiol valerate(0.4 mg.kg-1)had an antagonistic effect on the weight gain of osteoporosis rats induced by OVX,and significantly reduced the weight of rats(P<0.01).Different doses of ItgB-Gbv had no antagonistic effect on the weight gain of osteoporosis rats induced by OVX.2)Effects of ItgB-Gbv on concentration of Urine Calcium and Phosphorus in OVX Osteoporosis Rats:The whole day urine of rats after the last experiment was collected,and the concentration of Ca and P in the urine were determined,which were compared with those in the sham operation group(U-Ca=1.37±0.63 mmol·L-1;U-P=2.79±0.93mmol·L-1),model group U-Ca was 2.39 ± 0.69 mmol·L-1;U-P was 4.07±1.04 mmol·L-1,and the content was significantly increased(P<0.01);estradiol valerate(U-Ca=1.13±0.63 mmol·L-1;U-P=3.19±0.31 mmol·L-1)and ItgB-Gbv 1.25 mg·kg-1(U-Ca=0.59±0.31 mmol·L-1;U-P=3.23±1.70 mmol.L-1),ItgB-Gbv 2.5 mg.kg-1(U-Ca=1.53±0.91 mmol.L-1;U-P=3.20±1.91mmol.L-1)and ItgB-Gbv 3.75 mg.kg-1(U-Ca=1.61±1.07 mmol.L-1;U-P=3.21±1.52 mmol·L-1),has the effect of anti-osteoporosis rats induced by OVX urinary calcium Ga,P excretion increased(P<0.05 or P<0.01).3)Effect of ItgB-Gbv on serum calcium,phosphorus and alkaline phosphatase concentration in OVX rats:OVX-induced osteoporosis rats serum s-Ca was 2.28±0.162mmol·L-1;s-P was 2.39±0.201 mmol·L-1 and s-Ca was 2.54±0.109 mmol·L-1 in the sham operation group;s-P was 2.46±0.173 mmol·L-1,which was significantly decreased(P<0.05);estradiol valerate(s-Ca=3.02±0.117 mmol·L-1;s-P=2.31±0.20 mmol.L-1)and ItgB-Gbv 1.25mg.kg-1(s-Ca=2.40±0.129 mmol.L-1;s-P=2.47±0.365 mmol·L-1),ItgB-Gbv 2.5 mg·kg-1(s-Ca=2.43±0.113 mmol·L-1;s-P=2.39±0.221 mmol.L-1)and ItgB-Gbv 3.75mg.kg-1(s-Ca=2.37±0.155 mmol.L-1;s-P=2.29±0.108 mmol.L-1).The serum Ca content of osteoporosis rats treated with s-P=2.29±0.108 mmol·L-1 was significantly higher than that of model group(P<0.05 or P<0.01).Compared with the sham operation group,the P content in serum of OVX-induced osteoporosis rats was significantly decreased(P<0.05).In the treatment group,except for the low-dose group,the P content in other groups was decreased(P<0.05 or P<0.01).ALP in serum of OVX-induced osteoporosis rats was 139±27 pg/mL,which was significantly higher than 91±14 pg/mL in the sham operation group(P<0.01).Estradiol valerate was 109±20 pg/mL,ItgB-Gbv low,medium and high dose groups were 116±15 pg/mL,119±13 pg/mL and 120±17 pg/mL,respectively.Compared with the model group,the increase of ALP in OVX-induced osteoporosis rats treated with ItgB-Gbv at different doses and estradiol valerate could be significantly inhibited(P<0.05 or P<0.01),but it could not reach the level of the sham operation group after treatment.4)Effect of ItgB-Gbv on uterus index(IU)and serum estradiol(E2)in OVX rats:Compared with the sham operation group,the IU and s-E2 of the model group were 4.13±0.301 and 8.03±0.810 pg/mL,respectively.The IU and s-E2 of the model group were 1.33±0.210 and 4.08±0.439 pg/mL,respectively.The contents of uterine index(IU)and serum estradiol(E2)were significantly decreased(P<0.01),indicating that the modeling was successful.In estradiol valerate group,IU was 2.41±0.63,s-E2 was 7.13±0.901 pg/mL.Compared with the model group,IU and serum estrogen levels were significantly increased(P<0.01).ItgB-Gbv 1.25,2.50 and 3.75mg.kg-1 treatment group IU were 1.23±0.201,1.39±0.173 and 1.31±0.139,s-E2 were 5.69±0.481 pg/mL,6.11±0.803 pg/mL and 5.90±0.973 pg/mL.ItgB-Gbv had no obvious antagonistic effect on the decrease of uterine index and estrogen level in rats,and the uterine index and estrogen level were similar to those in the model group.5)Effect of ItgB-Gbv on bone mineral density in OVX osteoporotic rats:The BMD of femur in ovariectomized rats was 0.173±0.001 g/cm2,and that of lumbar L1-5 was 0.242±0.013 g/cm2,which were significantly lower than those in sham operation group(BMD of femur was 0.221±0.013 g/cm2,and BMD of lumbar L1-5 was 0.291±0.010 g/cm2;P<0.01),the decrease of BMD in long bone(femur)was more obvious than that in irregular bone(lumbar vertebra).The BMD of estradiol valerate group was 0.210±0.010 g/cm2,and that of lumbar L1-5 was 0.271±0.017 g/cm2.Compared with the model group,the BMD was significantly increased(P<0.01).Using ItgB-Gbv 1.25,2.5 and 3.75mg·kg-1 to treat OVX-induced osteoporosis rats,the BMD of femur were 0.194±0.010 g/cm2,0.193±0.013 g/cm2 and 0.197±0.007 g/cm2,respectively.BMD of lumbar L1-5 was 0.252±0.010 g/cm2,0.248±0.017 g/cm2 and 0.258±0.011 g/cm2,respectively.Compared with the model group,BMD of lumbar vertebrae and femur also increased(P<0.05).6)Effect of ItgB-Gbv on trabecular structure in ovariectomized rats:In the sham operation group,the trabecular bone of the femur was arranged neatly and densely,and the structure was connected with each other.The trabecular bone was coarse and the gap was small.Femoral pathology of OVX rats:bone trabecula of different thickness,number decreased significantly,thickness decreased,and became discontinuous,broken into button shaped fragments,bone marrow cavity became larger.Osteoporosis rats induced by OVX were treated with estradiol valerate.Compared with the model group,bone trabecula was more uniform,closely distributed and fractured.ItgB-Gbv 1.25,2.5 and 3.75 mg·kg-1 were used to treat osteoporosis rats induced by OVX.The number of bone trabeculae increased,the fracture decreased,the bone marrow cavity became smaller and the area of bone trabeculae increased.Conclutions:1.The ItgB-Gbv was obtained according to the previous isolation and purification protocol developed by the team of our lab..It specifically blocked integrin α2bβ3 and αvβ3 with affinity of 1.23uM and 650nM,respectively.2.ItgB-Gbv could inhibit RANKL-induced differentiation of RAW264.7 into mature OCs,which might be related to the binding of ItgB-Gbv to αvβ3.3.ItgB-Gbv could inhibit the formation of osteoporosis induced by retinoic acid of mice.ItgB-Gbv has therapeutic effect on osteoporosis of ovariectomized rats.These effects can possibly be attributed to the result of ItgB-Gbv blocking integrin αvβ3. |
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