二仙汤在绝经后骨质疏松症肾阳虚证治疗中的应用价值及作用机制研究

Applied values and mechanism of action of oral application of Erxian Tang(二仙汤)in treatment of postmeno-pausal osteoporosis with syndrome of kidney-yang deficiency:a clinical study

目的:探讨二仙汤在绝经后骨质疏松症肾阳虚证治疗中的应用价值,并探讨其作用机制。方法:将90例绝经后骨质疏松症肾阳虚证患者随机分为2组,每组45例,分别采用口服二仙汤联合碳酸钙D3和阿法骨化醇软胶囊(二仙汤组)与单纯碳酸钙D3和阿法骨化醇软胶囊(基础用药组)治疗。碳酸钙D3,每日1次,每次1片;阿法骨化醇软胶囊,每日1次,每次1粒;二仙汤,每日2次,每次1袋,早晚服用;均连续服用12周。分别于治疗前和治疗结束后,采用疼痛视觉模拟量表(visual analogue scale,VAS)评分评价腰背部疼痛情况,采用双能X线骨密度仪测定患者腰椎(L_(1)~L_(4))骨密度及股骨颈骨密度,采用酶联免疫吸附法测定血清骨钙素、Ⅰ型前胶原氨基端前肽(N-terminal propeptide of typeⅠprecollagen,PⅠNP)及Ⅰ型胶原羧基端肽β特殊序列(β C-terminal telopeptide of typeⅠcollagen,β-CTX)含量,采用化学发光法测定血钙及血磷含量,采用荧光定量PCR法测定血清miR-335-5p的表达量。结果:①受试者退出情况。共10例患者退出试验,其中二仙汤组3例因未能按时按计量服药退出,2例因失访退出;基础用药组1例因未能按时按计量服药退出,4例因失访退出。②腰背部疼痛VAS评分。治疗前,2组患者腰背部疼痛VAS评分比较,差异无统计学意义[(4.03±0.80)分,(3.90±0.93)分,t=0.645,P=0.521];治疗结束后,二仙汤组腰背部疼痛VAS评分低于基础用药组[(2.10±0.87)分,(2.98±1.10)分,t=-3.526,P=0.001],2组患者腰背部疼痛VAS评分均低于治疗前(t=14.198,P=0.000;t=7.656,P=0.000)。③骨密度。治疗前与治疗结束后,2组患者腰椎骨密度和股骨颈骨密度比较,组间差异均无统计学意义[腰椎:(0.886±0.040)g·cm^(-2),(0.880±0.030)g·cm^(-2),t=0.746,P=0.458;(0.888±0.040)g·cm^(-2),(0.878±0.030)g·cm^(-2),t=0.994,P=0.323。股骨颈:(0.763±0.070)g·cm^(-2),(0.767±0.070)g·cm^(-2),t=-0.263,P=0.794;(0.765±0.070)g·cm^(-2),(0.770±0.070)g·cm^(-2),t=-0.927,P=0.360];治疗结束后2组患者腰椎骨密度和股骨颈骨密度与治疗前比较,差异均无统计学意义(腰椎:t=-1.099,P=0.281;t=0.701,P=0.492。股骨颈:t=-1.640,P=0.109;t=-0.927,P=0.360)。④骨代谢生化指标。治疗前,2组患者血清骨钙素含量比较,差异无统计学意义[(14.09±3.97)ng·mL^(-1),(15.56±3.67)ng·mL^(-1),t=-1.070,P=0.092];治疗结束后,2组患者血清骨钙素含量的差异无统计学意义[(16.14±3.67)ng·mL^(-1),(15.58±4.74)ng·mL^(-1),t=0.602,P=0.549],二仙汤组患者血清骨钙素含量高于治疗前(t=-11.325,P=0.000),基础用药组患者血清骨钙素含量与治疗前的差异无统计学意义(t=0.045,P=0.964)。治疗前,2组患者血清PⅠNP含量比较,差异无统计学意义[(38.08±11.90)ng·mL^(-1),(36.90±9.80)ng·mL^(-1),t=0.484,P=0.630];治疗结束后,二仙汤组患者血清PⅠNP含量高于基础用药组[(45.96±13.38)ng·mL^(-1),(35.43±12.15)ng·mL^(-1),t=3.684,P=0.000],二仙汤组患者血清PⅠNP含量高于治疗前(t=-10.795,P=0.000),基础用药组患者血清PⅠNP含量与治疗前的差异无统计学意义(t=1.564,P=0.126)。治疗前与治疗结束后,2组患者血清β-CTX含量比较,组间差异均无统计学意义[(0.36±0.10)ng·mL^(-1),(0.36±0.09)ng·mL^(-1),t=0.140,P=0.889;(0.38±0.11)ng·mL^(-1),(0.37±0.10)ng·mL^(-1),t=0.499,P=0.619];治疗结束后2组患者血清β-CTX含量与治疗前比较,差异均无统计学意义(t=-1.279,P=0.209;t=-1.004,P=0.322)。治疗前与治疗结束后,2组患者血磷、血钙含量比较,组间差异均无统计学意义[(血磷:(1.27±0.14)mmol·L^(-1),(1.23±0.12)mmol·L^(-1),t=1.415,P=0.161;(1.25±0.08)mmol·L^(-1),(1.23±0.12)mmol·L^(-1),t=1.277,P=0.206。血钙:(2.33±0.08)mmol·L^(-1),(2.32±0.07)mmol·L^(-1),t=0.659,P=0.512;(2.34±0.06)mmol·L^(-1),(2.35±0.06)mmol·L^(-1),t=-0.514,P=0.608];治疗结束后2组患者血磷、血钙含量与治疗前比较,差异均无统计学意义(血磷:t=0.799,P=0.419;t=0.197,P=0.845。血钙:t=-0.401,P=0.690;t=-1.552,P=0.129)。⑤血清miR-335-5p表达量。治疗前,2组患者血清miR-335-5p的表达量比较,差异无统计学意义(1.04±0.72,1.06±0.66,t=-0.081,P=0.936);治疗结束后,二仙汤组血清miR-335-5p的表达量高于基础用药组(7.71±1.94,1.36±0.83,t=14.520,P=0.000),且高于治疗前(t=-17.289,P=0.000),基础用药组血清miR-335-5p的表达量与治疗前的差异无统计学意义(t=-1.279,P=0.216)。结论:口服二仙汤有利于缓解绝经后骨质疏松症肾阳虚证患者的腰背痛和促进骨形成,其作用机制可能与上调miR-335-5p的表达有关。

Objective:To explore the applied values of oral application of Erxian Tang(二仙汤,EXT)in treatment of postmenopausal osteoporosis(PMOP)with kidney-yang deficiency syndrome(KYDS),and to explore its mechanism of action.Methods:Ninety PMOP pa-tients with KYDS were enrolled in the study and were randomly divided into EXT group and basic medication group,45 cases in each group.The patients in EXT group were treated with oral applications of EXT(twice a day in the morning and evening respectively,1 bag at a time),calcium carbonate and Vitamin D3 tablets(once a day,1 tablet at a time)and alfacalcidol soft capsules(once a day,1 capsule at a time)for consecutive 12 weeks;while the ones in basic medication group were merely with oral applications of calcium carbonate and Vita-min D3 tablets and alfacalcidol soft capsules for consecutive 12 weeks.The low back pain was evaluated by using pain visual analogue scale(VAS)score,and the bone mineral density(BMD)of lumbar vertebra(LV)from L to L4 and femoral neck(FN),the serum levels of osteo-calcin(OCN),N-terminal propeptide of type I precollagen(P I NP)andβC-terminal telopeptide of type I collagen(β-CTX),the lev-els of serum calcium(Ca)and serum phosphorus(P)and the expression level of serum miR-335-5p were detected by using dual-energy X-ray absorptiometry(DEXA),enzyme linked immunosorbent assay(ELISA),chemiluminescence immunoassay(CLIA)and fluorescent quantitative PCR method respectively before the treatment and after the end of the treatment.Results:①Three patients in EXT group and 1 case in basic medication group dropped out of the study for failing to take medication as required,while 2 cases in EXT group and 4 cases in basic medication group dropped out of the study for losing to follow-up.②There was no statistical difference in low back pain VAS score between the 2 groups before the treatment(4.03±0.80 vs3.90±0.93 points,t=0.645,P=0.521).The low back pain VAS scores were lower in EXT group compared to basic medication group after the end of the treatment(2.10±0.87 vs 2.98±1.10 points,t=-3.526,P=0.001),and it decreased after the end of treatment compared to pretreatment in the 2 groups(t=14.198,P=0.000;t=7.656,P=0.000).③There was no statistical difference in BMD of LV from L to L4 and FN between the 2 groups before the treatment and after the end of the treatment(LV:0.886±0.040 Vs 0.880±0.030 g/cm(2),t=0.746,P=0.458;0.888±0.040 vs 0.878±0.030 g/cm(2),t=0.994,P=0.323.FN:0.763±0.070 vs 0.767±O.070 g/cm(2),t=-0.263,P=0.794;0.765±0.070 vs 0.770±0.070 g/cm(2),t=-0.927,P=0.360),and there was no statistical difference between the 2 timepoints in the 2 groups(LV:t=-1.099,P=0.281;t=0.701,P=0.492.FN:t=-1.640,P=0.109;t=-0.927,P=0.360).④There was no statistical difference in serum level of OCN between the 2 groups before the treatment and after the end of the treatment(14.09±3.97 vs 15.56±3.67 ng/mL,t=-1.070,P=0.092;16.14±3.67 Vs 15.58±4.74 ng/mL,t=0.602,P=0.549).The serum level of OCN was higher after the end of treatment compared to pretreatment in EXT group(t=-11.325,P=0.000),while the differences in serum level of OCN were not statisti-cally significant between the 2 timepoints in basic medication group(t=0.045,P=0.964).There was no statistical difference in serum lev-el of PI NP between the 2 groups before the treatment(38.08±11.90 Vs 36.90±9.80 ng/mL,t=0.484,P=0.630).The serum level of PINP was higher in EXT group compared to basic medication group after the end of treatment(45.96±13.38 vs 35.43±12.15 ng/mL,t=3.684;P=0.000),and it was higher after the end of treatment compared to pretreatment in EXT group(t=-10.795,P=0.000),while there was no statistical difference between the 2 timepoints in basic medication group(t=1.564,P=0.126).There was no statistical difference in serum level of β-CTX between the 2 groups before the treatment and after the end of treatment(O.36±0.10 Vs 0.36±0.09 ng/mL,t=0.140,P=0.889;0.38±0.11 vs0.37±0.10 ng/mL,t=0.499,P=0.619),and there was no statistical difference in serum level of β-CTX between the 2 timepoints in the2 groups(t=-1.279,P=0.209;t=-1.004,P=0.322).There was no statisti-cal difference in the levels of serum P and serum Ca between the 2 groups before the treatment and after the end of treatment(serum P:1.27±0.14 vs 1.23±0.12 mmol/L,t=1.415,P=0.161;1.25±0.08 vs1.23±0.12 mmol/L,t=1.277,P=0.206.serum Ca:2.33±0.08 vs 2.32±0.07 mmol/L,t=0.659,P=0.512;2.34±0.06 vs2.35±0.06 mmol/L,t=-0.514,P=0.608),and there was no sta-tistical difference in levels of serum P and serum Ca between the 2 timepoints in the 2 groups(serum P:t=0.799,P=0.419;t=0.197,P=0.845.serum Ca:t=-0.401,P=0.690;t=-1.552,P=0.129).⑤There was no statistical difference in the expression level of ser-um miR-335-5p between the 2 groups before the treatment(1.04±0.72 vs1.06±0.66,t=-0.081,P=0.936).The expression level of serum miR-335-5p was higher in EXT group compared to basic medication group after the end of treatment(7.71±1.94 vs 1.36±0.83,t=14.520,P=0.000),and it was higher after the end of treatment compared to pre-treatment in EXT group(t=-17.289,P=0.000),wheres there was no statistical difference in the expression level of serum miR-335-5p between the 2 timepoints in basic medi-cation group(t=-1.279,P=0.216).Conclusion:Oral application of EXT is helpful to relieve low back pain and promote bone formation in PMOP patients with KYDS,and its mechanism of action may be that it can up-regulate the expression of miR-335-5p.