Liuwei Dihuangtang Alleviates Myelin Injury in vHIP and Depression-like Behavior of Diabetes Mellitus Combined with Comorbid Depression Rats via AMPK/Akt/GSK3β/Nrf2 Pathway / 中国实验方剂学杂志

ObjectiveTo observe the effect of Liuwei Dihuangtang on depression-like behavior of diabetes mellitus combined with comorbid depression （DD） rats， so as to explore its action mechanism. MethodFifty male SD rats of SPF grade were fed with high fat diet and injected with low-dose streptozotocin （STZ） via tail vein for inducing diabetes. Afterwards， the diabetic rats were exposed to chronic unpredictable mild stress （CUMS） for 28 d. The successfully modeled DD rats were randomly divided into five groups： model group， fluoxetine （10 mg·kg-1·d-1） group， and low-， medium-， and high-dose （3.375， 6.75， 13.5 g·kg-1·d-1） Liuwei Dihuangtang groups， with 10 in each group. Another 10 rats were classified into the normal control group and treated with intragastric administration of normal saline for four weeks. The tail suspension test and open field test were conducted to evaluate the depressive-like phenotype of rats. The contents of malondialdehyde （MDA）， reactive oxygen species （ROS）， 8-hydroxy-2 deoxyguanosine （8-OHdG）， superoxide dismutase （SOD）， and glutathione （GSH） in ventral hippocampus （vHIP） were measured by enzyme-linked immunosorbent assay （ELISA）， and the myelin basic protein （MBP） expression in vHIP by immunofluorescence assay. The expression levels of MBP， myelin protein lipoprotein （PLP）， myelin oligodendrocyte glycoprotein （MOG）， phosphorylated adenosine 5'-monophosphate-activated protein kinase （p-AMPK）/AMPK， phosphorylated protein kinase B （p-Akt）/Akt， phosphorylated glycogen synthase kinase 3β （p-GSK3β）/GSK3β， and nuclear factor erythroid-2 related factor 2（Nrf2） were determined by Western blotting. ResultCompared with the normal control group， the model group exhibited significantly prolonged immobility in the tail suspension test （P<0.01） and shortened residence at the central area in the open field test （P<0.01）. The immobility time in the medium- and high-dose Liuwei Dihuangtang groups declined to different degrees as compared with that of the model group （P<0.01）， while the residence time at the central area was significantly increased （P<0.05， P<0.01）. Compared with the normal control group， the model group displayed down-regulated MBP， PLP， and MOG protein expression in vHIP （P<0.01）. Compared with the model group， Liuwei Dihuangtang at the low dose up-regulated the expression of MBP （P<0.05）， but did not obviously affect the expression of MOG and PLP. Fluoxetine and Liuwei Dihuangtang at the medium and high doses up-regulated the expression of MBP， PLP， and MOG （P<0.05， P<0.01）. Comparison with the normal control group revealed that the MBP fluorescence intensity in vHIP of the model group was significantly weakened （P<0.01）. After the intervention， the MBP fluorescence intensities in the medium- and high-dose Liuwei Dihuangtang groups and fluoxetine group were enhanced in contrast to that of the model group （P<0.05， P<0.01）. SOD and GSH in the model group were lower than those in the normal control group （P<0.01）， whereas the MDA， ROS， and 8-OHdG expression levels were higher （P<0.01）. Compared with the model group， Liuwei Dihuangtang at the medium and high doses and fluoxetine all down-regulated the expression levels of MDA， ROS， and 8-OHdG （P<0.05，P<0.01）， while up-regulated SOD and GSH expression （P<0.05，P<0.01）. The expression levels of p-AMPK， p-Akt， and Nrf2 in the model group were down-regulated as compared with those in the control group， and the expression of p-GSK3β was up-regulated （P <0.01）. As demonstrated by comparison with the model group， the protein expression of p-AMPK in the low-dose Liuwei Dihuangtang group was elevated （P<0.05）， while p-Akt and Nrf2 were slightly increased， exhibiting no statistical significant difference. However， the protein expression levels of p-AMPK， p-Akt， and Nrf2 in the medium- and high-dose Liuwei Dihuangtang groups and fluoxetine group were up-regulated， while those of p-GSK3β were down-regulated （P<0.05，P<0.01）. ConclusionLiuwei Dihuangtang improves the depressive-like behavior of DD rats， which may be related to its activation of the AMPK/Akt/GSK3β/NRF2 pathway， regulation of the oxidative stress in vHIP， and enhancement of myelin repair.

Modified Shuyuwan Mediated Mitochondrial Autophagy Improve APP/PS1 Mice Oxidative Stress Injury and Ability of Learning and Memory / 中国实验方剂学杂志

ObjectiveTo explore the effects and related mechanisms of modified Shuyuwan on the decline of learning and memory in Alzheimer's disease （AD） mice. MethodForty 5-month-old SPF APP/PS1 mice were randomly divided into model group， Donepezil group， modified Shuyuwan group， modified Shuyuwan+ chloroquine （CQ） group， 10 mice in each group， the same background wild type C57BL/6J ten mice were set as the normal group. Among them， the modified Shuyuwan group was given the modified Shuyuwan decoction （10 g·kg-1）， the Donepezil group was given the Donepezil hydrochloride solution （0.45 mg·kg-1）， the modified Shuyuwan + CQ group was CQ （10 mg·kg-1） was injected intraperitoneally on the basis of the modified Shuyuwan group， and the normal group and the model group were given the same amount of normal saline intragastrically， once a day， for a total of 35 days. After the administration， Morris water maze experiment and new object recognition experiment to detect the spatial memory ability of mice. TdT-mediated dUTP Nick-End Labeling（TUNEL） staining to detect the apoptosis level of mouse hippocampal CA1 neurons， biochemical detection of reactive oxygen species （ROS） and superoxide in mouse hippocampal neurons dismutase （SOD） levels. transmission electron microscopy to observe the ultrastructure of neuronal mitochondria in the CA1 region of mouse hippocampus. Western blot to detect mouse hippocampal mitochondrial autophagy adaptor protein （p62） and microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ）， PTEN-induced kinase 1 （PINK1）， E3 Ubiquitin Ligase（Parkin）protein expression level. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） detection of mouse hippocampal mitochondrial forkhead transcription factor O1 （FoxO1）， PINK1， Parkin mRNA expression level. ResultCompared with the normal group， the escape latency of the model group mice increased significantly， the number of crossing platforms and the retention time in the target quadrant decreased significantly， the relative resolution index decreased significantly， and the ability to recognize new objects was weakened （P<0.05）， neurons in the hippocampus CA1 area decreased. The number of dead cells increased significantly （P<0.05）， the level of ROS was significantly increased （P<0.01）， and the level of SOD was significantly decreased （P<0.01）， the morphology of hippocampal mitochondria was severely damaged， the expression of p62 and LC3Ⅱ proteins increased （P<0.01）， Parkin protein expression decreased （P<0.05）， and PINK1 protein expression increased （P<0.05）， FoxO1， PINK1， Parkin mRNA expressions all decreased （P<0.05）. Compared with the model group， the mice's escape latency was significantly shortened after the intervention of the modified Shuyuwan， the number of crossing platforms and the proportion of residence time in the target quadrant increased significantly， the relative resolution index increased significantly， and the ability to identify new objects was enhanced （P<0.05）. Apoptotic cells were significantly reduced （P<0.05）. ROS levels were significantly reduced （P<0.01）， and SOD levels were significantly increased （P<0.05， P<0.01）， mitochondrial morphology and various structures were significantly improved， p62， LC3Ⅱ protein expression decrease （P<0.05，P<0.01）， PINK1， Parkin protein expression increased （P<0.01）. FoxO1， PINK1， Parkin mRNA expression increased （P<0.05， P<0.01）. Compared with the modified Shuyuwan group， the evasion latency of mice in the modified Shuyuwan + CQ group increased significantly， the number of crossing platforms and the proportion of residence time in the target quadrant decreased， and the relative resolution index decreased （P<0.05）， the SOD level was significantly reduced （P<0.01）. The damage of mitochondrial morphology and structure increased again， the expression of p62 and LC3Ⅱ protein increased （P<0.05， P<0.01）， and the expression of PINK1 and Parkin decreased significantly（P<0.05， P<0.01）. FoxO1， PINK1， and Parkin mRNA expression was significantly reduced （P<0.05， P<0.01）. ConclusionModified Shuyuwan can effectively improve the oxidative stress damage and learning and memory ability of AD mice. The mechanism may be related to up-regulating the expression of FoxO1， PINK1， and Parkin factors， promoting mitochondrial autophagy， reducing oxidative stress， and protecting neuronal damage.

Liuwei Dihuangtang Alleviates Myelin Injury in Anterior Cingulate Cortex and Depression-like Behaviors of Rats with Diabetes Mellitus and Depression via Promoting Autophagy and Regulating Microglial Phenotype / 中国实验方剂学杂志

ObjectiveTo observe the effect of Liuwei Dihuangtang （LWDHT） on depression-like behaviors of rats with diabetes mellitus and depression （DD） and explore its mechanism. MethodThe diabetes mellitus （DM） model was induced by the high-fat diet and tail vein injection of low-dose streptozotocin （STZ） in 50 male Sprague-Dawley rats of SPF grade. Then the DD model was induced by chronic unpredictable mild stress （CUMS） for 28 days in DM rats. Fifty DD rats were randomly divided into model group， fluoxetine group （10 mg·kg-1·d-1）， and low-， medium-， and high-dose LWDHT groups （3.375， 6.75， 13.5 g·kg-1·d-1）， with 10 rats in each group. Another 10 healthy rats were assigned into a control group and received normal saline by gavage. After four weeks of drug intervention， the forced swimming assay was carried out to assess the depression-like behaviors of rats. The expression levels of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-4 （IL-4）， and interleukin-10 （IL-10） in the anterior cingulate cortex （ACC） were detected by enzyme-linked immunosorbent assay （ELISA）. Immunofluorescence was used to detect the expression of myelin basic protein （MBP） in ACC and the co-localization of ionized calcium-binding adapter molecule 1 （Iba1） with intracellular microtubule-associated protein 1 light chain 3 （LC3）. The protein expression levels of MBP， myelin proteolipid protein （PLP）， myelin oligodendrocyte glycoprotein （MOG）， Beclin-1， LC3， p62， and microglia （MG） phenotypic protein-related inducible nitric oxide synthase （iNOS）， and arginase 1 （Arg1） were detected by Western blot. ResultCompared with the control group， the model group showed shortened swimming time and prolonged immobility time （P<0.01）. Compared with the model group， the medium- and high-dose LWDHT groups showed reduced immobility time （P<0.05， P<0.01）. Compared with the control group， the model group showed decreased protein expression of MBP， PLP， and MOG in the ACC region （P<0.01）. Compared with the model group， the fluoxetine group and the medium- and high-dose LWDHT groups showed up-regulated protein expression of MBP， PLP， and MOG （P<0.05， P<0.01）. Compared with the control group， the model group showed decreased MBP fluorescence intensity in the ACC region （P<0.01）. Compared with the model group， the fluoxetine group and the medium- and high-dose LWDHT groups showed increased MBP fluorescence intensity in the ACC region （P<0.05， P<0.01）. Compared with the control group， the model group showed increased expression of iNOS （P<0.01） and slightly increased Arg1 protein expression. Compared with the model group， the medium- and high-dose LWDHT groups and the fluoxetine group showed down-regulated iNOS expression and up-regulated Arg1 protein expression （P<0.05， P<0.01）， but there was no significant difference between the fluoxetine group and the medium-，high-dose LWDHT groups. Compared with the control group， the model group showed increased expression levels of proinflammatory factors IL-1β and TNF-α in the ACC region （P<0.01） and slightly increased expression levels of anti-inflammatory factors IL-4 and IL-10. Compared with the model group， the fluoxetine group， and the medium- and high-dose LWDHT groups showed down-regulated expression of IL-1β and TNF-α （P<0.05， P<0.01） and up-regulated expression of IL-4 and IL-10 （P<0.05， P<0.01）. Compared with the control group， the model group showed reduced expression levels of Beclin-1 and LC3Ⅱ （P<0.01） and increased expression level of p62 （P<0.01）. Compared with the model group， the fluoxetine group and the medium- and high-dose LWDHT groups showed up-regulated Beclin-1 and LC3Ⅱ expression （P<0.01） and down-regulated p62 expression （P<0.01）. Compared with the control group， the model group showed decreased LC3+Iba1+ cells in the ACC region （P<0.01）. Compared with the model group， the fluoxetine group and the medium- and high-dose LWDHT groups showed increased LC3+Iba1+ cells （P<0.05， P<0.01）. ConclusionLWDHT can alleviate the depression-like behaviors in DD rats presumedly by promoting MG autophagy， regulating MG phenotypic changes， and increasing MG clearance of myelin sheath fragments. Meanwhile， MG phenotypic transformation also inhibits ACC inflammation in DD rats， improves the local microenvironment of oligodendrocyte proliferation and differentiation， and ultimately promotes the repair and remyelination of damaged myelin sheath.

Effect of Modified Shuyuwan on Neuroinflammation in APP/PS1 Dementia Mice Based on Nlrp3/ASC/Caspase-1 Signaling Pathway / 中国实验方剂学杂志

Objective:To observe the effect of modified Shuyuwan in amyloid precursor protein/ presenilin 1 （APP/PS1） dementia mice on cognitive and memory impairment and to explore its mechanism. Method:The 40 APP/PS1 mice were divided into model group （given Physiological saline）， low and high-dose modified Shuyuwan （14，64 g·kg^-1）group， and donepezil group （1 mg·kg^-1） and 10 wild mice were set as the blank control group （given Physiological saline）. All of the mice were administered intragastrically for 35 days. The memory and space exploration ability of mice was detected by Morris water maze， the morphology of mouse hippocampal neurons were observed by Nissl staining. The deposition of <italic>β </italic>amyloid 1-42（A<italic>β</italic>_1-42） in mouse hippocampus was detected by immunohistochemistry， and the expression of ionized calcium-binding adapter molecule 1（Iba1）， a marker of hippocampal microglia （MG） and Nitric oxide synthase（iNOS）， a marker of actived MG， were detected by immunofluorescence. The protein expression of NLR family pyrin domain containing 3（Nlrp3）， Apoptosis-associated speck-like protein containing a Caspase-recruitment domain （ASC）， cysteine protease-1（Caspase-1）and interleukin-1 beta （IL-1<italic>β</italic>） were detected by Western blot， and the expression of IL-1<italic>β</italic>， tumor necrosis factor-<italic>α</italic>（TNF-<italic>α</italic>）and interleukin-18 （IL-18） mRNA were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. Result:Compared with the blank control group， the memory and space exploration ability of the model group were significantly reduced （<italic>P</italic><0.05）， the number of hippocampal neurons decreased， the deposition of A<italic>β</italic>_1-42 increased， the markers of actived MG Iba1，iNOS increased， the protein expression of Nlrp3， ASC， Caspase-1， IL-1<italic>β</italic> increased significantly （<italic>P</italic><0.05）， and the mRNA expression of IL-1<italic>β</italic>， IL-18， and TNF-<italic>α</italic> increased significantly （<italic>P</italic><0.05）. Compared with model group， the Chinese medicine group can improve the APP/PS1 mice's space exploration ability and memory ability （<italic>P</italic><0.05）， increase the number of hippocampal neurons， reduce A<italic>β</italic>_1-42 deposition， reduce the activation of MG， and reduce the protein expression of Nlrp3， ASC， Caspase-1 and IL-1<italic>β</italic> （<italic>P</italic><0.05）， and reduced the expression of IL-1<italic>β</italic> mRNA （<italic>P</italic><0.05）. Conclusion:Modified Shuyuwan can reduce the expression of IL-1<italic>β</italic> and other inflammatory factors in the hippocampus of APP/PS1 mice by inhibiting the Nlrp3/ASC/Caspase-1 pathway， and relieve nerve inflammation and pathological injury of AD.

Xiaoyaosan Promotes Myelin Regeneration and Alleviates Depressive Symptoms in Vascular Dementia Mice with Depression via Promoting Microglia Polarization / 中国实验方剂学杂志

Objective:To investigate the effect of Xiaoyaosan on depressive behavioral phenotype in mice with vascular dementia （VaD） mice and its possible mechanism. Method:Sixty three-month-old male C57/BL6 mice were divided into the normal control group， model group， positive control group， as well as low-， medium-， and high-dose Xiaoyaosan groups. Mice in all groups except for the normal control group underwent bilateral carotid artery stenosis. Two weeks later， they were subjected to chronic restraint stress， 6 h per day， for inducing VaD complicated with depression. Mice in the low-， medium-， and high-dose Xiaoyaosan groups were treatment with intragastric administration of Xiaoyaosan decoction （5， 10， 20 g·kg^-1）， the ones in the positive control group with fluoxetine （10 mg·kg^-1）， and those in the normal control group and model group with an equal volume of normal saline for four weeks， during which the restraint stress was maintained. The depressive behavioral phenotype of mice was observed in sugar water preference test and tail suspension test. The fluorescence expression of myelin basic protein （MBP） in ventral hippocampus （vHIP） was detected by fluorescence immunoassay. The ultrastructure of myelin sheath in vHIP was observed by transmission electron microscopy. The protein expression levels of MBP， myelin oligodendrocyte glycoprotein （MOG）， myelin-associated glycoprotein （MAG）， triggering receptor expressed on myeloid cells-2 （TREM2）， inducible nitric oxide synthase （iNOS）， arginase 1 （Arg1）， interleukin-I<italic>β</italic> （IL-1<italic>β</italic>）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， interleukin-4 （IL-4）， and interleukin-10 （IL-10） were assayed by Western blot. Result:As revealed by behavioral test， compared with the normal control group， the model group exhibited prolonged immobility time and decreased percentage of sugar water preference （<italic>P</italic><0.01）. Compared with the model group， Xiaoyaosan significantly shortened the immobility time of mice （<italic>P</italic><0.05） and increased the percentage of sugar water preference （<italic>P</italic><0.01）. Western blot results showed that the protein expression levels of MBP， MOG， and MAG in vHIP of the model group were remarkably decreased as compared with those of the normal control group （<italic>P</italic><0.01）. The protein expression levels of MBP， MOG， and MAG in vHIP of the low-dose Xiaoyaosan group were increased in contrast to those in the model group （<italic>P</italic><0.05， <italic>P</italic><0.01）， while the protein expression of iNOS was decreased （<italic>P</italic><0.01）. The protein expression levels of MBP， MOG， MAG， TREM2， Arg1， IL-4， and IL-10 in the medium- and high-dose Xiaoyaosan groups were up-regulated （<italic>P</italic><0.05， <italic>P</italic><0.01）， whereas those of iNOS， IL-1<italic>β</italic>， and TNF-<italic>α</italic> were down-regulated （<italic>P</italic><0.01）. The immunofluorescence findings demonstrated that the mean fluorescence intensity of MBP in the model group declined in comparison with that in the normal control group （<italic>P</italic><0.01）， while the mean fluorescence intensities of MBP in the low-， medium-， and high-dose Xiaoyaosan groups were enhanced to different degrees （<italic>P</italic><0.01）. It was observed under the transmission electron microscope that the myelin structure of the model group was loosened and the dense layer was separated and irregularly arranged. Xiaoyaosan improved the structural integrity of myelin sheath and the looseness of lamellar structure. Conclusion:Xiaoyaosan ameliorates the depressive behavioral phenotype of VaD mice， which may be related to the up-regulation of TREM2， the induction of M2 polarization of microglia cells， the enhancement of their anti-inflammatory and phagocytic abilities， and the promotion of damaged myelin sheath regeneration.

Modified Shuyuwan Ameliorate Neuronal Apoptosis of APP/PS1 Mice Via Akt/GSK3β/Nrf2 Pathway / 中国实验方剂学杂志

Objective: To explore the effect and mechanism of modified Shuyuwan neuroprotection in APP/PS1 model mice. Method: Selecting 20 male APP/PS1 mice of 5 months old and 10 wild type mice.The mice were divided into blank group, model group and modified Shuyuwan group(14 g·kg-1·d-1),drug delivery for 28 days, and blank group and model group were given the same amount of normal saline,APP/PS1 background primary neuron model and wild type primary neurons were divided into blank group, model group and modified Shuyuwan group,tunicamycin group, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) inhibitor group.The blank group and model group were given 10% blank serum, the modified Shuyuwan group was given 5% modified Shuyuwan-containing serum, the tunicamycin group and the PI3K/Akt inhibitor group were respectively added with 2 mg·L-1 tunicamycin and 10 μmol·L-1 LY294002 on the basis of 5% modified Shuyuwan-containing serum.The spatial learning and memory ability of mice was measured by Morris water maze,and Western blot was used to detect nuclear factor erythroid-2 related factor 2(Nrf2) protein expression in hippocampus.Western blot was used to detect the protein expression of endoplasmic reticulum stress related proteins glucose regulatory protein 78(GRP78), protein kinase-like endoplasmic reticulum kinase(PERK),phosphorylation (p-)PERK and apoptosis expression of the pathway proteins eukaryotic translation initiation factor 2α(eIF2α),p-eIF2α,enhancer binding protein homologous protein(CHOP), and cysteinyl aspartate apecific proteinase 3 (Caspase-3),p-Akt, Akt, Glycogen Synthase kinase-3β(GSK3β),Nrf2. Result: In vivo experiment,compared with blank group, the learning and memory ability of APP/PS1 mice in the model group was impaired(PPP In vitro experiment,Western blot analysis showed that compared with the blank group, the expression of GRP78, p-PERK/PERK,p-eIF2α/eIF2α,CHOP, and cleaved Caspase-3 proteins was increased in the model group(Pα/eIF2α,CHOP, and cleaved Caspase-3 proteins(PPPβ was increased in the model group(Pβ was decreased after modified Shuyuwan-containing serum intervention(PPβ protein was inhibited by LY294002.(PPConclusion: Modified Shuyuwan can increase Nrf2 protein expression through PI3K/Akt/GSK3β signaling pathway, reduce neuronal apoptosis induced by endoplasmic reticulum stress,improve the learning and memory ability of APP/PS1 model mice.

Post-marketing study on clinical safety of ginkgo diterpene lactone meglumine injection in 6 300 patients with ischemic stroke / 中国中药杂志

To further evaluate the safety of ginkgo diterpene lactone meglumine injection in the clinical use in ischemic stroke patients. Clinical safety study was conducted in 82 clinical units and 6 300 cases were completed and included from June 2013 to December 2014 by using multicenter, prospective, open and uncontrolled design methods for clinical research. A total of 29 cases of adverse reactions were observed in the experiment. Adverse reaction ratio (ADR) was 0.46%, and about 86.21% (25 cases) of them was mild with transient response which could be alleviated or disappeared without intervention; about 13.79% (4 cases) was moderate, including 2 cases of headache, 1 case of dizziness and 1 case of rash; no serious adverse reactions were found. The adverse reactions occurred in this study were pre-known adverse reactions or common adverse reactions of Chinese medicine injection. The overall incidence of adverse reactions was low, and the risk was controllable.

Clinical research of early intervention of modified shuyu pill in vascular cognitive impairment no dementia / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe early intervention effects of Modified Shuyu Pill (MSP) on vascular cognitive impairment no dementia (VCIND).</p><p><b>METHODS</b>Totally 100 patients VCIND were randomly assigned to the treatment group (43 cases) and the control group (33 cases). On the basis of the treatment targeting risk factors of blood vessels, patients in the treatment group were treated by MSP, while those in the control group were treated by donepezil hydrochloride. The therapeutic course was 16 weeks. The neuropsychological scales [mini-mental state examination (MMSE) and Montreal cognitive assessment (MOCA) score] and Chinese medicine dementia syndromes scales were observed before and after treatment.</p><p><b>RESULTS</b>The MMSE and MOCA score of the two groups increased when compared with the same group before treatment (P < 0.01). But there was no statistical difference in MMSE or MOCA score after treatment between the two groups (P > 0.05). The Chinese medicine dementia syndromes scales significantly decreased in the treatment group when compared with before treatment (P < 0.01). But there was no statistical difference in Chinese medicine dementia syndromes scales in the control group between before and after treatment (P > 0.05). There was statistical difference in Chinese medicine dementia syndromes scales after treatment between the two groups (P < 0.01).</p><p><b>CONCLUSION</b>MSP could effectively intervene the progress of VCIND.</p>