Effects and Mechanisms of Xueshuantong on the Cognitive Function and Abnormal Neural Excitability in Mice with Alzheimer’ s Disease
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摘要:
目的 探究血栓通[主要有效成分为三七皂苷(panax notoginseng,PNS)]对阿尔茨海默症(Alzheimer’s disease,AD)模型小鼠认知功能及神经兴奋性的影响,并探讨其潜在分子机制。 方法 用APP/PS1小鼠作为AD研究动物模型,在小鼠淀粉样蛋白尚未检测到阶段(2月龄)开始每日以60 mg/kg对血栓通组(APP/PS1+PNS)行灌胃给药,每日1次,连续给药6个月(给药至8月龄);对照组小鼠予同等体积的0.9%氯化钠(APP/PS1+vehicle)灌胃处理,同月龄野生型小鼠予0.9%氯化钠灌胃处理作为正常对照组(WT+ vehicle),每组各15只。6个月后,新物体识别实验、Morris水迷宫实验检测小鼠的认知功能;EEG脑电检测、Western blot、细胞表面生物素化试验以检测各组小鼠皮质与海马中BACE1的活性、Nav1.1α的分布、表达以及Navβ2的表达与酶解情况(Navβ2的酶解片段Navβ2 full length及Navβ2-CTF表达检测)。 结果 新物体识别实验显示,与对照组APP/PS1小鼠相比,血栓通用药后APP/PS1小鼠的辨别指数(discrimination index,DI)上升(P < 0.05);Morris水迷宫检测结果发现,血栓通灌胃6个月后小鼠在探索实验中逃避潜伏期缩短(P < 0.05),撤除平台后在目标象限停留时间增加(P < 0.05)、穿梭平台次数增加(P < 0.05);EEG脑电检测结果发现,血栓通给药后减少了APP/PS1小鼠棘波放电出现的频率(P < 0.05)。血栓通给药后显著降低了BACE1蛋白水平的表达(P < 0.05),而全长片段Navβ2的蛋白水平显著上升(P < 0.05),并纠正了Nav1.1α在神经元内外的异常分布(P < 0.05)。 结论 血栓通可以改善AD模型小鼠的学习记忆能力、纠正大脑异常兴奋性,其作用机制可能与抑制BACE1的活性从而减少Navβ2由 APP/PS1诱导的过度酶解,纠正皮质、海马神经元Nav1.1α的异常表达与分布,调节神经元的兴奋性有关。 Abstract:Objective To explore the possible effects and the underlying molecular mechanisms of xueshuantong [The main active component is panax notoginseng (PNS)] on the cognitive function and neural excitability of mice with Alzheimer’ s disease (AD). Methods The APP/PS1 mice were used as an animal model for AD research, at the stage when amyloid protein was not detected in mice (2 months of age). Mice in the xueshuantong group (APP/PS1+PNS) were administered by gavage once a day at a dose of 60 mg/kg for six months (for 8 months of age). The mice of the control group were given 0.9% sodium chloride (APP/PS1+Vehicle) intragastric treatment of the same volume, while the wild-type mice of the same age were given 0.9% sodium chloride intragastric treatment as the normal control group (WT+Vehicle) (15 mice in each group, n=15). After six months, the cognitive function of the mice was evaluated by the Novel Object Recognition (NOR) task and Morris Water Maze (MWM) test. The activity of BACE1, the distribution and expression of Nav1.1α, as well as the expression and enzymatic hydrolysis of Navβ2 (Navβ2 full-length and Navβ2-CTF fragments) in cortex and hippocampus were detected by EEG, Western blot and cell surface biotinylation assay, respectively. Results The NOR task showed that compared with the mice in the APP/PS1+Vehicle group, the Discrimination index (DI) of mice in the APP/PS1 group was significantly increased after xueshuantong administration (P < 0.05). The MWM test found that, the escape latency of the mice in the xueshuantong group was shortened followed six months in gastric administration (P < 0.05), while the stay time in the target quadrant and the number of platforms significantly increased (P < 0.05) after the removal of the platform. The results of EEG recording showed that xueshuantong reduced the frequency of spike-wave discharges in APP/PS1 mice (P < 0.05). Furthermore, xueshuantong significantly reduced the expression of BACE1 (P < 0.05). In the APP+PNS group, the expression of Navβ2 full-length was increased (P < 0.05), as well as corrected the abnormal distribution of Nav1.1α inside and outside of neurons (P<0.05). Conclusion Treatment with xueshuantong can significantly improve the learning and memory ability and correct the abnormal excitability of the brain in AD model mice. The mechanism may be related to the inhibition of BACE1 activity, the reduction of APP/PS1-induced excessive enzyme digestion of Navβ2, the correction of the abnormal expression and distribution of Nav1.1α in cortical and hippocampal neurons, as well as the subsequent regulation of neuronal excitability. -
Key words:
- Xueshuantong /
- Alzheimer’ s disease /
- Cognition /
- BACE1 /
- The enzymolysis of Navβ2 /
- The distribution of Nav1.1α
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寻常型痤疮是皮肤科常见疾病,其病理改变为毛囊和皮脂腺慢性炎症,以青年男女常见,病因多与激素、皮脂过多,毛囊口上皮角化亢进等因素相关。本病好发于皮脂腺丰富的部位,尤其是颜面。而中重度寻常型痤疮,其皮损主要以囊肿、脓疱为主要表现,后期会出现瘢痕增生和色素沉着,严重影响患者的学习、工作及社交[1]。而有研究指出[2-3],中重度寻常型痤疮患者由于缺乏较为正确的疾病认知、继续不良的生活习惯等原因,导致本病的时好时坏,反复发作。另一方面,O2O即Online-to-Offline,是线上结合线下的意思。O2O式健康教育则是充分发挥线上和线下各自的优势,灵活运用多种线上线下健康教育手段,达到提高健康教育效果的目的[4-5]。而从2019年底到2022年底,我国面对爆发的新冠肺炎(COVID-19)疫情,始终坚持“动态清零”方针以及“人民至上、生命至上”信念是非常必要的。但是,部分防疫措施也客观上让患者减少了前往医院的就诊频率,同样也减少了接受相关健康教育的机会,对此O2O式健康教育具有深刻的现实意义;而随着2022年年底国家优化疫情防控方案,逐步放宽防控措施,并不意味着3 a中摸索出来的O2O式健康教育就可以直接摈弃,重新回归疫情前的健康教育模式。因此,本文选取疫情前以及疫情期间医院信息系统( hospital information system,HIS) 中的相关数据进行统计分析,以期为后续研究提供循证意义。
1. 资料与方法
1.1 一般资料
回顾性分析2018年4月至2023年1月中国人民解放军西部战区总医院HIS中中重度寻常型痤疮患者为研究对象,相关数据采用ETL (Extraction-Transformation-Loading)软件系统处理,本研究已获得单位伦理委员会批准。入选标准:(1)符合《中国临床皮肤病学》中关于寻常型痤疮的诊断标准[6],并按照PillSburv及国际改良痤疮分级法属于中度及重度;(2)年龄10~35岁。排除标准:(1)其他类型的痤疮;(2)伴有其他皮肤病,如特应性皮炎、银屑病等;(3)伴随严重脏器功能不全或严重免疫系统疾病者;(4)伴随恶性肿瘤患者;(5)代谢性疾病患者;(6)病历资料不完整。
1.2 方法
1.2.1 常规教育组
常规教育组患者在院治疗时进行口头宣教,并发放宣教手册,手册制作团队由1名副主任医师、1名副主任护师担任负责人,3名年资在5 a以上的主管护师为主干成员;先由团队制作中重度寻常型痤疮患者宣教手册初稿,基于德尔菲法进行2轮专家咨询,第1轮将初稿函询专家征求意见,根据相关意见修改后进行第2轮咨询并形成最终版本。手册包括疾病病因、发病机制、临床表现、皮肤护理、膳食指导、预防保健、心理健康等知识。手册以图文形式生动形象地向患者进行相关宣教。每周电话跟进其手册翻阅效果。
1.2.2 O2O组
O2O组进行线上线下健康教育 (1)建立O2O团队:团队同样由1名副主任医师、1名副主任护师担任负责人,3名年资在5 a以上的主管护师为主干成员。(2)建立线上资料库:主要以宣教短视频为主,辅以相关文章、图文等资料。组织团队成员拍摄、制作宣教短视频,视频内容同样包含有疾病病因、临床表现、皮肤护理、膳食指导、预防保健、药物指导、心理健康等知识,每个视频在4 min以内,以通俗易懂的形式只宣讲某一个宣教点。宣教短视频最后归档为两类:一类为基础性视频,针对所有中重度寻常型痤疮患者均适用,如“痤疮的危险因素”“用挤法来战痘,对吗”等等;一类为针对性视频,专门针对具体某一病因或相关问题的视频,如“正确看待雄激素”“什么是毛囊口上皮角化亢进”“别让青春的缺陷,由颜到心”“痘蔻年华我作主”“如何护肤不伤肤”等等。(3)患者就诊期间,执行线下为主、线上为辅的健康教育:线下健康教育同常规教育组,实施医护结合的口头宣教;同时组建微信群,以团队成员担任群主,患者入群后进行统一编号、备注,并登记每位患者的医嘱内容,不定期在群中推送相关视频、文章,内容主要是侧重于在院治疗期间的注意事项。(4)患者治疗结束后,执行线上为主、线下为辅的健康教育:①按照循序渐进、先易后难的原则,在微信群中推送基础性视频,并集中解答患者问题;再以私聊窗口一对一推送针对性视频,做到有的放矢;②无论哪一类视频的推送后,均有1~2道视频内容的小测试以问卷星链接发予患者,以保证患者认真观看;③要求患者每日进行清洁面部以及用药情况的打卡,管理人员以清单形式记录,未完成打卡者进行私聊窗口跟进,以保证患者依从性;④痤疮的发作与饮食有一定关系,要求患者在每周一和每周四各上传1次实时的膳食照片,群中医务人员进行点评;⑤在线下方面,不定期在科室举办小讲座,会中有医、护、营养等领域的专家从不同侧重点的科普宣教,邀请院外患者参加。
1.3 观察指标
统计2组下列指标:(1)失联率(常规教育组以电话失联、O2O组以微信失联为准)、对健康教育总体满意率(分为不满意、一般满意、喜欢满意、非常满意4个层次)、3个月内疾病复发率;(2)在治疗结束后3个月随访知识、态度以及行为调查量表(knowledge attitude practice,KAP),本量表包括3个维度,共计15个条目,分别赋值1~5分,分值越高则代表患者的疾病认知程度越高[7]。
1.4 统计学处理
应用 SPSS 22. 0 统计软件进行处理,计量资料均符合正态分布以(
$ \bar x $ ±s)表示,计数资料以例表示,采用 PSM 平衡与结局有关的协变量(年龄、性别、教育程度、病程),设定卡钳值为 0.02,采用 1∶1 最邻近匹配法匹配,匹配后组内比较采用配对样本t检验,组间比较采用两样本t检验,计数资料比较采用χ2检验,P < 0.05为差异有统计学意义。2. 结果
2.1 基线资料
根据是否进行O2O式健康教育分为常规教育组509例和O2O组 243例。由于2组人群基线资料不齐,见表1。因此采用倾向评分匹配(Propensity Score Matching,PSM)进行1∶1匹配,最终得到得到2组人群各241例,2组基线资料具有可比性(P > 0.05),见表2。
表 1 2组一般资料比较(未进行PSM) ($ \bar x \pm s$ )Table 1. Comparison of general information between two groups (Without PSM)($ \bar x \pm s$ )组别 n 平均年龄(岁) 性别(男/女) BMI指数(kg/m2) 病程(a) 教育程度(初中及以下/高中/大学/大学以上) O2O组 243 20.93 ± 1.24 133/110 23.35 ± 2.85 4.61 ± 0.42 75/78/79/11 常规教育组 509 23.30 ± 1.08 351/158 25.17 ± 3.12 3.25 ± 0.33 212/167/102/28 χ2/t 2.799 14.512 2.592 3.317 5.691 P 0.037* 0.0008* 0.048 0.026* 0.014* *P < 0.05。 表 2 2组一般资料比较(进行PSM后)($ \bar x \pm s$ )Table 2. Comparison of general information between two groups (After PSM)($ \bar x \pm s$ )组别 n 平均年龄(岁) 性别(男/女) BMI指数kg/m2 病程(a) 教育程度(初中及以下/高中/大学/大学以上) O2O组 241 20.92 ± 1.21 132/109 23.35 ± 2.81 4.60 ± 0.39 75/78/79/9 常规教育组 241 21.02 ± 1.15 136/105 25.47 ± 3.06 4.51 ± 0.34 75/80/78/8 χ2/t 0.265 0.134 0.553 0.331 0.012* P 0.902 0.714 0.609 0.840 0.997 *P < 0.05。 2.2 2组失联率、总体满意率、疾病复发率比较
O2O组失联率为7.88%,满意率为98.65%,疾病复发率为19.09%;常规教育组失联率为17.01%,满意率为84.5%,疾病复发率为65.56%, O2O组均优于常规教育组,差异有统计学意义(P < 0.05),见表3。
表 3 2组失联率、总体满意率、疾病复发率比较 [n(%)]Table 3. Comparison of drop-out rate,overall satisfaction rate,and disease recurrence rate between two groups [n (%)]组别 n 失联率 满意情况(n) 疾病复发率 不满意 一般满意 满意 非常满意 总体满意 O2O组 241 19(7.88)# 3 56 143 20 219(98.65)# 46(19.09)# 常规教育组 241 41(17.01) 31 128 36 5 169(84.5) 158(65.56) 与常规教育组相比,#P < 0.05。 2.3 2组知识、态度以及行为调查量表(KAP)得分比较
治疗前2组在知识得分、态度得分和行为得分相比,差异均无统计学意义(P > ;0.05)。治疗结束后3个月,2组3项得分均高于实施前,差异有统计学意义(P < 0.05);组间相比,O2O组在3项得分均高于常规教育组的3项得分,差异有统计学意义(P < 0.05),见表4。
表 4 2组知识、态度以及行为调查量表(KAP)得分结果比较 ($ \bar x \pm s$ )Table 4. Comparison of the results of two knowledge,attitude,and practice (KAP) scores($ \bar x \pm s$ )组别 n 知识、态度以及行为调查量表(KAP)得分 知识得分 态度得分 行为得分 治疗前 治疗结束后3个月 治疗前 治疗结束后3个月 治疗前 治疗结束后3个月 O2O组 241 6.22 ± 1.23 9.61 ± 1.39*# 11.19 ± 1.48 12.35 ± 2.08*# 9.26 ± 1.01 13.74 ± 2.15*# 常规教育组 241 6.17 ± 1.15 7.91 ± 1.28* 10.71 ± 1.30 9.56 ± 1.51* 9.10 ± 0.86 11.59 ± 1.76* t 1.621 6.154 2.057 5.158 1.851 4.861 P 0.574 0.001* 0.265 0.003* 0.468 0.008* 与治疗前相比,*P < 0.05;与常规教育组相比,#P < 0.05。 3. 讨论
本研究结果显示,O2O组失联率为7.88%,满意率为98.65%,疾病复发率为2.25%,3项指标均优于常规教育组。可见,O2O式健康教育可以提高患者对健康教育的满意度、减少复发率和失联率。O2O最先是一种电商模式,后来该理念逐渐延伸到教育、医疗领域。王玲等[8]采用基于“精准宣教”的家庭“O2O”宣教模式,发现可以提高老年结直肠癌患者的相关知识的知晓率;史逸秋等人[9]采用O2O教育管理模式可提高初始使用基础胰岛素治疗患者的血糖管理水平,均与本研究结果相似。O2O式健康教育的优势在于充分发挥线上和线下各自的优势:线上健康教育的优势在于可以打破空间限制,对居家患者进行远程干预和延续护理;而线下健康教育的优势在于让患者觉得亲近、真实、“看到见、摸的着”。而本研究另一处特色在于动态、合理调整线上线下的主次关系:患者在院接受治疗期间,执行线下为主、线上为辅的健康教育,其目的是充分利用与患者有限的接触时间以及面对面宣教的优势,并建立医患互信,同时让患者逐渐熟悉、过渡线上宣教模式;而患者治疗结束后主要采用线上健康教育,线下健康教育作为补充,最终达到达到提高健康教育效果的目的。
其次,O2O组在知识、态度和行为3项得分均高于常规教育组(P < 0.05),可见,O2O式健康教育可以提高患者知识、信念和行为能力。本研究中,线上健康教育主要时基于微信平台,微信是国内社交第一平台,患者熟悉,接受程度高。以微信群为载体,可以让患者聚集较为容易和方便。以微信群为载体,可以轻松实现宣教短视频推送、一对一沟通、线上讲座等等功能[10-12]。如果说微信是打破空间的限制,那么宣教短视频的推送,就是打破时间的限制。短视频这一形式,是近年来的热点技术,也是资本和资源追逐的风口[13]。而短视频不仅可以作为一种常规的娱乐工具,也为短视频这一形式进行健康教育提供可能性[14]。其次,短视频短小有趣,寓教于乐,不易引起学习疲劳;宣教短视频的拍摄和制作,虽然较为费时,但是具有一次拍摄、反复使用的优势,可以减少研究人员的工作量,提高健康教育的效率[15-17]。并且,本研究优化宣教短视频的推送环节,一是绝不盲目推送,而是从“共性与个性”的角度出发,制作出具有普适性推送的基础性视频和具有个性化推送的针对性视频,基础性视频以公域环境(微信群)推送,针对性视频在一对一的私域环境推送,这样其实是“精细护理”“个性化护理”的体现。其二,杜绝推送后就“撒手不管”,通过问卷星链接等形式健全反馈机制。其三,在线上管理方案中,做到“知、信、行”的兼顾,“知识、态度、行为”模式常简称“知信行”,即“knowledge,attitude(belief),practice”,是改变人类健康相关行为的模式之一。在“知识”方面,本研究通过健康宣教短视频提高痤疮的疾病认知,纠正错误认知;在“信念”方面,通过线上线下的心里干预、讲座、微信群中病友探讨等措施,提高患者的积极性;而在“行”方面,通过用药打卡、清洁面部打卡、膳食打卡来保证患者健康生活内容的贯彻和执行。
综上所述,O2O式健康教育不仅方便痤疮患者,易于患者的宣教知识接受,同样节约了医务工作者的时间和精力。本研究为回顾性分析,其数据源于医院信息系统,具有真实性、客观性。采用倾向性评分(PSM)能在一定程度上消除混杂因素。但是限于HIS资料以及选择偏倚、信息偏倚,可能对统计分析造成一定影响。今后将在本研究基础上,设置严谨的、前瞻的随机对照试验,从而获取高质量的循证证据。
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图 1 Morris水迷宫任务显示血栓通对APP/PS1小鼠空间学习记忆变化的影响
A:各组小鼠在训练期间从第1天到第6天的逃避潜伏期;B:探针实验测试期间在目标象限所花费的时间;C:探针实验中在目标平台的穿梭次数;D:探针实验中APP/PS1小鼠的游泳路径。($ \bar x \pm s $,n=15),*P < 0.05 vs. WT+Vehicle,#P<0.05 vs. APP/PS1+PNS。
Figure 1. Morris water maze task shows the effect of xueshuantong on spatial learning and memory changes in APP/PS1 mice
图 4 血栓通改变APP/PS1小鼠中Nav1.1α的分布和Navβ2的裂解
A:各组小鼠额叶皮层和海马中BACE1、Navβ2全长、Navβ2-CTF的蛋白电泳图;B:各组小鼠额叶皮层和海马中BACE1的蛋白表达比较;C:各组小鼠额叶皮层和海马中Navβ2全长的蛋白表达比较;D:各组小鼠额叶皮层和海马中Navβ2-CTF的蛋白表达比较;E:各组小鼠额叶皮层和海马中的Nav1.1α的总量、细胞外Nav1.1α和细胞内Nav1.1α的蛋白电泳图;F:各组小鼠额叶皮层和海马中Nav1.1α总量的蛋白表达比较;G:各组小鼠额叶皮层和海马中细胞外Nav1.1α的蛋白表达比较;H:各组小鼠额叶皮层和海马中细胞外Nav1.1α的蛋白表达比较。($ \bar x \pm s $,n=15),*P<0.05 vs. WT+ Vehicle,#P<0.05 vs. APP/PS1 + PNS。
Figure 4. Xueshuantong alters Nav1.1α distribution and Navβ2 cleavage in APP/PS1 mice
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