“Baihui”(DU20)-penetrating “Qubin”(GB7) acupuncture on blood–brain barrier integrity in rat intracerebral hemorrhage models via the RhoA/ROCK Ⅱ/MLC 2 signaling pathway

Background: Blocking the Rho A/ROCK Ⅱ/MLC 2(Ras homolog gene family member A/Rho kinase Ⅱ/myosin light chain 2) signaling pathway can initiate neuroprotective mechanisms against neurological diseases such as stroke, cerebral ischemia, and subarachnoid hemorrhage. Nevertheless, it is not clear whether and how disrupting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway changes the pathogenic processes of the blood–brain barrier(BBB) after intracerebral hemorrhage(ICH). The present investigation included the injection of rat caudal vein blood into the basal ganglia area to replicate the pathophysiological conditions caused by ICH. Methods: Scalp acupuncture(SA) therapy was performed on rats with ICH at the acupuncture point “Baihui”-penetrating “Qubin,” and the ROCK selective inhibitor fasudil was used as a positive control to evaluate the inhibitory effect of acupuncture on the Rho A/ROCK Ⅱ/MLC 2 signaling pathway. Post-assessments included neurological deficits, brain edema, Evans blue extravasation, Western blot, quantitative polymerase chain reaction, and transmission electron microscope imaging. Results: We found that ROCK Ⅱ acts as a promoter of the Rho A/ROCK Ⅱ/MLC 2 signaling pathway, and its expression increased at 6 h after ICH, peaked at 3 days, and then decreased at 7 days after ICH, but was still higher than the preintervention level. According to some experimental results, although 3 days is the peak, 7 days is the best time point for acupuncture treatment. Starting from 6 h after ICH, the neurovascular structure and endothelial cell morphology around the hematoma began to change. Based on the changes in the promoter ROCK Ⅱ, a 7-day time point was selected as the breakthrough point for treating ICH model rats in the main experiment. The results of this experiment showed that both SA at “Baihui”-penetrating “Qubin” and treatment with fasudil could improve the expression of endothelial-related proteins by inhibiting the Rho A/ROCK Ⅱ/MLC 2 signaling pathway and reduce neurological dysfunction, brain edema, and BBB permeability in rats. Conclusion: This study found that these experimental data indicated that SA at “Baihui”-penetrating “Qubin” could preserve BBB integrity and neurological function recovery after ICH by inhibiting Rho A/ROCK Ⅱ/MLC 2 signaling pathway activation and by regulating endothelial cell–related proteins.

青光安Ⅱ号方对DBA/2J小鼠视网膜中Rho/ROCK相关因子的影响

目的:观察青光安Ⅱ号方对DBA/2J小鼠视网膜中RhoA mRNA、ROCK mRNA、Caspase-3mRNA及Bcl-2 mRNA转录的影响。方法:使用48只DBA/2J小鼠随机平均分成模型组、青光安Ⅱ号汤剂组、青光安Ⅱ号有效组分低、中、高浓度组及阳性对照组(益脉康分散片组)6组,同时使用8只C57BL/6J小鼠作为空白组,DBA/2J小鼠喂养到38周后形成青光眼模型,干预4周后,采用实时荧光定量PCR(Quantitative Real-time,PCR)检测DBA/2J小鼠视网膜中RhoA mRNA、ROCK mRNA、Caspase-3 mRNA及Bcl-2 mRNA的转录情况。结果:干预4周后,在RhoA mRNA、ROCK mRNA和Caspase-3 mRNA的转录中,与空白组相比,其它6组的相对表达量均升高,模型组、益脉康分散片组及青光安Ⅱ号方有效组分低浓度组有统计学差异(P<0.05);与模型组相比较,其它6组的相对表达量均低于模型组,其中RhoA mRNA转录中的青光安Ⅱ号方汤剂组和青光安Ⅱ号方有效组分高浓度组有统计学差异(P<0.05);ROCK mRNA和Caspase-3 mRNA的转录中,模型组与青光安Ⅱ号方有效组分中、高浓度组统计学有差异(P<0.05);在Bcl-2 mRNA转录中,与空白组比较,其它6组相对表达量均下降,模型组、青光安Ⅱ号方汤剂组和青光安Ⅱ号方有效组分低浓度组统计学有差异(P<0.05);青光安Ⅱ号方有效组分中、高浓度组的Bcl-2 mRNA的相对表达量均高于模型组,差异有统计学意义(P<0.05)。结论:高浓度青光安Ⅱ号方可能是通过抑制Rho/ROCK信号通路而减弱了视网膜神经节细胞(RGCs)中Caspase-3的转录,激活了Bcl-2的表达,从而减弱了RGCs的凋亡。

Effect of QingguanganⅡon Rho/ROCK associated factors in the retina of DBA/2J mice

Objective:The effect of QingguanganⅡon the transcription of RhoA mRNA,ROCK mRNA,Caspase-3 mRNA and Bcl-2 mRNA in the retina of DBA/2J mice was observed.Methods:Forty-eight DBA/2J mice were randomly divided into six groups:model groups,Qingguangan II decoction group,low concentration,medium concentration and high concentration group of Qingguangan II effective ingredient and positive control group(Yimaikang tablet group),and eight C57BL/6 mice were used as blank group,DBA/2J mice were fed until 38 weeks before forming a glaucoma model,The transcription of RhoA mRNA,ROCK mRNA,Caspase-3 mRNA and Bcl-2 mRNA in the retinal of DBA/2J mice was detected using real-time fluorescence quantitative PCR(Quantitative Real-time PCR)after 4 weeks of intervention.Results:Four weeks after the intervention,In the transcription of the RhoA mRNA,ROCK mRNA and the Caspase-3 mRNA,Compared to the blank groups,Relative expression was increased in the other 6 groups,There are statistical differences in the model group,Yimaikang tablet group and low concentration group(P<0.05);In comparison to the model groups,The other 6 groups were lower than the model group,Among them,there are statistical differences between the effective groups of Qingguangan II decoction and high concentration group of Qingguangan II effective ingredient in RhoA mRNA transcription(P<0.05);In the transcription of the ROCK mRNA and the Caspase-3 mRNA,Statistics have differences between the model group and the effective component of the medium and high concentration group(P<0.05);In the Bcl-2 mRNA transcription,Compare them to blank groups,Relexpression expression decreased in the other 6 groups,Statistics have differences between model group,Qingguangan II decoction group and low concentration groups(P<0.05);The relative expression of Bcl-2 mRNA in high concentration group of effective component is higher than that of the model group,There are differences in statistics(P<0.05).Conclusion:The high concentration of QingguanganⅡprescription probably attenuated Caspase-3 transcription in retinal ganglion cells by inhibiting the Rho/ROCK signaling pathway and activated Bcl-2 expression by inhibiting ROCK signaling,which attenuated apoptosis in retinal ganglion cells.

腺苷蛋氨酸对过度激活的Rho-ROCK通路介导的糖尿病肝损伤的改善作用

目的 探讨腺苷蛋氨酸对过度激活的Rho-ROCK通路介导的糖尿病肝损伤的改善作用。方法 40只清洁级Wistar大鼠依据随机数字表法为4组：对照组、2型糖尿病组、Y27632干预组及腺苷蛋氨酸干预组,各10只。对照组大鼠给予正常饲粮,自由饮水;糖尿病组、Y27632干预组及腺苷蛋氨酸干预组大鼠每日给予高糖高脂饲料,自由饮水,并于第35日时一次性腹腔注射链脲佐菌素（35 mg/kg,腹膜内）;Y27632干预组于第9-12周给予Y27632 10 mg/kg进行治疗。腺苷蛋氨酸干预组于第9-12周给予腺苷蛋氨酸10 mg/kg进行治疗。分析各组大鼠胰岛素抵抗系数、血脂水平及Rho A、ROCK1/2和基质金属蛋白酶的表达变化。同时分析各组大鼠肝脏组织细胞凋亡相关蛋白胱天蛋白酶（caspase）-3及caspase-9的表达变化。结果 糖尿病组、Y27632干预组和腺苷蛋氨酸干预组大鼠的胰岛素抵抗指数稳态模型、血糖、低密度脂蛋白胆固醇较对照组明显升高（P〈0.01）;糖尿病组、Y27632干预组和腺苷蛋氨酸干预组大鼠的总胆固醇高于对照组（P〈0.01）;糖尿病组、Y27632干预组和腺苷蛋氨酸干预组大鼠的高密度脂蛋白胆固醇低于对照组（P〈0.01）;糖尿病组、Y27632干预组和腺苷蛋氨酸干预组大鼠的羟脯氨酸高于对照组（P〈0.01）。糖尿病组大鼠肝脏组织的Rho及ROCK1/2蛋白的表达、MMP-2及MMP-9的表达及促凋亡蛋白相关蛋白caspase-3和caspase-9的表达较对照组明显升高（P〈0.01）,而Y27632干预组和腺苷蛋氨酸干预组上述异常表达得到一定的恢复（P〈0.01）。结论 腺苷蛋氨酸通过抑制肝脏组织中过度激活的Rho/ROCK信号转导通路改善小剂量SZT合并高糖高脂饮食复制的2型糖尿病大鼠肝脏组织损伤。

BKca下调RhoA/ROCK信号通路改善糖尿病大鼠阴茎勃起功能

目的:观察大电导钙激活钾通道(big conductance Ca2+-activated K+channel,BKca)对糖尿病勃起功能障碍(diabetes mellitus-induced erectile dysfunction,DMED)大鼠阴茎海绵体平滑肌Rho A/ROCK信号通路的影响。方法:实验大鼠分为空白对照组8只,DMED组8只,NS1619组(DMED治疗组)7只,NS1619组大鼠采用特异性BKca激活剂(NS1619)阴茎海绵体注入2周。观察各组勃起行为,并电刺激检测阴茎海绵体内压(intracavernous pressure,ICP)/平均动脉压(mean arterial pressure,MAP),取各组阴茎海绵体平滑肌检测肌张力,采用Western blot和定量PCR(real-time PCR)方法检测Rho A、ROCK1、ROCK2表达,反应Rho A/ROCK信号通路差异,同时检测MYPT-1表达反应肌球蛋白轻链磷酸酶(myosin light chain phosphatase,MLCP)磷酸化程度。结果:成功构建DMED大鼠模型,NS1619组大鼠与DMED组大鼠比较,勃起次数和ICP/MAP明显改善(P=0.025、0.024),阴茎海绵体平滑肌舒缩顺应性提高(P=0.031、0.024)并且Rho A、ROCK2以及肌球蛋白磷酸酶靶向结合亚基(myosin phosphatase target subunit,MYPT)-1蛋白和m RNA表达水平下调(P=0.029、0.003、0.002),但仍未达到正常对照组大鼠水平(P=0.018、0.040、0.003),ROCK1表达无明显差异(P=0.533)。结论:DMED大鼠因Rho A/ROCK信号通路上调和MLCP磷酸化增强导致阴茎海绵体平滑肌舒缩顺应性降低以致勃起功能障碍发生,激活BKca可以通过下调Rho A/ROCK信号通路和抑制MLCP磷酸化,改善勃起功能。

Catharmus tinctorius volatile oil promote the migration of mesenchymal stem cells via ROCK2/Myosin light chain signaling

MSC transplantation has been explored as a new clinical approach to stem cell-based therapies for bone diseases in regenerative medicine due to their osteogenic capability. However, only a small population of implanted MSC could successfully reach the injured areas. Therefore, enhancing MSC migration could be a beneficial strategy to improve the therapeutic potential of cell transplantation. Catharmus tinctorius volatile oil(CTVO) was found to facilitate MSC migration. Further exploration of the underlying molecular mechanism participating in the pro-migratory ability may provide a novel strategy to improve MSC transplantation efficacy. This study indicated that CTVO promotes MSC migration through enhancing ROCK2 mRNA and protein expressions. MSC migration induced by CTVO was blunted by ROCK2 inhibitor, which also decreased myosin light chain(MLC) phosphorylation.Meanwhile, the si RNA for ROCK2 inhibited the effect of CTVO on MSC migration ability and attenuated MLC phosphorylation,suggesting that CTVO may promote BMSC migration via the ROCK2/MLC signaling. Taken together, this study indicates that C.tinctorius volatile oil could enhance MSC migration via ROCK2/MLC signaling in vitro. C. tinctorius volatile oil-targeted therapy could be a beneficial strategy to improve the therapeutic potential of cell transplantation for bone diseases in regenerative medicine.

Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.

白术内酯Ⅰ通过SGLT1/NHE3通路治疗腹泻的机制研究

目的:探讨白术内酯Ⅰ通过SGLT1/NHE3通路治疗腹泻的机制。方法:Caco-2细胞建立体外肠道吸收模型,将细胞分别设为空白对照组、抑制剂(SGLT1抑制剂根皮苷、P38MAPK抑制剂SB203580、Ezrin抑制剂NSC668394)组、白术内酯Ⅰ组、白术内酯Ⅰ+抑制剂组,采用2-NBDG测定白术内酯Ⅰ对Caco-2细胞葡萄糖摄取的影响;Western Blot法检测SGLT1、p-P38MAPK、p-MAPKAPK2、p-AKT2、p-Ezrin、MLCK、p-MLC、NHE3等蛋白表达,荧光定量PCR检测SGLT1、MLCK、NHE3 mRNA表达。结果:白术内酯Ⅰ促进Caco-2细胞葡萄糖摄取,上调SGLT1、NHE3、MLCK的表达;此外,白术内酯Ⅰ增加了MLCK和P38MAPK/Ezrin信号通路中的蛋白磷酸化。结论:白术内酯Ⅰ可激活SGLT1促进Caco-2细胞葡萄糖摄取,通过P38MAPK/Ezrin信号通路上调其下游靶点NHE3和MLCK促进水钠吸收是其治疗腹泻的机制之一。

重组人红细胞生成素对高糖诱导人肾小管上皮细胞增殖及凋亡的影响及其可能机制

目的探讨重组人红细胞生成素(rh EPO)对高糖诱导的正常人肾小管上皮(HK-2)细胞增殖及凋亡的影响及其可能机制。方法将体外培养的HK-2细胞按随机数字表法分为空白对照组、高糖诱导组(高糖终浓度为30mmol/L)、甘露醇对照组(甘露醇浓度为24.5 mmol/L)、rh EPO对照组(rh EPO终浓度为20 U/m L)、不同浓度rh EPO干预组(高糖终浓度为30 mmol/L+rh EPO终浓度分别为5、10、20 U/m L)及Rho激酶抑制剂(Y27632)组(Y27632终浓度为30μmol/L+高糖终浓度为30 mmol/L),各组均刺激24 h。应用RT-PCR法检测各组HK-2细胞Rho A、ROCK1 m RNA的表达;MTT法测定细胞增殖,流式细胞技术分析细胞凋亡。结果高糖诱导组Rho A及ROCK1m RNA表达较空白对照组显著升高(P<0.05),不同浓度rh EPO干预组Rho A m RNA及ROCK1 m RNA的表达较高糖诱导组显著减少(P<0.05),高糖诱导组及不同浓度rh EPO干预组Rho A m RNA与ROCK1 m RNA表达呈正相关。rh EPO可明显促进HK-2细胞增殖(P<0.05),而高糖可诱导正常人肾小管上皮细胞凋亡,加入不同浓度rh EPO或Y27632干预后,其凋亡明显受抑制(P<0.05),且在实验rh EPO浓度范围内,rh EPO促进增殖及抑制凋亡的作用呈现浓度依赖性。结论 rh EPO可促进高糖诱导的HK-2细胞增殖,抑制高糖诱导的HK-2细胞凋亡,其机制可能与阻断Rho A/ROCK信号通路有关。

rHuEPO对肾间质纤维化过程中炎症因子的影响及其可能作用机制

目的临床治疗虽可延缓肾间质纤维化进展,却无法逆转肾功能丧失。探讨重组人促红细胞生成素(recombinant human erythropoietin,r Hu EPO)对肾间质纤维化过程中炎症因子的影响及其可能作用机制。方法将体外培养的HK-2细胞随机分为7组:空白对照组、r Hu EPO对照组(20 U/m L r Hu EPO)、清蛋白刺激组(5 mg/m L清蛋白)、5 U/m L r Hu EPO干预组(5 mg/m L清蛋白+5 U/m L r Hu EPO)、10 U/m L r Hu EPO干预组(5 mg/m L清蛋白+10 U/m L r Hu EPO)、20 U/m L r Hu EPO干预组(5 mg/m L清蛋白+20 U/m L r Hu EPO)、Rho激酶抑制组(10μmol/L Y27632+5 mg/m L清蛋白),各组均作用24 h。观察各组细胞形态的变化;RT-PCR检测各组细胞Rho A、ROCK1 mRNA及白细胞介素-6因子(interleukin-6,IL-6)mRNA含量水平;ELISA检测细胞上清液中肿瘤坏死因子(tumor necrosis factor,TNF-α)、IL-6蛋白的含量表达。结果空白对照组、r Hu EPO干预组显示鹅卵石或者铺路石样形态,清蛋白刺激组表现出细长梭状改变,呈现纤维细胞样外观。5、10、20 U/m L r Hu EPO干预组细胞向鹅卵石样复转,Rho激酶抑制组细胞形态呈椭圆形、细胞间隙稍增大;与空白对照组比较,清蛋白刺激组Rho A、ROCK1 mRNA及IL-6 mRNA显著升高(P<0.05),而5、10、20 U/m L r Hu EPO干预组逐渐下调(P<0.05),且与r Hu EPO浓度负相关;与清蛋白刺激组比较,Rho激酶抑制组ROCK1 mRNA、IL-6 mRNA表达下调(P<0.05),但Rho A mRNA表达差异无统计学意义(P>0.05)。ELISA结果显示:清蛋白刺激组上清液TNF-α、IL-6蛋白[(1 347.54±41.52)ng/L、(884.62±0.73)pg/L]表达较空白对照组[(452.32±33.23)ng/L,(95.12±0.32)pg/L]显著增高(P<0.05),5、10、20 U/m L r Hu EPO干预组、Rho激酶抑制组TNF-α表达[(1 003.32±3.42)、(821.32±21.32)、(590.15±7.68)、(488.13±65.03)ng/L)]较清蛋白刺激组[(1 347.54±41.52)ng/L]下降(P<0.05)、IL-6蛋白表达[(656.68±0.55)、(422.35±0.22)、(217.32±0.35)、(309.49±0.21)pg/L]亦较清蛋白刺激组[(884.62±0.73)pg/L]下降(P<0.05),5、10、20 U/m L r Hu EPO干预组组间两两比较差异均有统计学意义(P<0.05)。结论 r Hu EPO可能通过减少炎症因子的产生来抑制清蛋白诱导的HK-2细胞转分化过程,其作用机制部分涉及Rho A/ROCK信号通路。

Houshiheisan and its components promote axon regeneration after ischemic brain injury

Houshiheisan,a classic prescription in traditional Chinese medicine,contains Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari,Radix Platycodonis,Rhizoma Atractylodis macrocephalae,Poria,Rhizoma Zingiberis,Radix Angelicae sinensis,Radix et Rhizoma Ginseng,Radix Scutellariae and Concha Ostreae.According to traditional Chinese medicine theory,Flos Chrysanthemi,Radix Saposhnikoviae,Ramulus Cinnamomi,Rhizoma Chuanxiong,Radix et Rhizoma Asari and Radix Platycodonis are wind-dispelling drugs;Rhizoma Atractylodis macrocephalae,Poria,Rhizoma Zingiberis,Radix Angelicae sinensis and Radix et Rhizoma Ginseng are deficiency-nourishing drugs.A large number of randomized controlled trials have shown that Houshiheisan is effective in treating stroke,but its mechanism of action is unknown.Axonal remodeling is an important mechanism in neural protection and regeneration.Therefore,this study explored the effect and mechanism of action of Houshiheisan on the repair of axons after cerebral ischemia.Rat models of focal cerebral ischemia were established by ligating the right middle cerebral artery.At 6 hours after model establishment,rats were intragastrically administered 10.5 g/kg Houshiheisan or 7.7 g/kg wind-dispelling drug or 2.59 g/kg deficiency-nourishing drug.These medicines were intragastrically administered as above every 24 hours for 7 consecutive days.Houshiheisan,and its wind-dispelling and deficiency-nourishing components reduced the neurological deficit score and ameliorated axon and neuron lesions after cerebral ischemia.Furthermore,Houshiheisan,and its wind-dispelling and deficiency-nourishing components decreased the expression of proteins that inhibit axonal remodeling：amyloid precursor protein,neurite outgrowth inhibitor protein A（Nogo-A）,Rho family small GTPase A（Rho A） and Rho-associated kinase 2（Rock2）,and increased the expression of growth associated protein-43,microtubule-associated protein-2,netrin-1,Ras-related C3 botulinum toxin substrate 1（Rac1） and cell division cycle 42（Cdc42）.The effect of Houshiheisan was stronger than wind-dispelling drugs or deficiency-nourishing drugs alone.In conclusion,Houshiheisan,and wind-dispelling and deficiency-nourishing drugs promote the repair of axons and nerve regeneration after cerebral ischemia through Nogo-A/Rho A/Rock2 and Netrin-1/Rac1/Cdc42 signaling pathways.These effects are strongest with Houshiheisan.

体外沉默ICMT基因对人唾液腺腺样囊性癌细胞侵袭和迁移的影响

目的 探讨异戊二烯基半胱氨酸羧基甲基转移酶(isoprene cysteine carboxymethyl transferase,ICMT)基因对唾液腺腺样囊性癌(salivary adenoid cystic carcinoma,SACC)细胞迁移和侵袭的影响及相关机制,为SACC的分子靶向治疗提供实验依据。方法 体外培养腺样囊性癌细胞SACC-LM和SACC-83,采用脂质体载体瞬时转染的方法,将ICMT siRNA转染至人SACC-LM和SACC-83细胞中(实验组),并分别设置空白对照组和阴性对照组(转染NC-siRNA)。通过qRT-PCR检测转染后各组细胞中ICMT和RhoA的m RNA表达并明确沉默效率;Western blot检测各组的ICMT、膜RhoA、总RhoA、Rho关联含卷曲螺旋结合蛋白激酶1(Rho associations contain curly helical binding protein kinase 1,ROCK1)、基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)蛋白表达;通过CCK-8实验检测SACC细胞的增殖能力;通过比较细胞划痕实验的相对愈合面积和Transwell实验细胞穿膜数目,分别检测SACC细胞的迁移和侵袭能力。结果 SACC-LM和SACC-83细胞转染ICMT-siRNA后,实验组相较于空白对照组和阴性对照组,ICMT mRNA和蛋白表达显著下降(P<0.05),但RhoA mRNA和RhoA总蛋白表达均无显著性差异(P>0.05);膜RhoA、ROCK1、MMP-2、MMP-9的蛋白表达显著下降(P<0.05),细胞增殖能力明显下降(P<0.05),迁移和侵袭能力明显降低(P<0.05)。结论 体外沉默ICMT基因可有效抑制人SACC-LM和SACC-83细胞迁移及侵袭能力,其机制可能与RhoA-ROCK信号通路有关。