Intervention of Bushen Yizhi formula on cognitive disorder rat and the related mechanism of the protective function on neural network

OBJECTIVE Bushen Yizhi formula,constructed by Prof LIAO Shi-long′research group(Guangzhou University of chinese medicine),is the combination of clinical experience and modern pharmacological research,and mainly focuses on the etiology and pathogenesis of AD for treating both cause and symptoms.In this research,the pharmacodynamic study protocol of Bushen Yizhi formula was designed according to the characteristics and indications function of herbs in the formula,and was conformed to preclinical pharmacological research of traditional Chinese medicine for AD,The Technical Requirements of Pharmacology and Toxicology Research in New Drug Development Enacted by State Food and Drug administration also was guiding principle.METHODS A preliminary randomized double-blind clinical trail with 141 cases of AD showed that the efficiency of Bushen Yizhi formula was 61.9%and the increased mean of MMSE was 3.17,but the specific mechanism remains unclear.This study was aimed to reveal the preventive and therapeutic effect of Bushen Yizhi formula on AD rats and its related mechanism.In this research,forebrain-injected IBO-induced AD rats(cholinergic neuron lesion),senescence accelerated mouse,scopolamineinduced learning and memory deficiency rats(mild cognitive impairment)were all established.The learning and memory ability was tested with Morris water maze.Then formation of age pigment,extent of neuronal loss,activation of astrocytes,content of NTFs and degree of oxidized stress damage were determined by morphology,mmunohistochemical and molecular biology methods.RESULTS As the application of Bushen Yizhi formula,the learning and memory ability in all three groups were significantly improved,the formation of age pigment and the content of ACH in cortex and hippocampus were reduced,the activation of astrocytes and release of inflammatory factor(TNF-αand IL-1β)were inhibited,and the antioxidases(CAT,SOD,GSH-PX)were up-regulated and MDA was down-regulated.CONCLUSION Bushen Yizhi formula can prevent and treat AD rats,which might be achieved by anti-inflammatory,antioxidant and protecting cholinergic neuron.

Bushen Yizhi Formula regulates the IRE1αpathway to alleviate endoplasmic reticulum stress in an Alzheimer’s disease rat model

While the Bushen Yizhi Formula can treat Alzheimer’s disease(AD),the yet to be ascertained specific mechanism of action was explored in this work.Methods:Different concentrations of the Bushen Yizhi Formula and amyloid-beta peptide(Aβ)were used to treat rat pheochromocytoma cells(P12)and human neuroblastoma cells(SH-SY5Y).Cell morphological changes were observed to determine the in vitro cell damage.Cell Counting Kit(CCK)-8 assay and flow cytometry were employed to identify cell viability and apoptosis/cell cycle,respectively.Western blotting and immunohistochemistry were employed to measure the expressions of endoplasmic reticulum stress(ERS)-related proteins(GRP78 and CHOP),p-IRE1α,IRE1α,ASK1,p-JNK,JNK,Bax,Bcl-2,XBP-1,and Bim.Fura 2-acetoxymethyl ester(Fura-2/AM)was used to determine the intracellular calcium(Ca^(2+))concentration.Also,an AD model was constructed by injecting Aβinto the CA1 area of the hippocampus in Sprague Dawley rats.AD model rats were gavaged with different concentrations of Bushen Yizhi Formula for 14 consecutive days.The Morris water maze experiment was conducted to test the learning and memory of rats.Hematoxylin&Eosin(H&E)and Terminal-deoxynucleotidyl Transferase(TdT)-mediated dUTP Nick-End Labeling(TUNEL)staining were done to determine histopathological changes in the brain.Results:Bushen Yizhi Formula relieved the Aβ-induced effects including cell injury,decreased viability,increased apoptosis,G0/G1 phase cell cycle arrest,upregulation of GRP78,CHOP,p-IRE1α,p-JNK,Bax,XBP-1 and Bim,as well as down-regulation of Bcl-2.These results were also seen with IRE1αsilencing.While Aβsuppressed the learning and memory abilities of rats,the Bushen Yizhi Formula alleviated these effects of Aβ.Brain nerve cell injury induced by Aβcould also be treated with Bushen Yizhi Formula.Conclusion:Bushen Yizhi Formula could influence ERS through the IRE1αsignaling pathway to achieve its therapeutic effects on AD.

Mechanism of treating recurrent abortion with “Peiyuan Bushen Antai formula” based on network pharmacology and molecular docking

Objective:To study the effective compound action target signal pathway of Peiyuan bushenan taifang(PYBSATF)has achieved good clinical efficacy in the treatment of recurrent spontaneous abortion(RSA),but its mechanism has not been clarified because of its complex components.In this study,network pharmacology is applied to study the effective compounds,targets and signal pathways of PYBSATF in the treatment of RSA,so as to reveal its pharmacological mechanism of action in the treatment of recurrent spontaneous abortion.Methods:The Traditional Chinese Medicine Systems Pharmacology database(TCMSP)and CNKI are used to obtain the main compounds and drug action targets of PYBSATF.Genecards and the Online Mendelian Inheritance of Man databases(OMIM)are searched to collect the known genes related to RSA,so as to construct compound-target network and screen out the common target proteins and main active compounds.We also use string database to construct a visual protein-protein interaction network(PPI).Cluster Profiler and R software were used to analyze the common targets of drugs and diseases for GO function analysis and KEGG pathway enrichment analysis.Finally,the compound and the protein sequences were conducted according to the node parameters,so that the core protein and core compounds are used to perform molecular docking.Results:186 potential active components and 65 predicted action targets of PYBSATF were screened out.At the same time,1658 genes were also screened out to be closely related to RSA,among which 65genes overlaped with PYBSATF targets and were considered to be related to way of treatment.PPI network showed that VEGFA,IL6,EGFR,MAPK8 and ESR1were the core targets of PYBSATF for the treatment of RSA.GO and KEGG enrichment analysis obtained 93 biological processes of cells(P<0.01)and 87 signaling pathways(P<0.01).PYBSATF played a pharmacological role through a variety of pathways including anti-inflammatory,anti-apoptosis,promoting proliferation and angiogenesis.Molecular docking showed that most active components and key targets of PYBSATF had strong efficiency.Conclusion:Through the study of network pharmacology,it predicted that PYBSATF might treat RSA through multiple targets and multiple signal pathways.Significantly,the predictive targets may be potential targets for treatment of RSA.

Expression of Nrf2/ARE pathway in diabetic nephropathy and renal protection of Qihuang Bushen Xiezhuo Formula

Objective:To investigate the effect of Qihuang Bushen Xiezhuo Formula on the expression of the nuclear factor E2 related factor 2(Nrf2)/antioxidant response element(ARE)signaling pathway of human glomerular mesangial cells(HMC)in high glucose medium and its protective effect on oxidative stress.Methods:The HMC were cultured in vitro to prepare normal rat serum.The rats were intragastrically administered with irbesartan and Qihuang Bushen Xiezhuo Formula.The serum containing the drugs was prepared after the blood concentration was reached.The rats were divided into the control group,the high glucose group,the irbesartan group and the Qihuang Bushen Xiezhuo Formula group.The HMC of the control group was cultured with normal rat serum(10%serum concentration)medium,while that of the high glucose group was cultured with high glucose medium of rat serum(10%serum concentration).The irbesartan group and the Qihuang Bushen Xiezhuo Formula group were respectively treated with 10%irbesartan and 10%Qihuang Bushen Xiezhuo Formula in serum high glucose medium.After 48 h of culture,the relevant indicators were collected and detected.The mRNA expression levels of Nrf2,gamma-glutamylcysteine synthetase(γ-GCS)and superoxide dismutase(SOD)in each group were detected by real-time PCR.The expressions ofγ-GCS and SOD were detected by immunohistochemistry.The protein expressions ofγ-GCS and SOD in HMC of each group were observed by Western Blot.Results:The expressions of Nrf2,γ-GCS,SOD mRNA and protein in experimental cells of each group were low in the control group,while those in the high glucose group were decreased as compared with the control group(P<0.05).After interference of irbesartan and Qihuang Bushen Xiezhuo Formula the expressions were increased,and the increase in Qihuang Bushen Xiezhuo Formula group was more significant(P<0.05).Conclusion:Qihuang Bushen Xiezhuo Formula can improve HMC oxidative stress injury in high glucose culture,and its mechanism may be achieved by activating Nrf2 and its related downstream proteinsγ-GCS and SOD.

补肾益冲方对卵巢储备功能减退大鼠卵巢组织形态及性激素的影响

目的:研究补肾益冲方对卵巢储备功能减退(DOR)模型大鼠卵巢功能的影响。方法:选取雌性未孕SD大鼠40只,随机分为空白组、模型组、补肾益冲方组、补佳乐组。除空白组外,其余大鼠采用雷公藤多苷片混悬液灌胃制备DOR大鼠模型,造模成功后分别给予补肾益冲方及补佳乐进行相应的治疗干预。药物干预结束后,采用酶联免疫吸附试验法(ELISA)检测大鼠血清中抗苗勒管激素(AMH)、促卵泡生成素(FSH)、雌二醇(E_(2))、促黄体生成素(LH)的水平;HE染色观察大鼠卵巢组织的病理学形态变化。结果:与空白组比较,模型组大鼠血清FSH和LH水平升高(P<0.05),AMH和E_(2)水平降低(P<0.05);卵巢中原始卵泡和生长卵泡的数量减少,闭锁卵泡增加;卵巢间质增生并纤维化。补肾益冲方干预后,DOR大鼠血清中FSH和LH激素水平降低(P<0.05),AMH和E_(2)激素水平升高(P<0.05);卵巢组织形态得到改善,原始卵泡和生长卵泡增多,闭锁卵泡减少。结论:补肾益冲方对DOR大鼠卵巢组织形态、卵母细胞的数量和质量具有改善作用,能调节AMH、FSH、E_(2)和LH从而提高DOR大鼠的卵巢储备功能。

补肾活血方调控NF-κB信号通路对膝骨关节炎小鼠软骨组织炎症及细胞凋亡的影响

目的观察补肾活血方(BSHXF)对膝骨关节炎(KOA)小鼠软骨组织炎症及细胞凋亡的影响,探讨BSHXF治疗KOA的作用机制。方法将24只雄性BALB/c小鼠按体重随机分为假手术组、KOA模型组、KOA模型+BSHXF组,每组8只。造模、灌胃干预后,小鼠取血,ELISA检测血清IL-6、iNOS、COX2水平;提取小鼠膝关节软骨组织,HE染色及番红O-固绿染色观察软骨组织病理学情况,TUNEL染色检测软骨组织细胞凋亡情况,Western blot检测Cleaved-Caspase-9、Bcl-2、Bax蛋白表达,qRT-PCR检测CollagenⅡ、Aggrecan、ADAMTS-5 mRNA表达,Western blot检测核转录因子-κB(NF-κB)信号通路关键蛋白p-IκBα、IκBα、p-p65、p65的表达。结果与假手术组相比,KOA模型组小鼠膝关节软骨细胞病理改变明显;血清炎症细胞因子IL-6、iNOS和COX2的水平升高,软骨组织细胞凋亡率增加,Cleaved-Caspase-9、Bax蛋白表达水平升高,Bcl-2蛋白表达降低,CollagenⅡ、Aggrecan的mRNA表达水平降低,ADAMTS-5 mRNA表达升高,p-IκBα/IκBα、p-p65/p65比值升高。与KOA模型组相比,KOA模型+BSHXF组膝关节软骨损伤改善、病理变化减轻,炎症细胞因子IL-6、iNOS和COX2的水平降低,软骨组织细胞凋亡率降低,Cleaved-Caspase-9、Bax蛋白表达降低,Bcl-2蛋白表达升高,CollagenⅡ、Aggrecan的mRNA表达升高,ADAMTS-5 mRNA表达降低,p-IκBα/IκBα、p-p65/p65比值降低。结论BSHXF可抑制NF-κB信号通路,减少炎性因子释放、抑制软骨细胞凋亡及细胞外基质降解,从而缓解KOA疾病进展。

补肾健脾方治疗卵巢储备功能减退相关不孕脾肾两虚证临床观察

目的:研究补肾健脾方治疗卵巢储备功能减退相关不孕脾肾两虚证的临床疗效。方法:纳入60例卵巢储备功能减退相关不孕脾肾两虚证患者,采用随机数字法分成治疗组和对照组,每组30例。治疗组患者给予补肾健脾方治疗,对照组患者给予芬吗通治疗,两组均连续治疗6个周期。检测性激素指标[基础卵泡刺激素(bFSH)、基础黄体生成素(bLH)、血清雌二醇(E2)、抗米勒管激素(AMH)]、超声指标[卵巢体积、窦卵泡(AFC)数量],比较两组患者中医证候积分、总有效率、妊娠率、流产率,并监测安全性指标。结果:治疗组总有效率为79.31%(23/29),对照组总有效率为82.14%(23/28),差异无统计学意义(P>0.05)。经过6个月经周期治疗后,两组治疗后中医证候积分、bFSH均较治疗前下降(P<0.05),AMH较治疗前升高(P<0.05),且治疗组优于对照组(P<0.05)。治疗组治疗后bLH较治疗前下降(P<0.05);对照组治疗前后bLH比较,差异无统计学意义(P>0.05)。两组治疗后E2较治疗前有明显升高(P<0.05),两组患者治疗后E2比较,差异无统计学意义(P>0.05)。治疗组治疗后卵巢体积明显增大(P<0.05),窦卵数量明显增多(P<0.05),治疗组明显优于对照组(P<0.05)。治疗组妊娠率[34.48%(10/29)]高于对照组[21.43%(6/28)](P<0.05),治疗组流产率[10.00%(1/10)]低于对照组[33.30%(2/6)(P<0.05)]。两组患者治疗前后安全性指标未发现异常,未发生不良反应。结论:补肾健脾方能安全有效地治疗卵巢储备功能减退相关不孕脾肾两虚证,改善卵巢相关激素水平,提高妊娠率,降低流产率,缓解临床症状。

补肾活血膏方对肾虚血瘀证强直性脊柱炎患者的临床疗效

目的 探讨补肾活血膏方对肾虚血瘀证强直性脊柱炎患者的临床疗效。方法 167例患者随机分为对照组(常规治疗,55例)、暴露组(常规治疗+补肾活血汤剂,54例)、高暴露组(常规治疗+补肾活血汤剂+补肾活血膏方,58例),疗程2年。检测临床疗效、BASDAI评分、ASDAS-CRP、BASFI评分、脊柱痛评分、PGA评分、BASMI评分、ASQoL评分、SPARCC评分、肾虚血瘀证评分、ESR、CRP、IL-6、TNF-α、IL-17、IL-23、IL-35、NLR、PLR、安全性指标变化。结果 高暴露组ASAS40、ASAS5/6、BASDAI50例数多于暴露组和对照组(P<0.05)。治疗后,高暴露组BASDAI评分、ASDAS-CRP、BASFI评分、脊柱痛评分、PGA评分、BASMI评分、SPARCC评分、ASQoL评分、肾虚血瘀证评分、ESR、CRP、IL-6、TNF-α、IL-17、IL-23低于其他2组(P<0.05),IL-35更高(P<0.05)。经Logistic回归分析校正混杂因素后,补肾活血汤、补肾活血膏方降低BASDAI评分和ASDAS-CRP(P<0.05),提高临床疗效(P<0.05)。3组未发现严重不良反应。结论 补肾活血膏方可安全有效地抑制肾虚血瘀证强直性脊柱炎患者炎症,降低疾病活动度,减轻骨髓水肿,改善临床症状,提高关节功能和生活质量。

基于JNK通路探讨补肾活血方对大鼠骨关节炎软骨细胞凋亡的影响

目的 探讨补肾活血方(Bushen Huoxue Formula,BSHXF)通过c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)信号通路对大鼠骨关节炎(Osteoarthritis,OA)软骨细胞凋亡的影响。方法 采用白细胞介素-1β (interleukin-1β,IL-1β)诱导体外分离培养的大鼠软骨细胞构建体外OA模型,并使用不同浓度的BSHXF干预处理。使用MTT法测定细胞活力;CCK-8法检测细胞增殖能力;流式细胞术评估细胞凋亡情况;Western bolt检测凋亡相关蛋白裂解的胱天蛋白酶-3(Cleaved Caspase-3)、B淋巴细胞瘤-2(B-cell lymphoma 2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2-associated X protein,Bax)水平和JNK、磷酸化c-Jun氨基末端激酶(phosphorylated c-Jun N-terminal kinase,p-JNK)蛋白水平。试剂盒检测细胞中活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)、谷胱甘肽(Glutathione,GSH)水平;ELISA法检测细胞上清液中促炎因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)含量。结果 (1)与对照组相比,模型组软骨细胞活力显著降低,细胞凋亡率显著升高(P<0.01);模型组Cleaved Caspase-3、Bax、p-JNK蛋白表达水平显著升高(P<0.01),Bcl-2蛋白表达水平显著降低(P<0.01)。与模型组相比,BSHXF治疗组软骨细胞活力升高(P<0.05),凋亡率显著降低(P<0.01);BSHXF治疗组Cleaved Caspase-3、Bax、p-JNK蛋白表达水平显著降低(P<0.01),Bcl-2蛋白表达水平升高(P<0.01)。(2)与对照组相比,模型组细胞中ROS和MDA的相对含量显著升高(P<0.01),GSH的相对含量显著降低(P<0.01);模型组细胞中TNF-α和IL-6的含量均显著升高(P<0.01)。与模型组相比,BSHXF治疗组细胞中ROS和MDA的相对含量显著降低(P<0.01),GSH的相对含量显著升高(P<0.01);BSHXF治疗组细胞中TNF-α和IL-6的含量均显著降低(P<0.01)。(3)与溶剂对照组相比,激活剂对照组的p-JNK、Cleaved Caspase-3、Bax蛋白水平显著上调(P<0.05),Bcl-2蛋白表达水平显著降低(P<0.01),细胞增殖能力下降(P<0.05),细胞凋亡率显著增加(P<0.01);BSHXF治疗组和溶剂对照组两组间细胞增殖、凋亡率和凋亡相关蛋白表达(Cleaved Caspase-3、Bax、Bcl-2)情况差异均无统计学意义(P>0.05)。(4)与溶剂对照组相比,激活剂对照组的ROS和MDA水平显著升高(P<0.01),而GSH水平显著降低(P<0.01),TNF-α、IL-6含量升高(P<0.05);BSHXF治疗组和溶剂对照组两组间ROS相对含量、MDA、GSH水平差异均无统计学意义(P>0.05)。结论 BSHXF通过抑制JNK信号通路激活抑制IL-1β诱导的OA软骨细胞凋亡,改善氧化应激和炎症反应。

补肾活血方对脂多糖诱导的软骨细胞炎症损伤的影响

目的观察补肾活血方(BSHXF)含药血清对脂多糖(LPS)诱导的软骨细胞炎症及凋亡的影响,探讨BSHXF治疗骨关节炎的作用机制。方法制备空白血清及BSHXF含药血清。采用LPS诱导C28/I2人正常软骨细胞,构建软骨细胞炎症模型。将细胞分为空白组、LPS模型组、LPS+BSHXF组,空白组以含10%空白血清的培养基培养,LPS模型组以含10μmol·L^(-1 )LPS和10%空白血清培养基培养,LPS+BSHXF组以含10μmol·L^(-1 )LPS和10%含药血清培养基培养。各组均干预48 h后,CCK-8法检测细胞活力,LDH法检测细胞毒性,ELISA法检测细胞培养基上清液中IL-6、iNOS和COX2的水平,TUNEL染色法检测细胞凋亡情况,Western blot法检测细胞凋亡相关蛋白Cleaved-Caspase-9、Bcl-2、Bax的表达及核转录因子-κB(NF-κB)信号通路关键蛋白p-IκBα、IκBα、p-p65、p65的表达。结果与空白组相比,LPS模型组软骨细胞活力降低,细胞上清液中LDH水平升高,IL-6、iNOS和COX2的水平升高,细胞凋亡率增加,Cleaved-Caspase-9及Bax蛋白表达升高、Bcl-2蛋白表达降低,p-IκBα/IκBα、p-p65/p65比值升高。与LPS模型组相比,LPS+BSHXF组软骨细胞活力增加,细胞上清液中LDH水平降低,IL-6、iNOS和COX2的水平降低,细胞凋亡率降低,Cleaved-Caspase-9、Bax蛋白表达降低,Bcl-2蛋白表达升高,p-IκBα/IκBα、p-p65/p65比值降低。结论BSHXF可提升LPS诱导的损伤软骨细胞的活性,减轻LPS对软骨细胞的毒性,减少炎症因子释放、抑制软骨细胞凋亡,其机制可能是通过抑制NF-κB信号通路,进而发挥软骨保护作用。

补肾安胎方治疗肾虚型早期先兆流产患者的临床疗效及对其Th1/Th2相关因子、Th17/Treg细胞平衡的影响

目的探讨补肾安胎方治疗肾虚型早期先兆流产临床疗效及对Th1/Th2相关因子、Th17/Treg细胞平衡的影响。方法选取2021年1月—2022年12月期间南京市妇幼保健院收治的126例肾虚型早期先兆流产患者为研究对象,按随机数字表法分为对照组和观察组,每组各63例。对照组给予口服地屈孕酮治疗,观察组在对照组基础上增加补肾安胎方治疗。连续治疗14 d后,观察比较两组患者临床疗效及治疗前后中医证候积分、性激素[人绒毛膜促性腺激素(Human chorionic gonadotropin,β-HCG)、孕酮(Progesterone,P)、雌二醇(Estradiol,E_(2))、卵泡刺激素(Follicle stimulating hormone,FSH)]、Th1/Th2相关因子[白细胞介素-2(Interleukin-2,IL-2)、白细胞介素-4(Interleukin-4,IL-4)、白细胞介素-10(Interleukin-10,IL-10)、干扰素-γ(Interferonγ,INF-γ)]、Th17/Treg水平。结果治疗后观察组临床总有效率92.06%(58/63)明显高于对照组79.37%(50/63),差异有统计学意义(P<0.05)。治疗后两组患者阴道流血时间、出血量、小腹疼痛或坠胀、腰酸痛、头晕耳鸣、夜尿频数、两膝酸软积分及总积分均较治疗前降低,差异有统计学意义(P<0.05);且观察组阴道流血时间、出血量、小腹疼痛或坠胀、腰酸痛、头晕耳鸣、夜尿频数、两膝酸软积分及总积分均明显低于对照组,差异有统计学意义(P<0.05)。治疗后两组患者性激素β-HCG、P、E_(2)、FSH水平均较治疗前升高,差异有统计学意义(P<0.05);且观察组性激素β-HCG、P、E_(2)、FSH水平均明显高于对照组,差异有统计学意义(P<0.05)。治疗后两组患者血清IL-4、IL-10水平较治疗前升高,IL-2、INF-γ水平较治疗前降低,差异有统计学意义(P<0.05);且观察组IL-4、IL-10水平高于对照组,IL-2、INF-γ水平低于对照组,差异有统计学意义(P<0.05)。治疗后两组患者血清Th17及Th17/Treg水平较治疗前降低,差异有统计学意义(P<0.05);且观察组血清Th17及Th17/Treg水平较明显低于对照组,差异有统计学意义(P<0.05)。两组患者Treg水平治疗前后比较,差异无统计学意义(P>0.05)。结论在地屈孕酮治疗基础上结合补肾安胎方治疗,可显著缓解肾虚型早期先兆流产患者临床症状,并可改善性激素水平及免疫状态。

补肾活血方治疗老年缓慢性心律失常随机对照研究

目的观察补肾活血方对老年缓慢性心律失常肾虚血瘀证患者的临床疗效及安全性。方法选取就诊于中国中医科学院广安门医院心血管科门诊及病房的63例老年缓慢性心律失常患者,按照随机数字表法分为治疗组32例和对照组31例。治疗组予补肾活血方,200 mL/次,2次/d;对照组予参松养心胶囊,3粒/次,3次/d。两组患者疗程均为4周。对比两组患者中医证侯积分、临床疗效、24小时动态心电图、常规心电图、心功能、生活质量评分及安全性指标。结果与对照组比较,治疗组中医证候总积分,心悸、胸闷、畏寒肢冷、头晕耳鸣等单项证候积分均降低(P<0.05);24小时动态心电图总心率、平均心率及最慢心率均升高(P<0.05,P<0.01);生理和社会功能等方面生活质量评分升高(P<0.05)。在临床疗效、常规心电图及心功能方面,治疗组与对照组相比无统计学意义(P>0.05)。结论补肾活血方治疗老年缓慢性心律失常肾虚血瘀证患者具有良好的临床疗效及安全性。

补肾化瘀方加减对肾虚血瘀型子宫内膜异位症术后复发的预防作用与机制研究

[目的]探讨补肾化瘀方加减对肾虚血瘀型子宫内膜异位症术后复发的预防作用与机制。[方法]选择于2019年1月—2021年1月在衡水市第四人民医院治疗的子宫内膜异位症患者320例,按随机数字表法随机分组,常规西医组160例,给予患者常规西医治疗,补肾化瘀方组160例,在常规西医治疗同时给予患者补肾化瘀方加减治疗,治疗前后检测两组患者血清性激素[促黄体生成素(LH)、孕激素(P)、雌激素(E2)],肿瘤坏死因子-α(TNF-α),血管内皮生长因子(VEGF),抗子宫内膜抗体(EMAb)水平,通过语言描述评分法(VRS)评价患者痛经及非经期盆腔痛情况,给予患者中医证候评分,比较两组临床疗效,记录两组患者2年复发率及治疗期间两组患者不良反应发生情况。[结果]补肾化瘀方组患者痛经及非经期盆腔痛评分低于常规西医组(P<0.05),补肾化瘀方组TNF-α、VEGF、EMAb水平较常规西医组低(P<0.05),补肾化瘀方组患者LH、P、E2含量较常规西医组明显低(P<0.05),补肾化瘀方组患者中医证候评分较常规西医组明显降低(P<0.05),补肾化瘀方组患者总有效率(97.50%)较常规西医组(88.13%)高(P<0.05),常规西医组2年复发率为12.90%,补肾化瘀方组2年复发率为3.21%,两组2年复发率比较,差异有统计学意义(P<0.05),补肾化瘀方组开始复发时间长于常规西医组(P<0.05);治疗期间两组患者均无明显不良反应发生。[结论]补肾化瘀方加减治疗肾虚血瘀型子宫内膜异位症术后疗效显著,可有效缓解患者痛经及非经期盆腔痛,降低复发率,其作用机制可能与其抑制机体炎症,降低VEGF、EMAb水平,改善性激素水平有关。

基于JAK/STAT3通路探讨补肾活血方改善单侧输尿管梗阻大鼠炎症和肾纤维化

目的:观察补肾活血方(BSHXF)对单侧输尿管梗阻大鼠(unilateral ureteral obstruction,UUO)肾纤维化和炎症的治疗效果,揭示其抗炎及延缓肾纤维化的机制。方法:32只8周龄雌性SD大鼠根据随机数字表分为假手术组(Sham组)、单侧输尿管梗阻组(UUO组)、补肾活血方低剂量组(BSHXF-L组)和补肾活血方高剂量组(BSHXF-H组)。造模后第2天开始,连续灌胃干预21 d,BSHXF-L组和BSHXF-H组给予相应剂量补肾活血方汤剂,Sham组和UUO组给予与BSHXF组等体积的生理盐水。干预结束后,记录24 h尿量,全自动生化分析仪检测血肌酐、尿素氮、尿蛋白、尿肌酐和肾功能指数;采用HE和Masson染色检测肾脏病理损伤和纤维化程度;通过免疫组化染色分析各组TGF-β和TNF-α;Western blotting检测各组TGF-β_(1)、α-SMA、JAK、STAT3、p-STAT3、TNF-α、Caspase-3和Bax的蛋白表达情况。结果:与UUO模型组相比,BSHXF-L组和BSHXF-H组能明显明显改善UUO大鼠的肾功能,降低大鼠肾组织中TGF-β_(1)和α-SMA蛋白表达水平,抑制JAK、STAT3和p-STAT3表达,减少炎症细胞浸润和纤维组织以及胶原的增生,显著降低大鼠的肾脏指数和TNF-α等炎症指标。结论:补肾活血方可通过抑制JAK/STAT3通路激活,改善UUO大鼠肾功能,减轻其肾脏炎症水平、肾脏指数,延缓肾纤维化进程。

基于相关细胞凋亡途径探讨补肾健脾方调控大鼠卵巢储备功能减退的机制研究

目的观察补肾健脾方对环磷酰胺诱导的卵巢储备功能减退大鼠模型卵巢功能的影响,并基于肿瘤坏死因子诱导相关细胞凋亡途径探讨补肾健脾方调控大鼠卵巢储备功能减退的机制研究。方法60只动情周期正常的健康雌性SD大鼠,随机分成正常组10只和造模组50只,造模组采用环磷酰胺75 mg/kg单次腹腔注射造模。造模成功的大鼠再随机分为模型组、西药组[戊酸雌二醇0.103 mg/(kg·d)+黄体酮胶囊2.575 mg/(kg·d)]、补肾健脾方低剂量组[3.21 g/(kg·d)]、补肾健脾方中剂量组[6.43 g/(kg·d)]、补肾健脾方高剂量组[12.85 g/(kg·d)],每组10只。正常组及模型组均予等体积蒸馏水灌胃,每组干预1次/d,干预14 d。检测各组大鼠动情周期、体质量、卵巢脏器指数、子宫脏器指数;ELISA法检测大鼠血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、γ干扰素(interferon-γ,IFN-γ)含量;免疫荧光法检测大鼠卵巢颗粒细胞内B细胞淋巴瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)蛋白表达情况;HE染色检测卵巢组织病理情况;Western blot法检测卵巢磷酸化蛋白激酶B(posphorylated-proteinkinase B,p-AKT)、磷酸化哺乳动物雷帕霉素靶蛋白(p-mammalian target of rapamycin,p-mTOR)、自噬蛋白苄氯素-1(Beclin-1)表达情况。结果与正常组比较,模型组大鼠动情周期紊乱,可见卵巢结构紊乱、各级卵泡数量不同程度的减少、未见成熟卵泡,卵巢间质中见到明显炎细胞浸润;大鼠体质量、卵巢脏器指数和子宫脏器指数无明显变化;血清TNF-α、IFN-γ水平升高(P<0.05,P<0.01);卵巢颗粒细胞Bcl-2、Bax、Beclin-1蛋白表达水平升高(P<0.05,P<0.01),p-AKT、p-mTOR蛋白表达水平降低(P<0.01)。与模型组比较,西药组及补肾健脾方低、中剂量组血清TNF-α、IFN-γ水平降低(P<0.05,P<0.01);补肾健脾方中剂量组Bax蛋白表达水平降低(P<0.05);补肾健脾方高剂量组Bcl-2蛋白阳性表达升高(P<0.05);西药组、补肾健脾方低剂量组Beclin-1蛋白表达水平降低(P<0.05,P<0.01),p-AKT、p-mTOR蛋白表达水平升高(P<0.01)。与西药组比较,补肾健脾方高剂量组TNF-α水平升高(P<0.01);补肾健脾方低、高剂量组IFN-γ水平降低(P<0.05);补肾健脾方低剂量组Bax蛋白表达水平升高(P<0.05),补肾健脾方中剂量组Bcl-2蛋白水平表达升高(P<0.05);补肾健脾方中、高剂量组Beclin-1蛋白表达水平升高(P<0.05,P<0.01),p-AKT、p-mTOR蛋白表达水平降低(P<0.01)。结论补肾健脾方可改善卵巢病理变化、提高卵巢储备功能,其机制可能通过抑制肿瘤坏死因子TNF-α、IFN-γ表达,增强Bcl-2蛋白表达水平,降低Bax蛋白表达水平,激活mTOR通路抑制大鼠卵巢颗粒细胞的自噬与凋亡,从而改善环磷酰胺造模所致的大鼠卵巢储备功能减退的影响。

补肾活血方对去势骨质疏松小鼠骨-血管形成偶联调节作用的实验研究

目的:基于骨-血管形成偶联调节作用探讨补肾活血方对去势骨质疏松小鼠骨量及骨代谢的影响。方法:选取40只雌性ICR小鼠按随机数字表法分为假手术组(10只)和去势组(30只)。去势组小鼠行卵巢去势手术后常规饲养8周,随机抽取3只小鼠证实骨质疏松造模成功后,按随机数字表法分为模型组和中药组,每组各10只。假手术组及模型组予以等量生理盐水灌胃。中药组成模后采用补肾活血方(8.6 mg/kg)灌胃干预,8周后处死小鼠收集样本。进行Micro-CT、TRAP染色、免疫荧光染色(EMCN、CD31)、骨代谢指标检测。结果:Micro-CT显示模型组较假手术组骨小梁显著减少,中药组较模型组骨小梁增加。定量分析结果显示,模型组骨体积分数(BV/TV)、骨小梁数目(Tb.N)、骨小梁厚度(Tb.Th)低于假手术组,而中药组BV/TV、Tb.N、Tb.Th高于模型组,差异具有统计学意义(P<0.05)。骨组织TRAP染色及定量分析显示,模型组TRAP阳性表达的数目高于假手术组,中药组阳性表达的数目少于模型组,差异具有统计学意义(P<0.05)。模型组干骺端H血管的比例相比假手术组显著减少,中药组干骺端H血管的比例较模型组增加,差异具有统计学意义(P<0.05)。与假手术组相比,模型组PINP水平降低,TRACP水平升高,差异具有统计学意义(P<0.05);中药组PINP水平较模型组升高,而TRACP降低,差异具有统计学意义(P<0.05)。结论:补肾活血方可通过促进骨-血管形成,增加骨生成,减少骨吸收,提高去势骨质疏松小鼠的骨量。

补肾运脾方联合生长激素治疗脾肾不足型生长激素缺乏症患儿的临床效果

目的 探讨补肾运脾方联合生长激素治疗脾肾不足型生长激素缺乏症患儿的临床效果。方法 选取2021年9月至2023年3月仪征市中医院收治的100例脾肾不足型生长激素缺乏症患儿,按照随机数字表法分为对照组(n=50)与观察组(n=50)。对照组采用生长激素治疗,观察组采用补肾运脾方联合生长激素治疗。比较两组临床疗效、生长发育情况和代谢指标。结果 观察组总有效率高于对照组(P <0.05)。治疗后,观察组生长发育情况优于对照组(P<0.05)。治疗后,观察组生长激素、胰岛素样生长因子-1水平均高于对照组(P<0.05)。结论 补肾运脾方联合生长激素治疗脾肾不足型生长激素缺乏症患儿的临床效果较好,可促进患儿生长发育,改善代谢指标,值得临床推广。

补肾化痰方含药血清对HepG2肝癌细胞增殖、迁移的实验研究

目的:研究补肾化痰方(BHF)对小鼠脏器指数及BHF含药血清对HepG2肝癌细胞增殖、迁移的影响。方法:将小鼠随机分为对照组、BHF高剂量组、BHF中剂量组、BHF低剂量组,灌胃7 d后,采血制备含药血清,处死后取材测定各组小鼠脏器指数。体外培养HepG2肝癌细胞,设置空白组、对照组、BHF高剂量组、BHF中剂量组、BHF低剂量组,每组设3个浓度(20%、10%、5%),3个复孔。各组加入相应的含药血清,培养24、48、72 h后,采用CCK8法测算细胞增殖抑制率。采用Transwell细胞培养24 h后镜下观察各组细胞的形态学改变。进行细胞划痕实验,检测各组HepG2细胞迁移的水平。结果:在20%血清浓度下,与对照组比较,24 h时BHF高、低剂量组细胞增殖抑制率显著增加(P<0.05)。72 h时BHF各剂量组细胞增殖抑制率均显著增加(P<0.05),且高、低剂量组均高于中剂量组(P<0.05)。在10%血清浓度下,与对照组比较,24 h时BHF低剂量组细胞增殖抑制率显著增加(P<0.05),72 h时BHF各剂量组细胞增殖抑制率均显著增加(P<0.05),且高剂量组显著高于低剂量组(P<0.05)。在5%血清浓度下,与对照组比较,48 h时BHF高剂量组显著增加(P<0.05)。72 h时BHF各剂量组细胞增殖抑制率均显著增加(P<0.05),且高剂量组显著高于中、低剂量组(P<0.05)。与空白组比较,72 h时BHF高剂量组划痕迁移率显著降低(P<0.05)。结论:BHF含药血清对HepG2肝癌细胞增殖、迁移具有抑制作用,且高剂量组表现最优。

肾虚血瘀证大鼠模型的建立及补肾活血方补肾益精活血化瘀功效研究

目的:通过建立肾虚血瘀证大鼠模型并基于方证相应研究补肾活血方的补肾益精、活血化瘀功效。方法:将48只雌性SD大鼠随机分为正常对照组、模型组、阳性药组、补肾活血方低剂量组(0.72 g/kg)、补肾活血方中剂量组(1.44 g/kg)、补肾活血方高剂量组(2.89 g/kg),每组8只。除正常对照组外,各组从第1天开始,上午灌胃造模药羟基脲溶液(450 mg/kg),连续14 d,从第4天上午开始加用肾上腺素0.3 mg/(kg·d)皮下注射至第14天,制备大鼠肾虚血瘀模型。观察大鼠的一般状态,检测子宫和卵巢脏器指数、卵泡数目、黄体数量,血清雌二醇(E_(2))、卵泡刺激素(FSH)、抑制素B(INHB)、红细胞(RBC)、血红蛋白(HGB)、红细胞比容(HCT)、血栓素(TXB-2)、内皮素(ET-1)的水平,血浆红细胞压积、红细胞聚集指数、红细胞变形指数。结果:与正常对照组比较,模型组子宫指数和卵巢指数均下降(P<0.001),卵泡数目下降(P<0.05),黄体数目上升(P<0.05),血清E_(2)、FSH含量均降低,INHB含量升高(P<0.001),RBC、HGB、HCT含量均降低(P<0.001),血浆红细胞压积、红细胞聚集指数、红细胞变形指数均升高(P<0.01,P<0.001),血清ET-1、TXB-2含量均升高(P<0.001)。与模型组比较,给药组卵巢指数均上升(P<0.01,P<0.001),而子宫指数除补肾活血方高剂量组,其他给药组均上升(P<0.05);给药组卵泡数目均上升(P<0.05,P<0.01,P<0.001),补肾活血方各剂量组黄体数量降低(P<0.05);给药组血清E_(2)、FSH均升高(P<0.01,P<0.001)、INHB含量均降低(P<0.001),补肾活血方各剂量组RBC、HGB、HCT含量均升高,差异有统计学意义;给药组血清ET-1、TXB-2含量均降低(P<0.001),血浆红细胞压积和红细胞聚集指数均降低(P<0.001),红细胞变形指数除了补肾活血方高剂量组,其余给药组均降低(P<0.05)。结论:首次通过灌胃羟基脲结合皮下注射肾上腺素建立大鼠肾虚血瘀证模型,补肾活血方可以改善卵巢功能和激素水平,具有补肾益精、补血功效,且通过改变血液流变学的异常,改善内皮细胞功能,降低血栓素,从而达到活血化瘀之功。

补肾中药复方对高盐摄入去卵巢大鼠骨代谢及ENaCα、NCC和ClC-3表达的影响

目的:探讨补肾中药复方(Bushen formulae,BHF)对高盐摄入去卵巢大鼠骨代谢的影响及其可能的作用机制。方法:80只SPF级SD雌性大鼠按随机数字表法分为假手术组、模型组、中高盐组、高盐组、补肾方组、补肾方+生理盐水组、补肾方+中高盐组、补肾方+高盐组,每组各10只,造模后给予不同饮食、补肾中药复方干预,中高盐饮食、高盐饮食中氯化钠添加浓度分别为2%w/w、8%w/w,补肾中药复方干预剂量为7.8 g·kg^(-1)·d^(-1),每日1次,连续给药12周。检测各组骨密度、骨微结构、骨参数、骨代谢生物标志物、骨组织病理变化情况以及采用Western blot检测骨组织上皮钠离子通道α(ENaCα)、钠-氯同向转运体(NCC)、电压门控性氯离子通道3(ClC-3)蛋白的表达。结果:与假手术组相比,模型组大鼠骨密度降低,骨微结构破坏;与模型组相比,中高盐、高盐饮食组大鼠骨微结构破坏更为明显,同时高盐组骨形成标志物骨钙素(BGP)、Ⅰ型原胶原氨基端前肽(PINP)水平显著上升(P<0.05),骨吸收标志物Ⅰ型胶原氨基端肽(NTX)、Ⅰ型胶原羧基端肽(CTX)、抗酒石酸酸性磷酸酶(TRACP)均显著增加(P<0.05),骨代谢处于高转换状态;高盐饮食加速骨小梁结构的破坏,Western blot结果显示高盐饮食引起股骨组织ENaCα、ClC-3蛋白表达水平下降(P<0.05),NCC蛋白表达水平升高(P<0.05),补肾中药复方干预后能够不同程度的调节高盐引起的相关离子通道的表达。结论:补肾中药复方能够不同程度的调节高盐引起的相关离子通道ENaCα、ClC-3、NCC的表达,对骨代谢失衡具有一定的改善和治疗作用。