Effect on Danggui Shaoyao Powder on mitophagy in rat model of Alzheimer's disease based on PINK1-Parkin pathway / 中国中药杂志

This study investigated the mechanism of Danggui Shaoyao Powder(DSP) against mitophagy in rat model of Alzheimer's disease(AD) induced by streptozotocin(STZ) based on PTEN induced putative kinase 1(PINK1)-Parkin signaling pathway. The AD rat model was established by injecting STZ into the lateral ventricle, and the rats were divided into normal group, model group, DSP low-dose group(12 g·kg~(-1)·d~(-1)), DSP medium-dose group(24 g·kg~(-1)·d~(-1)), and DSP high-dose group(36 g·kg~(-1)·d~(-1)). Morris water maze test was used to detect the learning and memory function of the rats, and transmission electron microscopy and immunofluorescence were employed to detect mitophagy. The protein expression levels of PINK1, Parkin, LC3BⅠ/LC3BⅡ, and p62 were assayed by Western blot. Compared with the normal group, the model group showed a significant decrease in the learning and memory function(P<0.01), reduced protein expression of PINK1 and Parkin(P<0.05), increased protein expression of LC3BⅠ/LC3BⅡ and p62(P<0.05), and decreased occurrence of mitophagy(P<0.01). Compared with the model group, the DSP medium-and high-dose groups notably improved the learning and memory ability of AD rats, which mainly manifested as shortened escape latency, leng-thened time in target quadrants and elevated number of crossing the platform(P<0.05 or P<0.01), remarkably activated mitophagy(P<0.05), up-regulated the protein expression of PINK1 and Parkin, and down-regulated the protein expression of LC3BⅠ/LC3BⅡ and p62(P<0.05 or P<0.01). These results demonstrated that DSP might promote mitophagy mediated by PINK1-Parkin pathway to remove damaged mitochondria and improve mitochondrial function, thereby exerting a neuroprotective effect.

Baicalin inhibits LPS/IFN-γ-induced inflammation via TREM2/TLR4/NF-κB pathway in BV2 cells / 中国中药杂志

This study investigated the mechanism of baicalin on lipopolysaccharide(LPS)/interferon γ(IFN-γ)-induced inflammatory microglia based on the triggering receptor expressed on myeloid cells 2(TREM2)/Toll-like receptor 4(TLR4)/nuclear factor kappaB(NF-κB) pathway. Specifically, LPS and IFN-γ were used to induce inflammation in mouse microglia BV2 cells. Then the normal group, model group, low-dose(5 μmol·L~(-1)) baicalin group, medium-dose(10 μmol·L~(-1)) baicalin group, high-dose(20 μmol·L~(-1)) baicalin group, and minocycline(10 μmol·L~(-1)) group were designed. Cell viability was detected by CCK-8 assay and cell morphology was observed under bright field. The expression of interleukin-1β(IL-1β), interleukin-4(IL-4), inducible nitric oxide synthase(iNOS), interleukin-6(IL-6), interleukin-10(IL-10), and arginase-1(Arg-1) mRNA was detected by real-time quantitative PCR, the protein expression of tumor necrosis factor-α(TNF-α), IL-1β, TREM2, TLR4, inhibitor kappaB-alpha(IκBα), p-IκBα, NF-κB p65 and p-NF-κB p65 by Western blot, and transfer of NF-κB p65 from cytoplasm to nucleus by cellular immunofluorescence. Compared with the normal group, most of the BV2 cells in the model group tended to demonstrate the pro-inflammatory M1 amoeba morphology, and the model group showed significant increase in the mRNA levels of IL-1β, IL-6, and iNOS, decrease in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), rise of the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.01), reduction in TREM2 protein expression, and increase in the expression of NF-κB p65 in nucleus. Compared with the model group, baicalin groups and minocycline group showed the recovery of BV2 cell morphology, significant decrease in the mRNA levels of IL-1β, IL-6 and iNOS, increase in the mRNA levels of IL-4, IL-10, and Arg-1(P<0.01), reduction in the protein expression of TNF-α, IL-1β, TLR4, p-IκBα, and p-NF-κB p65(P<0.05), rise of TREM2 protein expression, and decrease in the expression of NF-κB p65 in nucleus. In summary, these results suggest that baicalin can regulate the imbalance between TREM2 and TLR4 of microglia and inhibit the activation of downstream NF-κB, thus promoting the polarization of microglia from pro-inflammatory phenotype to anti-inflammatory phenotype.

Analysis of Neuroprotective Effect of Danggui Shaoyaosan on APPswe/PS1ΔE9 Transgenic Mice Based on Circular RNA Sequencing / 中国实验方剂学杂志

Objective:To investigate the neuroprotective effects of Danggui Shaoyaosan （DSS） on APP_swe/PS1_ΔE9transgenic （APP/PS1） mice and its mechanism related to circular RNA （circRNA）. Method:Totally twenty 6-month-old APP/PS1 mice were divided into model group and DSS group， and 10 C57BL/6 wild-type mice were set as the normal control group. The normal group and model group received the same volume of normal saline， and DSS group received drug by gavage administration， all for 8 weeks. The differentially expressed circRNA of APP/PS1 mice before and after DSS intervention was analyzed by circRNA sequencing to construct circRNA-miRNA mRNA interaction network. The results of cricRNA sequencing were then verified by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of phosphoinositide 3-kinase （PI3K）， p-PI3K， protein kinase B1 （Akt1）， p-Akt1， B lymphocytoma-2 （Bcl-2）， and Bcl-2-Associated X protein （Bax） in the hippocampus were detected by immunoblotting （Western blot）. The protein expression of Caspase-3 in the hippocampus was detected by immunohistochemistry and the level of apoptosis in the hippocampus was detected by the TUNEL method. Result:Compared with the model group， there were 90 differentially expressed circRNA after intervention with DSS， of which 46 were up-regulated and 44 down-regulated. Compared with the normal group， the expression levels of circRNA1398 and circRNA1399 in the model group decreased， and the expression levels of miR-103-3p， miR-153-3p， miR-143-3p， and miR-143-5p increased. Compared with the model group， the expression levels of circRNA1398 and circRNA1399 in the DSS group were up-regulated， while the expression levels of miR-103-3p， miR-153-3p， miR-143-3p， and miR-143-5p were down-regulated. Compared with the normal group， the expression of p-PI3K， Akt1， p-Akt1， and Bcl-2 in the model group decreased （<italic>P</italic><0.05，<italic> P</italic><0.01）， and the expression of Bax and Caspase in the model group increased （<italic>P</italic><0.01）. Compared with the model group， the expression of p-PI3K， Akt1， p-Akt1， and Bcl-2 in the hippocampus of the DSS group increased （<italic>P</italic><0.01）， and the protein expression of Bax and Caspase decreased （<italic>P</italic><0.01）. Compared with the normal group， the apoptosis level in the hippocampus of the model group increased， with an apoptosis rate of （43.76±2.92）%. Compared with the model group， the apoptosis rate of DSS group was （24.64±3.39）%. Conclusion:DSS can activate PI3K/Akt pathway and inhibit apoptosis in hippocampal neurons of APP / PS1 mice， and play a neuroprotective role. The specific mechanism may be related to the regulation of circRNA1398 and circRNA1399 expression and the corresponding miRNA expression.

Danggui Shaoyaosan Inhibits Neuroinflammation in AD Rats by Regulating NLRP3/Caspase-1 Pathway / 中国实验方剂学杂志

Objective:To investigate the neuroprotective effect of Danggui Shaoyaosan （DSS） in a rat model of amyloid-<italic>β</italic>-peptide_1-42 （A<italic>β</italic>_1-42）-induced Alzheimer's disease （AD） as well as its regulatory effect on NOD-like receptor protein 3 （NLRP3）/cysteinyl aspartate-specific protease-1 （Caspase-1） signaling pathway. Method:The AD animal model was established via intracerebral injection of A<italic>β</italic>_1-42 and treated with different concentrations of DSS after the division of rats into the sham operation group， model group， as well as the high-， medium-， and low-dose DSS groups. Morris water maze test was conducted to determine the learning and memory abilities of rats. The morphology and function of neurons were detected by hematoxylin-eosin （HE） staining and Golgi staining， followed by immunofluorescence co-localization of NLRP3 inflammasome activation. The mRNA expression levels of interleukin （IL）-1<italic>β</italic> and IL-18 were measured by Real-time polymerase chain reaction （Real-time PCR）， and the protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β </italic>were assayed by Western blot. Result:Compared with the sham operation group， the model group exhibited significantly decreased learning and memory abilities （<italic>P</italic><0.01）， impaired neuronal morphology and function， up-regulated IL-1<italic>β</italic> and IL-18 mRNA expression， enhanced NLRP3 inflammasome activation， and elevated NLRP3， Caspase-1， and IL-1<italic>β</italic> protein expression （<italic>P</italic><0.01）. Compared with the model group， DSS at both medium and high doses remarkably improved the learning and memory abilities of AD rats （<italic>P</italic><0.05， <italic>P</italic><0.01）， restored neuronal morphology and function， down-regulated the mRNA expression levels of inflammatory factors IL-1<italic>β</italic> and IL-18， reduced the activation of NLRP3 inflammasomes， and lowered the protein expression levels of NLRP3， Caspase-1， and IL-1<italic>β</italic> （<italic>P</italic><0.01）. Conclusion:DSS inhibits inflammasome activation and neuroinflammatory response possibly by regulating the NLRP3/Caspase-1 signaling pathway， thus exerting the neuroprotective effect.

Extract of Naotaifang, a compound Chinese herbal medicine, protects neuron ferroptosis induced by acute cerebral ischemia in rats / 中西医结合学报

OBJECTIVE@#Our previous research showed that Naotaifang (a compound traditional Chinese herbal medicine) extract (NTE) has clinically beneficial effects on neurological improvement of patients with acute cerebral ischemia. In this study, we investigated whether NTE protected acute brain injury in rats and whether its effects on ferroptosis could be linked to the dysfunction of glutathione peroxidase 4 (GPX4) and iron metabolism.@*METHODS@#We established an acute brain injury model of middle cerebral artery occlusion (MCAO) in rats, in which we could observe the accumulation of iron in neurons, as detected by Perl's staining. Using assay kits, we measured expression levels of ferroptosis biomarkers, such as iron, glutathione (GSH), reactive oxygen species (ROS) and malonaldehyde (MDA); further the expression levels of transferrin receptor 1 (TFR1), divalent metal transporter 1 (DMT1), solute carrier family 7 member 11 (SLC7A11) and GPX4 were determined using immunohistochemical analysis, real-time quantitative polymerase chain reaction and Western blot assays.@*RESULTS@#We found that treatment with NTE reduced the expression levels of TFR1 and DMT1, reduced ROS, MDA and iron accumulation and reduced neurobehavioral scores, relative to untreated MCAO rats. Treatment with NTE increased the expression levels of SLC7A11, GPX4 and GSH, and the number of Nissl bodies in the MCAO rats.@*CONCLUSION@#Taken together, our data suggest that acute cerebral ischemia induces neuronal ferroptosis and the effects of treating MCAO rats with NTE involved inhibition of ferroptosis through the TFR1/DMT1 and SCL7A11/GPX4 pathways.

Effect of Danggui Shaoyaosan Ameliorates in Alleviating H2O2-induced SH-SY5Y Cells Oxidative Stress Through Modulation of NF-κB Pathway / 中国实验方剂学杂志

Objective::To investigate the regulatory effect of Danggui Shaoyaosan (DSS)-containing serum on oxidative stress and inflammation in H2O2-induced SH-SY5Y cells. Method::Methyl thiazolyl tetrazolium(MTT) assay was used to determine the cell activity and construct the H2O2-induced cell damage model with the optimal time and dose. Normal group, model group and high, medium and low-dose DSS groups(2.5%, 5%, 10%) were set up. MTT method was used to detect cell activity, spectrophotometry anti-oxidation indexes of malonaldehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) and glutathione (GSH). Real-time quantitative PCR (Real-time PCR) was used to detect tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) mRNA expressions. And immunofluorescence test was adopted to detect nuclear transcription factor-κB(NF-κB) p65 nuclear translocation of the DSS after the intervention. Result::After 24 h intervention with 250 μmol·L-1 H2O2, SH-SY5Y cell viability was about 55%, which was the best modeling condition. After high, medium and low-dose DSS intervention on H2O2-damaged cell model, compared with the model group, the cell activity showed a dose-dependent increase (P<0.05), MDA was significantly reduced (P<0.05), and antioxidant indexes CAT, SOD and GSH were significantly increased (P<0.05). H2O2 could significantly increase SH-SY5Y cell inflammatory factor TNF-α, IL-6 and IL-1β mRNA expressions, and promote activation of cytoplasmic NF-κB and nuclear translocation. DSS-containing serum showed a dose-dependent inhibition of NF-κB p65 from nuclear, and reduced inflammatory factor levels, such as TNF-α, IL-6 and IL-1β. Conclusion::DSS-containing serum can significantly reduce the oxidative damage in H2O2-induced SH-SY5Y cells by improving their antioxidant status, and reduce the inflammatory response by inhibiting NF-κB signaling pathway.

Effect of Jiawei-Naotai formula on ATF4/CHOP/Puma pathway of ovariectomized rats with cerebral ischemia / 中国病理生理杂志

AIM:To investigate the effects of Jiawei-Naotai formula (JWNTF) on ATF4/CHOP/Puma pathway in hippocampal neurons of ovariectomized female rats with cerebral ischemia.METHODS:The female rats were randomly divided into sham group, model group, JWNTF group and positive control group.The rats, expect in the sham group, were ovariectomized.The rats in each group were intragastric administration 11 days after ovariectomy.The rats in sham group and model group were given a gavage of 0.9％Na Cl, while the rats in other groups were administrated by corresponding therapy intragastrically for 3 d.The regional cerebral ischemia model was established by middle cerebral artery occlusion (MCAO) suture method 14 days after ovariectomy.The behaviors of the rats were evaluated 24 h after cerebral ischemia.The mRNA levels of Bax, Bcl-2 and caspase-3 were detected by RT-qPCR, and the protein expression of Bax, Bcl-2, caspase-3, ATF4, CHOP and Puma was determined by Western blot.RESULTS:Compared with sham group, the neurobehavioral scores significantly increased in other groups (P<0.05).Compared with model group, the neurobehavioral scores were significantly decreased in positive control group and JWNTF group (P<0.05).The protein expression of Bax, caspase-3, ATF4, CHOP and Puma, and the mRNA expression of Bax and caspase-3 in the hippocampus were much higher, and Bcl-2 was lower in model group than those in sham group (P<0.05).JWNTF significantly reduced the protein expression of Bax, caspase-3, ATF4 and CHOP, and the mRNA expression of Puma, Bax and caspase-3, and markedly increased the expression of Bcl-2 at mRNA and protein levels compared with model group.CONCLUSION:The JWNTF protects against brain damage induced by cerebral ischemia, which may be related to inhibitiing the expression of ATF4/CHOP/Puma pathway-related molecules at mRNA and protein levels.

Effect of Jiawei Naotaifang on cerebral infarction area and level of estrogen of ovariectomized rats with cerebral ischemia and its correlation / 中国药理学通报

Aim To investigate the effects of Jiawei Naotaifang on cerebral infarction area, pathological changes of brain tissue and estrogen level of focal cere-bral Iischemia in female ovariectomized rats, and cor-relation between estrogen levels and cerebral infarction area. Methods SD rats were randomly divided into sham operation group, ovariectomized group, cerebral ischemia group,model group,and drug groups(estro-gen group, Jiawei Naotaifang high dose group, Jiawei Naotaifang middle dose group, Jiawei Naotaifang low dose group). The rats in the ovariectomized group, model group, drug groups were ovariectomized, elev-enth days after the ovariectomy. The rats in the drug groups were given intragastric administration for three days. The rats in the model group, cerebral ischemia group and drug groups were prepared for cerebral is-chemia models. Neurological function scores were scored 24 hours after the success of the model, serum levels of estrogen were detected, and the brain was stained with 2, 3, 5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin staining(HE), TTC staining was used to measure the area of cerebral in-farction, and HE staining was used to observe the pathological changes of brain tissues. Results Com-pared with cerebral ischemia group,cerebral infarction area of rats in the model group increased significantly, the estrogen level was lower and the necrosis and py-knosis of cortical and hippocampus cells of rats in the model group were more obvious. Compared with model group,the cerebral infarction area of rats in the drug groups was reduced,the estrogen levels were elevated, especially in Jiawei Naotaifang high dose group and es-trogen group. The cell morphology of rats,in the estro-gen group,Jiawei Naotaifang high dose group and mid-dle dose group, was improved obviously. Cerebral in-farction area was negatively correlated with the level of estrogen. Conclusions The cerebral infarction area of cerebral ischemia in female ovariectomized rat is signif-icantly correlated with the level of estrogen. Jiawei Naotaifang can reduce the damage and alleviate brain injury of cerebral ischemia in female ovariectomized rats,which may be related to the improvement of estro-gen level.

Analysis on medication rules for treatment of dementia by ancient physicians based on data mining methods / 中国中药杂志

To analyze the medication features and regularity of prescriptions of Chinese medicine in treating patients with dementia based on ancient medical records. In the article, we retrieved the ancient medical records related to the treatment of dementia (from the Han Dynasty to the late Qing period) in Chinese Medical Classics, Chinese Ancient Medical Books and digital library, and then set up a medical records normalized database. The medication features and prescription rules for dementia were analyzed by frequency statistics and association rules (Apriori algorithm, improved mutual information algorithm and complex system entropy clustering). Finally, a total of 156 prescriptions were selected, involving 123 Chinese herbs, with a total frequency of 11 747 for the herbs, and 8 core prescriptions were mined. After the association rules between the frequency and prescriptions for the treatment of dementia were determined, we found that the most commonly used herbs included Fuling (Poria), Yuanzhi (Polygalae Radix), Renshen (Ginseng Radix et Rhizoma), Shichangpu (Acori Tatarinowii Rhizoma), Gancao (Glycyrrhizae Radix et Rhizoma), Danggui (Angelicae Sinensis Radix), Maidong (Ophiopogonis Radix), Baizhu (Bletillae Rhizoma), Dihuang (Rehmanniae Radix) and Ganjiang (Zingiberis Rhizoma); the frequently-used drugs compatibility was mainly for tonifying Qi-blood, regulating Yin and Yang and inducing resuscitation. The drugs were mainly of warm nature and sweet (mild) flavor, and the channel tropism of drugs mainly distributed to the heart, liver, spleen and kidney. The core prescriptions were composed of Renshen (Ginseng Radix et Rhizoma), Fuling (Poria), Yuanzhi (Polygalae Radix), Shichangpu (Acori Tatarinowii Rhizoma), and Baizhu(Bletillae Rhizoma). In conclusion, high frequency herbs and core prescriptions reflect the prescriptions by ancient physicians mainly focus on Qi-replenishing, spleen-invigorating and heart-nourishing, but also reflect the prescription rules of nourishing Yin, enriching blood, eliminating phlegm and warming Yang for the treatment of dementia. The medication features and prescription rules for the treatment of dementia obtained by association rules are useful to guide the clinical practice of Chinese medicine in treatment of dementia.

Effect of Jiawei Naotaifang on Activation of Extracellular Signal-regulated Kinase 1/2 and c-Jun N-terminal Kinase in Ovariectomized Rats after Cerebral Ischemia / 中国康复理论与实践

Objective To investigate the effect of Jiawei Naotaifang on neuronal apoptosis and the mechanism in ovariectomized rats with cerebral ischemia. Methods Female Sprague-Dawley rats(n=40)were randomly divided into sham group(n=10),model group(n=10),es-trogen group(n=10)and Jiawei Naotaifang group(n=10).The model group,estrogen group and Jiawei Naotai-fang group were ovariectomized.Eleven days after ovariectomy,the estrogen group and Jiawei Naotaifang group were given estrogen and Jiawei Naotaifang respectively intragastrically for three days.14 days after ovariecto-my,the model group,estrogen group and Jiawei Naotaifang group were modeled cerebral ischemia with Langa's method.24 hours after modeling,the apoptosis rate of neurons was detected with TUNEL,and the activation of extracellular signal-regulated kinase 1/2(ERK1/2)and c-Jun N-terminal kinase(p-JNK)in hippocampus were de-tected with Western blotting. Results Compared with the model group, the apoptosis rates decreased in Jiawei Naotaifang group and the estrogen group(P<0.001),with more activation of ERK1/2(P<0.01)and less activation of JNK(P<0.01). Conclusion Jiawei Naotaifang can protect neuron from apoptosis by promoting the activation of ERK1/2 and inhibiting the activation of p-JNK.