Xiongzhi Dilong decoction interferes with calcitonin gene-related peptide(CGRP)-induced migraine in rats through the CGRP/iNOS pathway

Objective: To evaluate the effects of Xiongzhi Dilong decoction(XZDLD) and its wind medicine on calcitonin gene-related peptide(CGRP)-induced migraine and explore the mechanism through the CGRP/inducible nitric oxide synthase(iNOS) pathway.Methods: Rats were divided into control, model, XZDLD, XZDLD(external wind), XZDLD(internal wind),and olcegepant groups. CGRP was injected into the dura mater to induce a migraine. The frequency of head scratching, cage climbing, and facial grooming was observed. The pain threshold, the levels of CGRP,pituitary adenylate cyclase activating polypeptide(PACAP), substance P(SP), and the plasma protein extravasation(PPE) ratio were measured. The phosphorylation levels of p38, extracellular signalregulated kinase 1/2(ERK1/2), and expression of iNOS were detected by western blot.Results: Compared with the model group, the three modified XZDLD groups showed reduced frequency of head scratching and cage climbing in the first 30 min(all P <.05). Facial grooming frequency was reduced in XZDLD and XZDLD(external wind) groups(P =.0003 and P =.0131, respectively). External wind medicine played a more important role in increasing mechanical pain threshold than internal wind medicine. Moreover, compared with the model group, the three modified XZDLD groups demonstrated reduced plasma levels of CGRP and PACAP(all P <.05). No difference in the SP level was observed among the six groups. XZDLD reduced PPE ratio. XZDLD and XZDLD(external wind) groups suppressed the CGRP/iNOS pathway by inhibiting the p-p38/p38 ratio and the expression of iNOS. No difference in pERK1/2/ERK1/2 ratio was detected among the six groups.Conclusion: XZDLD increases pain threshold, downregulates the expression of CGRP and PACAP, and reduces PPE ratio by inhibiting the CGRP/iNOS pathway. External wind medicine is more effective than internal one on improving facial grooming and head scratching, increasing the mechanical pain threshold, and inhibiting the expression of iNOS.

Therapeutic Effect and Mechanism of Jipei Dilong Ointment on Acute Soft Tissue Injury in Rats

[Objectives]This study was conducted to observe the therapeutic effect of Jipei Dilong Ointment on rats with acute soft tissue injury caused by heavy objects and to explore its action mechanism.[Methods]Thirty six rats were randomly divided into six groups(control group,model group,high-dose Jipei Dilong Ointment group(JP-H),medium-dose Jipei Dilong Ointment group(JP-M),low-dose Jipei Dilong Ointment group(JP-L)and diclofenac group).Except for the Control group,other groups were subjected to modeling of acute soft tissue injury by the weight impact method.All administration was performed once a day for nine consecutive days.The local appearance score and activity disorder score were determined after soft tissue injury in rats.HE staining was used to detect the pathological changes of injured soft tissues in rats.RT-PCR was used to detect the relative mRNA expressions of Bax,Bcl-2,MMP-9 and TIMP-1 in injured soft tissues of rats.Western Blot was used to detect the protein expressions of MMP-9,TIMP-1,TLR4,MyD88 and NF-κB p65 in injured soft tissues of rats.Results were statistically analyzed.[Results]Compared with the model group,Jipei Dilong Ointment could significantly improve the appearance symptoms such as swelling and ecchymosis in the injured area and the movement function of the affected limb(P<0.05).It could also improve the infiltration of inflammatory cells and widening of the intermuscular space caused by injury.Among them,the JP-H group and the diclofenac group had more significant curative effects.After 9 d of administration,each administration group could significantly up-regulate the ratio of Bcl-2/Bax mRNA expression level(P<0.05 or P<0.01),and the ratio of MMP-9/TIMP-1 mRNA expression level showed a downward trend(P>0.05).The expression level of NF-κB p65 protein in each administration group was significantly decreased(P<0.01).The protein expression levels of TLR4 and MyD88 and the ratio of MMP-9/TIMP-1 protein expression level in each administration group decreased to varying degrees.Among them,the JP-H group and diclofenac group significantly decreased(P<0.05).[Conclusions]Jipei Dilong Ointment has the functions of relieving pain,swelling and inflammation.It could improve the local appearance,functional activity and tissue morphology of affected limbs in rats,and has a therapeutic effect on acute soft tissue injury in rats.Its mechanism of action might be related to the inhibition of TLR4/MyD88/NF-κB p65 signaling pathway and the regulation of Bcl-2/Bax and MMP-9/TIMP-1 balance.

Protective Effect and Molecular Mechanism of Shui People's Classic Prescription Jipei Dilong Ointment on Osteoarthritis in Rats

[Objectives]To explore the protective effect and molecular mechanism of Shui People s Classic Prescription Jipei Dilong Ointment on knee osteoarthritis in rats.[Methods]72 SPF male SD rats were divided into control group,model group,Jipei Dilong Ointment high,medium and low dose groups,and positive drug Diclofenac group,with 12 rats in each group.Except the control group,all other groups were injected with 0.2 mL of 2%papain and 0.3%L-cysteine mixture into knee joint cavity to establish osteoarthritis model,while the control group was injected with the same amount of normal saline.The Lequesne MG score was used to determine the success of the model.After successful modeling,external administration was given for 4 weeks.The histopathological changes in articular cartilage and synovium were observed by HE staining;the levels of TNF-α,IL-1βand COX-2 in serum were detected by ELISA,and the relative expression of MMP-9 and TIMP-1 mRNA in cartilage was detected by qRT-PCR;the relative expression of MMP-9,TIMP-1,TLR4,MyD88 and NF-κB in rat cartilage was detected by Western-blot.[Results]Compared with the model group,Jipei Dilong Ointment could significantly reduce the Lequesne MG score and Mankin s score of arthritic rats(P<0.01);significantly improve the pathological changes in articular cartilage and synovium,reduce tissue edema,necrosis,inflammatory cell infiltration and fibrous tissue proliferation;reduce the expression of MMP-9 mRNA(P<0.01)in different degrees and increase the expression of TIMP-1 mRNA(P<0.01);reduce the relative expression of MMP-9,TLR4,MyD88 and NF-κB protein in different degrees(P<0.01),and significantly increase the relative expression of TIMP-1 protein in cartilage(P<0.01).[Conclusions]Jipei Dilong Ointment can improve the joint injury of osteoarthritis rats,and the mechanism may be related to the regulation of TLR4/MyD88/NF-κB signaling pathway and the ratio of MMP-9/TIMP-1.

A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine:A case study of Amynthas earthworms as"Guang Dilong"

For wild natural medicine,unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials,which affects the efficacy and safety of clinical medication.DNA barcoding as an effective species identification tool is limited by its low sample throughput nature.In this study,combining DNA minibarcode,DNA metabarcoding and species delimitation method,a novel biological sources consistency evaluation strategy was proposed,and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as"Guang Dilong"and 25 batches of proprietary Chinese medicines.Besides Amynthas aspergillum as the authentic source,8 other Molecular Operational Taxonomic Units(MOTUs)were elucidated.Significantly,even the subgroups within A.aspergillum revealed here differ significantly on chemical compositions and biological activity.Fortunately,this biodiversity could be controlled when the collection was limited to designated areas,as proved by 2796"decoction pieces"samples.This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control,and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.

Effects of Wuwei Dilong Decoction on Inflammatory Cells and Cytokines in Asthma Model Guinea Pigs

Objective： To explore the effects and the mechanism of Wuwei Dilong Decoction （五味地龙汤 Schisandra Fruit and Earthworm Decoction） for treatment of asthma. Methods： The asthma guinea pig model was established with spray of ovalbumin （OVA）. Fifteen days later, the guinea pigs were administered by intra-gastric perfusion of Wuwei Dilong Decoction once a day for 8 consecutive days. Blood samples were taken for testing the total leucocytes, eosinophil （EOS）, lymphocytes, interferon-γ （IFN-γ） and leukotriene B4 （LTB4）. Results： In the asthma model group, the total leucocytes, EOS and lymphocytes were all increased, with significant differences as compared with the different dosage Wuwei Dilong Decoction groups （P〈0.01 or P〈0.05）. The serum LTB4 in the asthma model group was significantly increased and IFN-γ decreased. After administration of Wuwei Dilong Decoction of the large, medium and small dosages, LTB4 decreased, while IFN-7 increased （P〈0.05 or P〈0.01）. Conclusion： Wuwei Dilong Decoction can inhibit infiltration and diffusion of the inflammatory cells in the asthma model guinea pigs, and regulate LTB4 and IFN-γ, which is probably one of the important mechanisms of Wuwei Dilong Decoction for relieving asthma.

地龙舒腰汤治疗腰椎间盘突出症椎间孔镜术后残留症状的临床观察

目的观察地龙舒腰汤对腰椎间盘突出症经皮椎间孔镜术后残留症状的临床疗效,为临床上优化治疗及缓解患者症状提供参考。方法将诊断为腰椎间盘突出症并行经皮椎间孔镜术,术后残留腰腿部症状的患者60例,按照随机数字表法分为对照组30例与观察组30例。对照组患者予以常规西药及腰背肌功能锻炼治疗,观察组在对照组的基础上,服用地龙舒腰汤治疗,连续服药14 d。比较2组患者手术前、术后第2周疼痛视觉模拟量表(VAS)、日本骨科学会评估腰椎治疗量表(JOA)及腰椎功能障碍指数量表(ODI)。结果术后第2周2组患者疼痛程度及腰椎功能障碍指数均低于术前,且观察组均低于对照组,差异均具有统计学意义(P<0.05);手术后2组患者JOA评分均高于术前,且观察组高于对照组,差异均具有统计学意义(P<0.05);观察组总有效率为96.70%高于对照组的73.33%,差异具有统计学意义(P<0.05)。结论地龙舒腰汤改善腰椎间盘突出症经皮椎间孔镜术后残留症状有较好疗效。

地龙冬花汤加减治疗儿童哮喘急性发作期合并肺炎支原体感染临床研究

目的:观察地龙冬花汤加减治疗痰浊郁肺型儿童哮喘急性发作期合并肺炎支原体(MP)感染的临床疗效。方法:选取50例痰浊郁肺型哮喘急性发作期合并MP感染患儿,采用随机数字表法分为对照组和治疗组,每组25例。对照组给予阿奇霉素干混悬剂与布地奈德福莫特罗粉吸入剂治疗,治疗组在对照组基础上加用地龙冬花汤加减治疗。2组疗程均为8周。比较2组临床疗效、中医证候积分、呼气峰流速占预计值的百分比(PEF%pred)及第1秒用力呼气容积占预计值的百分比(FEV1%pred)。结果:治疗8周后,治疗组总有效率96.00%,与对照组88.00%比较,差异无统计学意义(P>0.05)。2组中医证候积分均较治疗前降低,治疗组中医证候积分低于对照组,差异均有统计学意义(P<0.05)。2组PEF%pred、FEV1%pred均较治疗前升高,治疗组PEF%pred、FEV1%pred均高于对照组,差异均有统计学意义(P<0.05)。结论:地龙冬花汤加减能够改善痰浊郁肺型哮喘急性发作期合并MP感染患儿的临床症状、体征和肺功能,有效控制哮喘发作。

地龙舒腰汤治疗退行性腰椎管狭窄症(肾虚血瘀型)临床疗效观察

目的:探究地龙舒腰汤治疗退行性腰椎管狭窄症(肾虚血瘀型)的临床疗效。方法:选取2021年12月—2022年12月收治的退行性腰椎管狭窄症患者68例,按随机数字表法将患者分为治疗组与对照组,每组各34例,对照组予以现代医学常规治疗,治疗组在对照组基础上予以地龙舒腰汤治疗,持续治疗28d。比较2组患者治疗前后的疼痛视觉模拟评分法(VAS评分)、日本骨科学会腰椎评估治疗分数评分(JOA评分)及中医证候积分。结果:治疗前,2组患者疼痛程度、腰椎评估治疗分数及中医证候积分比较,差异均无统计学意义;治疗后2组患者疼痛程度及中医证候积分均低于治疗前,且治疗组均低于对照组,差异均具有统计学意义;治疗后2组患者JOA评分均高于治疗前,且治疗组高于对照组,差异具有统计学意义;治疗组有效率为97.06%高于对照组的88.23%,差异具有统计学意义。结论:地龙舒腰汤能有效缓解退行性腰椎管狭窄症患者的临床症状,有效减轻疼痛,改善其腰椎功能,提高患者的生活质量,值得在临床推广。

地龙舒腰汤加味对腰椎间盘突出症后路术后残余根性腰腿痛(气滞血瘀型)临床疗效观察

目的:观察地龙舒腰汤加味治疗腰椎间盘突出症(LDH)后路后残余神经根症状(气滞血瘀型)临床疗效。方法:选取2022年12月—2023年9月腰椎后路椎板减压植骨融合内固定术后残余根性症状(气滞血瘀型)患者60例,按照随机数字表法分为治疗组与对照组,每组30例。术后常规补液、换药预防感染,对照组在此基础上服用塞莱昔布胶囊与甲钴胺片,治疗组在对照组基础上服用地龙舒腰汤加味治疗,连续服用药物4周。比较2组患者在接受治疗后2周、4周、6周的视觉模拟评分法(Visual Analogue Scale,VAS),腰椎JOA(Japanese Orthopaedic Association scores)评分,分析手术单节段与多节段及年龄因素对患者疼痛(VAS)评分改善的影响判定地龙舒腰汤加味采用治法是否有效并评估有效率。结果:治疗后2周、4周、6周治疗组VAS评分明显低于对照组;腰椎JOA评分,治疗组的评分明显高于对照组;2组2周、4周、6周VAS评分,单节段低于多节段评分;治疗组2周、4周、6周,单节段与多节段的评分均低于对照组单节段与多节段的评分;年龄与VAS评分下降分值之间存在负相关,治疗后4周、6周,治疗组VAS下降分值高于对照组;治疗组有效率高于对照组,差异均有统计学意义。结论:地龙舒腰汤加味采用温经通络、活血化瘀兼以补益气血、补益肝肾法,能够有效改善LDH术后腰椎功能,缓解术后根性腰腿疼痛。

地龙药材蛋白质电泳鉴定的初步研究

目的探讨聚丙烯酰胺凝胶电泳技术对地龙药材进行鉴别的可行性,为地龙的品种鉴定以及药材品质评价提供参考依据。方法采用聚丙烯酰胺凝胶电泳技术,对不同来源、不同品种、不同加工方法的地龙药材进行可溶性蛋白质电泳研究。结果电泳图谱中广地龙在分子质量66.214.4 ku之间存在5条清晰、稳定的条带,可初步认定其为广地龙的特征鉴别条带。不同品种的地龙在蛋白质电泳图谱谱型和谱带的强弱、蛋白质相对含量上均有一定的差异;地龙鲜品和干品在蛋白质谱带的强弱上有一定差异。结论聚丙烯酰胺电泳方法可以用于不同品种地龙的鉴定以及品质评价。

芎芷地龙汤对偏头痛模型动物血液和脑干中β-EP、CGRP的影响

目的研究芎芷地龙汤对偏头痛模型体内β-内啡肽(β-EP)、钙素基因相关肽(CGRP)的影响。方法健康雄性SD大鼠随机分为生理盐水组、模型组、舒马普坦组、中药治疗组,每组12只。皮下注射硝酸甘油(NTG)注射剂复制实验性偏头痛动物模型,经药物干预后在4h、6h两个时间点测定各组血液和脑干中β-EP、CGRP的含量。结果中药治疗组和舒马普坦组血浆和脑组织中β-EP含量升高,两个时间点的含量与模型组比较有显著性差异(P<0.05,P<0.01)。中药治疗组和舒马普坦组血浆和脑组织中CGRP含量降低,两个时间点的含量与模型组比较有显著性差异(P<0.01)。结论芎芷地龙汤可以下调由硝酸甘油诱导的CGRP异常升高的水平,可以上调由硝酸甘油诱导的β-EP异常减少的水平。

地龙规范化生产的研究概况

综合分析近年来地龙规范化生产的相关研究,重点针对地龙的种质资源调查、品种鉴定、人工养殖技术、采集加工和质量控制等关键问题进行评述。地龙规范化养殖相关研究取得了一定进展,但仍然存在着野生药用资源不足,药材品种混杂以及药材品质优劣参差不齐等问题。特别是缺少规范化养殖、产地加工和饮片炮制等关键技术的专项深入研究。因此,加强这方面的研究是今后研究的主攻方向和发展策略。

地龙有效成分对高糖下HMC细胞炎症因子分泌影响

目的观察地龙有效成分含药血清对HMC细胞炎症因子分泌的影响,并探讨其可能的作用机制。方法地龙有效成分及福辛普利干预高糖刺激HMC模型细胞,并设立正常组和高汤刺激组进行对照,应用MTT法检测HMC细胞生长抑制水平,酶联免疫法检测细胞培养液中TNF-α、IL-6和MCP-1的含量,应用RT-qPCR技术检测细胞样本的TNF-α、IL-6和MCP-1 mRNA表达水平。结果地龙有效成分组、福辛普利组HMC细胞生长平均抑制率下降(P<0.05);地龙有效成分、福辛普利干预后,细胞培养液中的TNF-α、IL-6和MCP-1含量均有下降趋势,与高糖刺激组比较,地龙有效成分组培养液中细胞TNF-αmRNA表达水平下降,MCP-1 mRNA表达水平上升,福辛普利组TNF-α、IL-6 mRNA表达水平明显下降。结论地龙有效成分对高糖下HMC细胞的干预作用可能是抑制细胞炎症因子分泌。

地龙及其注射液指纹特征谱研究

采用薄层扫描法建立了地龙药材及其注射液的指纹特征谱,为控制其质量提供了依据。

芎芷地龙汤不同时间预防给药对偏头痛动物模型痛阈及血管活性物质的影响

目的:研究芎芷地龙汤不同时间预给药对偏头痛动物模型痛阈及血管活性物质的影响。方法:将健康雄性SD大鼠,随机分为生理盐水组、模型组、舒马普坦组、中药治疗组,每组8只,给药干预后,皮下注射硝酸甘油注射剂10 mg/kg制作偏头痛动物模型,在皮下注射药物后2 h后测定,电子式机械测痛仪测定机械痛阈值,温度刺激仪测定温度痛阈值;硝酸还原法进行测定血浆NO浓度;ELISA法测定血浆CGRP、Neurokinin A、Endothelin-1(ET-1)、TXB2、6-Keto-PGF1α含量。结果:给予硝酸甘油后,模型组与生理盐水组比较,机械痛阈(1 d P<0.01,3 d P<0.05,7 d P<0.05)和温度痛阈(1 d P<0.01,3 d P<0.01,7 d P<0.01)均降低,血浆NO浓度升高(1 d P<0.01,3 d P<0.01,7 d P<0.01),血浆ET-1浓度降低(1 d P<0.05,3 d P<0.05,7 d P<0.01),血浆CGRP含量降低(1 d P<0.01,3 d P<0.01,7 d P<0.05),血浆NKA含量升高(1 d P<0.01,3 d P<0.01,7 d P<0.01),血浆TXB2含量降低(1 d P<0.01,3 d P<0.01,7 d P<0.05),血浆6-Keto-PGF1α含量降低(1 d P<0.05,3 d P<0.05,7 d P<0.01);预防给药后,芎芷地龙汤组和模型组比较,机械痛阈和温度痛阈均显著升高(3d P<0.01,7d P<0.05),NO浓度显著降低(3 d P<0.01,7 d P<0.01),血浆ET-1浓度升高(3 d P<0.01,7 d P<0.05),血浆CGRP浓度升高(7d P<0.01),NKA含量有所降低(1 d P<0.05,3 d P<0.05,7 d P<0.01),血浆TXB2含量有所增高(3d P<0.05,7 d P<0.01),血浆6-KetoPGF1α有所增高(3d P<0.05,7 d P<0.05)。结论:预防给药3天、7天,芎芷地龙汤均可以提高动物病理状态下的痛阈值,可减弱由NTG诱发的NO的升高水平,可升高由NTG诱发的TXB2降低水平,缓解由NTG诱发的6-Keto-PGF1α的降低水平,升高由NTG诱发的ET-1含量降低水平,减弱由NTG诱发的NKA的释放水平;预防给药7天后,芎芷地龙汤可以升高由NTG诱发的血浆CGRP降低水平。整体来分析,预防给药7天效果好于预防给药3天、1天。

地龙注射液HPLC指纹图谱的研究

目的建立地龙注射液中游离氨基酸的HPLC指纹图谱。方法采用2,4-二硝基氟苯柱前衍生化法,用HPLC法进行梯度洗脱分析地龙注射液中游离的氨基酸。结果通过优化衍生条件,得到分离度、重复性均较好的HPLC指纹图谱,确定了14个共有峰,以缬氨酸为参照峰,用中药指纹图谱相似度软件计算不同批次样品的相似度均在0.97以上。结论本研究建立的指纹图谱,可用于地龙注射液的质量控制。

射干地龙颗粒的含量测定及制剂稳定性研究

本试验旨在建立射干地龙颗粒中次野鸢尾黄素的含量测定方法及考察该制剂的稳定性,为制定该制剂质量标准中含量测定方法及保质期提供依据。采用反相高效液相色谱(RP-HPLC)法,色谱柱:GL Sciences Inertsil ODS-3(150mm×4.6mm,5μm),流动相:乙腈-0.2%磷酸溶液(39∶61),流速:1mL/min,检测波长:266nm,柱温:30℃,进样量:10μL;根据药典,采用强光照射试验、加速试验及长期稳定性试验考察射干地龙颗粒中次野鸢尾黄素含量的稳定性。试验结果表明,次野鸢尾黄素的含量在0.3422～86.60μg/mL范围内与峰面积呈良好的线性关系,r=0.9995(n=10),平均加样回收率为99.80%,RSD为1.50%(n=9);强光照射试验、加速试验及长期稳定性试验结果表明,该产品稳定性良好;此方法简便、灵敏、重现性好,可用于射干地龙颗粒中次野鸢尾黄素的含量测定,且该制剂的稳定性良好。

五味地龙汤对哮喘豚鼠炎症细胞和细胞因子的影响

目的探讨五味地龙汤治疗哮喘的作用及其机制。方法采用卵白蛋白喷雾引喘制作动物模型,致敏15天后开始灌胃给药,每日1次,连续8天。取血分别进行白细胞总数、嗜酸性粒细胞(EOS)、淋巴细胞分类计数、γ-干扰素(IFN-γ)和二羟酸白三烯(LTB4)测定。结果哮喘模型组白细胞总数、EOS、淋巴细胞均显著升高,与五味地龙汤各剂量组比较,差异均具有统计学意义(P<0.01或P<0.05)。哮喘模型组动物血清LTB4含量明显升高,IFN-γ水平明显降低,给予五味地龙汤后,大、中、小3个剂量组均使血清LTB4含量降低、IFN-γ上升(P<0.05或P<0.01)。结论五味地龙汤能抑制哮喘状态下的炎症细胞浸润和扩散,对LTB4和IFN-γ有调节作用,其可能是五味地龙汤治疗哮喘的重要机制之一。