益气化瘀补肾方对大鼠肾虚型颈椎病的作用机制

Mechanism of Yiqi Huayu Bushen Recipe in treating cervical syndrome with kidney deficiency in rats

目的:研究益气化瘀补肾方对大鼠肾虚型颈椎病的作用机制。方法:选择3月龄雌性SPF级SD大鼠30只,随机分为正常对照组、肾虚型颈椎病模型组(简称模型组)和益气化瘀补肾方组,每组10只。除正常对照组不施加任何手术外,采用由动静力失衡大鼠颈椎间盘退变模型和去卵巢肾虚模型复合法建立肾虚型颈椎病模型。手术3个月后,益气化瘀补肾方组大鼠予益气化瘀补肾方灌胃治疗1个月。放射免疫法检测血清雌二醇(estradiol,E_2)和血浆环磷酸腺苷(cyclic adenosine monophosphate, cAMP)、环磷酸鸟苷(cyclic guanosine monophosphate,cGMP)含量;锥板法检测血液流变学;流式细胞仪检测血小板表面α-颗粒膜糖蛋白(alpha-granular membrane protein,CD62p)表达;HE染色观察颈椎间盘组织病理学改变,免疫组织化学检测颈椎间盘Ⅱ型胶原蛋白表达情况,并通过实时荧光定量聚合酶链式反应法检测聚集蛋白聚糖(aggrecan-1,Agcl)、Ⅱ型前胶原基因(typeⅡprocollagen gene,Col2a1)、基质金属蛋白酶抑制剂-1(tissue inhibitor of metalloproteinase-1,TIMP-1)、基质金属蛋白酶-13(matrix metalloproteinase-13,MMP-13)mRNA表达。结果:与模型组比较,益气化瘀补肾方组大鼠血清E_2含量升高(P<0.05);血浆cAMP、cGMP含量有所升高,但差异无统计学意义;全血低切黏度、低切还原黏度和CD62p表达百分率下降(P<0.05或P<0.01);颈椎间盘退变有所改善,Miyamoto评分下降(P<0.05),Ⅱ型胶原表达增加;Agcl、Col2a1和TIMP-1 mRNA表达增加(P<0.05,P<0.01),MMP-13 mRNA表达下降(P<0.01)。结论:益气化瘀补肾方可通过调节免疫系统、代谢系统、凝血系统和内分泌系统等指标,改善肾虚型颈椎病证候指标,并延缓椎间盘退变。

Objective： To research the action mechanism of Yiqi Huayu Bushen Recipe （YHBR）, a compound of traditional Chinese herbal medicine, in treating cervical syndrome （CS） with kidney deficiency in rats. Methods： A total of 30 three-month-old female Sprague-Dawley rats were randomly divided into normal control group, CS with kidney deficiency model group （untreated group） and YHBR group, with 10 rats in each group. Rats in the normal control group received no treatment, while rats of the other two groups underwent resection of both cervical muscles and ovaries to establish the model of CS with kidney deficiency. Three months after surgery, rats in the YHBR group were intragastricly administered YHBR for one month. Another one month later, all rats were sacrificed. The content of serum estradiol （E2） was detected by radio-immunoassay; contents of plasma cyclic adenosine monophosphate （cAMP） and cyclic guanosine monophosphate （cGMP） were tested, and the ratio of cAMP/cGMP was also calculated. Hemorheology was detected by Weissenberg＇s method; expression of alpha-granular membrane protein （CD62p） was detected by flow cytometry; HE staining was used to detect the histopathology of cervical intervertebral disc degeneration; type Ⅱ collagen protein was detected by immunohistochemistry and aggrecan-1 （Agcl）, type Ⅱ procollagen gene, Col2al, tissue inhibitor of metalloproteinase-1 （TIMP-1） and matrix metalloproteinase- 13 （MMP-13） mRNAs were detected by fluorescent quantization polymerase chain reaction. Results： Compared with the untreated group, rats in the YHBR group showed an obvious increase in serum E2 content （P〈0.05）, and an increase in plasma cAMP and cGMP content without significant difference; hemorheological parameters and percentage of CD62p expression were significantly decreased in the YHBR group （P〈0. 05, P〈0. 01）. YHBR could improve the degeneration of cervical intervertebral discs, decrease the Miyamoto scores （P〈0. 05）, and increase the type Ⅱ collagen. The expressions of Agcl, Col2al and TIMP-1 mRNAs were significantly increased and MMP-13 mRNA significantly decreased in YHBR group （P〈 0.05, P〈0.01）. Conclusion： YHBR may improve CS with kidney deficiency and delay the degeneration of cervical intervertebral disc by regulating the immune-metabolism system, coagulation system and endocrine system.