左归丸对去卵巢大鼠骨组织中TGF-β1、Smad4信号转导通路的研究

【作者】 吕海波；

【导师】 任艳玲；

【作者基本信息】 辽宁中医药大学 ， 方剂学， 2011， 硕士

【摘要】 目的:基于“肾藏精,主骨生髓”的理论,本实验采取去卵巢造模方法建立大鼠绝经后骨质疏松症(PMOP)模型,以肾虚为PMOP的主要发病原因出发,研究左归丸对PMOP大鼠的防治作用,并从TGF-β/Smad信号转导途径探讨左归丸防治PMOP大鼠的作用机制。材料与方法:选取91只SPF级4.5月龄SD雌性大鼠,按体重随机分层,每层按数量比暂分为三组:正常(Normal)组13只;假手术(Sham)组13只;其余65只采取双侧背部卵巢切除术进行PMOP造模。造模21d后,将去卵巢大鼠按体重随机分为五组:模型空白(OVX)组、尼尔雌醇(N)组、左归丸高剂量[ZG(H)]组、左归丸中剂量[ZG(M)]组、左归丸低剂量[ZG(L)]组,每组均为13只。然后开始灌胃给药:左归丸各剂量组用左归丸混悬液(高、中、低)进行灌胃;尼尔雌醇组为阳性对照组,灌服尼尔雌醇混悬液;正常组、假手术组和模型空白组均灌服蒸馏水,灌胃期间,各组大鼠每周称重1次,并根据体重调整给药量。实验大鼠连续给药120d后取材,截止取材时,除去灌胃期间死亡的大鼠,取材大鼠共82只。取材前禁食禁水24h,大鼠经腹主动脉取血,室温静置2h后,4℃,3500r·min-1离心30min,取血清,分装,于-70℃冰箱保存,除去溶血血清,用于骨代谢指标及性腺激素水平检测的血清,每组大鼠均为8只;取大鼠左后肢股骨远端1/3,剔除软组织及筋膜,置于4%的多聚甲醛固定液中固定保存,用于病理形态学测定;取大鼠左后肢股骨近端1/3部分,剔除软组织及筋膜,用DEPC水处理过的铝箔纸包裹好,做好标记,迅速置入液氮中速冻,之后放入-70℃冰箱保存,以备RT-PCR方法检测骨组织中TGF-β1、Smad4m RNA的表达;取大鼠右后肢股骨,剔除骨组织上附着的肌肉筋膜,保留骨膜,生理盐水冲洗后,用盐水浸湿过的纱布将其包好,放入保鲜袋中,置-20℃冰箱冻存,用于骨密度(BMD)的检测。结果:1、左归丸对骨组织形态计量学参数的影响(1)骨组织形态学检测光镜下观察,正常组、假手术组大鼠骨小梁完整,连接紧密,有规则,相互之间成网状,骨髓腔较小;模型空白组大鼠骨小梁较紊乱,变细,缺少连接,可见到骨小梁断端。左归丸组(高、中、低)及尼尔雌醇组骨小梁有较显著的增生,骨小梁排列整齐,较粗壮,以尼尔雌醇组和左归丸高剂量组较为显著。(2)骨组织形态计量学测定模型空白组大鼠与假手术组比较,骨小梁面积百分比(%Tb·Ar)、骨小梁厚度(Tb·Th)明显降低,骨小梁分离度(Tb·Sp)明显升高,差异显著(P<0.01);去卵巢大鼠灌服左归丸及尼尔雌醇后,与模型空白组比较,左归丸高、中、低剂量组及尼尔雌醇组大鼠的%Tb·Ar、Tb·Th明显升高,Tb·Sp明显降低,差异显著(P<0.01);各用药组之间无显著性差异(P>0.05)。2、左归丸对去卵巢大鼠离体股骨近端1/3骨密度的影响模型空白组大鼠与假手术组相比,离体股骨近端骨密度显著下降(P<0.01);去卵巢大鼠灌服左归丸及尼尔雌醇后,与模型空白组比较,左归丸高、中、低剂量组及尼尔雌醇组大鼠离体股骨近端骨密度显著升高(P<0.01),各用药组之间无显著性差异(P>0.05)。3、左归丸对骨代谢指标的影响(1)左归丸对骨形成指标血清BGP的影响模型空白组大鼠与假手术组相比,血清BGP含量显著升高(P<0.01);去卵巢大鼠灌服左归丸及尼尔雌醇后,与模型空白组比较,左归丸高、中、低剂量组及尼尔雌醇组大鼠血清BGP含量显著下降(P<0.01),其中以左归丸高、中剂量组的下降趋势较强,各用药组之间无显著性差异(P>0.05)。(2)左归丸对骨吸收指标血清TRAP的影响模型空白组大鼠与假手术组相比,血清TRAP含量显著升高(P<0.01);去卵巢大鼠灌服左归丸及尼尔雌醇后,与模型空白组比较,左归丸高、中、低剂量组及尼尔雌醇组大鼠血清TRAP含量显著下降(P<0.01),各用药组之间无显著性差异(P>0.05)。4、左归丸对血清性腺激素的影响模型空白组大鼠与假手术组相比,血清雌二醇(E2)明显下降(P<0.01),促卵泡生成素(FSH)、促黄体生成素(LH)明显升高(P<0.01);灌服左归丸各剂量后, E2水平升高(P<0.05),而FSH、LH明显下降(P<0.05),各用药组之间无显著性差异。5、左归丸对骨组织中TGF-β1、Smad4 mRNA表达的影响去卵巢后,与假手术组比,模型空白组大鼠骨组织中TGF-β1、Smad4 mRNA水平明显降低(P<0.01);灌服左归丸各剂量组后,骨组织中TGF-β1、Smad4 mRNA水平显著上升(P<0.01),各用药组之间无显著性差异(P>0.05)。结论:1、切除大鼠双侧卵巢可成功复制PMOP模型。2、左归丸可以有效防治PMOP。3、骨组织中TGF-β1、Smad4 mRNA表达水平的下调可能是PMOP发生的重要机制之一。4、左归丸能够上调骨组织中TGF-β1、Smad4 mRNA的表达,纠正PMOP骨代谢失衡,其防治PMOP的作用机制可能与其干预TGF-β1、Smad4 mRNA的表达有关。更多还原

【Abstract】 Purpose:To research the effect of Zuogui Pill on preventing and curing postmenopausal osteoporosis (PMOP),through the PMOP modeled rats Ovariectomized. Consequently,to dwas cuss the therapeutic mechanwas m on signal transduction pathway of TGF-β/Smad. All those practice have been made on baswas of the theory of kindey storing the body essence and in charge of bone and generating marrow. Material and method:Divided 91 SD female rats at 4.5 months to 3 groups temporarily:Normal group with 13 rats,Sham group with 13 rats,for the rest 65 rats were eccoped both ovaries to establish PMOP models. 21 days later,divided the model rats into 5 groups randomly:OVX,N,ZG(H),ZG(M),ZG(L),13 rats per group. ZG(H),ZG(M), ZG(L) were ig. with Zuogui Pill suspl.(high,middle,low dose),N was ig. with Nilestriol as postive control group,Normal,Sham,OVX were ig. with dwas tilled water. Rats were weighten per week to regulate the dose. 120d later,except the dead rats during ig.,84 rats were cut off food and water for 24h,then killed,collected blood from abdominal aorta that was standing for 2h and centrifuged to get serum to test hebin level,stored at -70℃,the left hind limb at the 1/3 of dwas tal femur was taken to test test the expression of TGF-β1、Smad4 mRNA in bone tissues,which should be removed soft tissue and fascia,than treated by DEPC and packaged with aluminium foil,quick freezed in liquid nitrogen and stored at -70℃;The femoral bone of right hind limb was taken to determine the bone mineral density (BMD),which should be removed muscls and fascia but peritosteum,water flushed and packaged with saline water sinked gauze in refreshing bags,stored at -20℃.Results:1. The effect of Zuogui Pill on morphometry parameter of bone(1) Histomorphology of bone tissue Under the light microscope, the bone trabecula of Normal, Sham is complete, close linked in order,net shaped with small cavitas medullaris; bone trabecula of OVX changes to be disorder,thinner,relatively sparse,lack of connection,and with more fracture;the medicine treated rats of N,ZG(H),ZG(M),ZG(L) has more hyperplasy,linked in order and strong,specially the N and ZG(L).(2) Morphometry of bone tissueCompared with Sham, the %Tb·Ar and Tb·Th of OVX rats decreased,Tb·Sp increased significently(P<0.01);compared with OVX,%Tb·Ar and Tb·Th of medicine treated rats increased,and Tb·Sp decreased significantly(P<0.01) without differences between every two groups(P>0.05).2. The effect of Zuogui pill on the BMD of right hind limb at the 1/3 of proximate caput femorisCompared with Sham, the BMD of proximate caput femoris of OVX rats decreased significantly (P<0.01);compared with OVX,the the BMD of proximate caput femoris of medicine treated rats increased significantly (P<0.01),without differences between every two groups (P>0.05).3. The effect of Zuogui Pill on bone metabolism(1) The effect on BGPCompared with Sham,BGP of OVX increased significantly(P<0.01);compared with OVX,BGP of medicine treated rats decreased significantly(P<0.01),specially the ZG(M),ZG(L) but differences (P>0.05).(2) The effect on TRAPCompared with Sham,TRAP of OVX increased significantly (P<0.01);compared with OVX, BGP of medicine treated rats decreased significantly (P<0.01),without differences between every two groups(P>0.05).4. The effect of Zuogui Pill on sex hormonesCompared with Sham,E2 of OVX decreased significantly(P<0.01),FSH,LH increased significantly(P<0.01); compared with OVX,E2 of medicine treated rats increased(P<0.05),and FSH,LH increased(P<0.05) without differences between every two groups. 5. The effect of Zuogui Pill on expression of TGF-β1、Smad4 mRNACompared with Sham, expression of TGF-β1、Smad4 mRNA of OVX decreased significantly(P<0.01);compared with OVX,the expression of medicine treated rats increased significantly(P<0.01),without differences between every two groups(P>0.05).Conclusion:1. Eccope the both ovaries of rats can establish the PMOP models.2. Zuogui Pill can prevent and cure PMOP effectively.3. The down regulation of TGF-β1 and Smad4 mRNA in bone tissues may be the one important machanism of PMOP attacking.4. Zuogui Pill can up regulate the TGF-β1 and Smad4 mRNA in bone tissues, rebalance the bone metabolism, and that may be related to the mechanism of therapy action.更多还原