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1.
Article in Chinese | WPRIM | ID: wpr-906265

ABSTRACT

Objective:To investigate the neuroprotective effects of Danggui Shaoyaosan (DSS) on APP<sub>swe</sub>/PS1<sub>ΔE9 </sub>transgenic (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.

2.
Article in Chinese | WPRIM | ID: wpr-906045

ABSTRACT

Objective:To explore the protective effect and the mechanism of Danggui Shaoyaosan(DSS) on angiotensin Ⅱ (AngⅡ)/transient receptor potential cation channel 6 (TRPC6) pathway in nephrotic syndrome (NS) rats. Method:In animal experiments, doxorubicin (4 mg·kg<sup>-1</sup> for the 1<sup>st</sup> week and 2 mg·kg<sup>-1</sup> for the 2<sup>nd</sup> week) was injected twice to the tail vein of rats to induce NS model in 160 rats, which were then randomly divided into model group (normal saline), losartan group (30 mg·kg<sup>-1</sup>·d<sup>-1</sup>), and low-(4.3 g·kg<sup>-1</sup>·d<sup>-1</sup>), medium-(8.6 g·kg<sup>-1</sup>·d<sup>-1</sup>), and high-dose (17.2 g·kg<sup>-1</sup>·d<sup>-1</sup>) DSS groups. Besides, a normal group was also set. After intervention for four weeks, ultrastructure changes of the kidney were identified by transmission electron microscopy (TEM). The 24-hour urine protein was detected by kits. Radioimmunoassay was used to detect the content of AngⅡ and Calcineurin (CaN) in plasma. Western blot was used to detect the protein expression of TRPC6, angiotensin Ⅱ type 1 receptor (AT1R), podocyte slit diaphragm-specific protein (Nephrin), and cysteine-aspartic acid protease-3 (Caspase-3) in the renal cortex. Immunohistochemistry was used to detect the expression of TRPC6 and AT1R in the slit diaphragm. In cell experiments, AngⅡ stimulated MPC5 podocytes. The cells were randomly divided into a normal group, an AngⅡ group, an AngⅡ+SAR7334 (TRPC6-specific inhibitor) group, an AngⅡ+5%DSS group, an AngⅡ+10%DSS group, and an AngⅡ+15%DSS group. Western blot was used to detect the protein expression of TRPC6, AT1R, Nephrin, and Caspase-3 in podocytes. Result:Compared with the normal group, the model group showed increased 24-hour urine protein content (<italic>P</italic><0.01) and AngⅡ and CaN in plasma (<italic>P</italic><0.01), incomplete glomerular structure, the extensive fusion of podocyte process with elevated fusion rate (<italic>P</italic><0.01), increased expression distribution of AT1R and TRPC6 in the renal cortex, and up-regulated protein expression of AT1R, TRPC6, and Caspase-3 in renal tissues (<italic>P</italic><0.01), and reduced Nephrin protein expression (<italic>P</italic><0.01). Compared with model group, the losartan group and the high-dose DSS group exhibited decreased 24-hour urine protein content (<italic>P</italic><0.01) and the content of AngⅡ and CaN in plasma (<italic>P</italic><0.01), improved glomerular structure, reduced fusion rate of podocyte process (<italic>P</italic><0.01), diminished expression distribution of TRPC6 and AT1R in the renal cortex, declining protein expression of AT1R, TRPC6 and Caspase-3 in renal tissues (<italic>P</italic><0.01), and elevated Nephrin protein expression (<italic>P</italic><0.01). Additionally, compared with the normal podocytes, AngⅡ-stimulated podocytes showed increased protein expression of AT1R, TRPC6 and Caspase-3 (<italic>P</italic><0.01), and decreased expression of Nephrin (<italic>P</italic><0.01). Compared with the AngⅡ group, the AngⅡ+SAR7334 group displayed reduced protein expression of AT1R, TRPC6, and Caspase-3 (<italic>P</italic><0.01) and increased protein expression of Nephrin (<italic>P</italic><0.01). Conclusion:DSS can improve the pathological characteristics of NS presumedly by inhibiting the interaction between AngⅡ and TRPC6 in podocytes and improving the structural integrity of podocytes to repair the damage of glomerular molecular barrier and slow down the progression of NS-induced proteinuria.

3.
Article in Chinese | WPRIM | ID: wpr-906044

ABSTRACT

Objective:To investigate the neuroprotective effect of Danggui Shaoyaosan (DSS) in a rat model of amyloid-<italic>β</italic>-peptide<sub>1-42</sub> (A<italic>β</italic><sub>1-42</sub>)-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><sub>1-42</sub> 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.

4.
Article in Chinese | WPRIM | ID: wpr-905984

ABSTRACT

Objective:To explore<italic> </italic>the efficacy and mechanism of Danggui Shaoyaosan on rats of nonalcoholic fatty liver disease (NAFLD). Method:Sixrty SPF SD male rats were randomly divided into normal group, model group,essentiale (0.144 g·kg<sup>-1</sup>) and low, middle and high-dose of Danggui Shaoyaosan groups (2.44, 4.88, 9.76 g·kg<sup>-1</sup>). High fat diet were fed to bulid the NAFLD model, and each treatment group was given corresponding drugs at the same time. After 8 weeks, the serum and liver tissue were collected to detect the contents or activities of total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), aspartic acid aminotransferase (AST), tumor necrosis factor-<italic>α</italic> (TNF-<italic>α</italic>) and interleukin-10 (IL-10) in serum, the contents of TC, TG and free fatty acid (FFA) in liver tissue, Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) and Western blot were used to observe the gene and protein expressions of Toll-like receptor 4 (TLR4), myeloid different factory 88 (MyD88) and c-Jun n-terminal kinase (JNK) and the protein expression of phosphorylation JNK(p-JNK) in liver tissue. Hematoxylin-eosin (HE) staining and Oil red staining to observe the pathological morphological changes of liver. Result:Compared with control group, the contents or activities of TC, TG, ALT, AST and TNF-<italic>α</italic> in serum, the contents of TC, TG and FFA in liver and the gene and protein expressions of TLR4, MyD88, JNK, and the protein expression of p-JNK in liver tissue of model group were distinctly increased (<italic>P</italic><0.01), the content of IL-10 was significantly decreased (<italic>P</italic><0.01). Compared with model group, the contents or activities of TC, TG, ALT, AST and TNF-<italic>α</italic> in serum, the contents of TC, TG and FFA in liver and the mRNA and protein expressions of TLR4, MyD88 and JNK, and the protein expression of p-JNK in liver tissue of Danggui Shaoyaosan groups were significantly reduced (<italic>P</italic><0.05,<italic>P</italic><0.01), the content of IL-10 in serum of Danggui Shaoyaosan groups was distinctly increased(<italic>P</italic><0.05,<italic>P</italic><0.01), HE staining and Oil red staining show that the degree of liver steatosis was alleviated obviously by Danggui Shaoyaosan. Conclusion:Danggui Shaoyaosan has a better treatment on NAFLD by inhibiting TLR4/MyD88/JNK pathway and alleviating the inflammation response.

5.
Article in Chinese | WPRIM | ID: wpr-905956

ABSTRACT

Objective:To observe the improving effect of Danggui Shaoyaosan on diminished ovarian reserve (DOR) in rats triggered by Tripterygia wilfordii polyglycoside tablet combined with stress, and to explore the role of transforming growth factor-<italic>β</italic><sub>1 </sub>(TGF-<italic>β</italic><sub>1</sub>)/Smads signaling pathway in such improvement. Method:Forty-eight female SD rats with normal sexual cycle were selected and randomly divided into a normal group (<italic>n</italic>=8) and a modeling group (<italic>n</italic>=40), and the ones in the modeling group were given Tripterygium wilfordii polyglycoside tablets (50 mg·kg<sup>-1</sup>) combined with random stress for 15 d. After successful modeling, the rats were randomized into the model group, low-, medium-, and high-dose (3.96, 7.92, 15.84 g·kg<sup>-1</sup>) Danggui Shaoyaosan groups, and estradiol valerate group (0.09 mg·kg<sup>-1</sup>), with eight in each group. Under the premise of stress exposure, they were separately gavaged with the normal saline, low-, medium- and high-dose Danggui Shaoyaosan, and estradiol valerate for 15 successive days. The estrous cycle of rats in each group was observed daily. After intervention, the rats were sacrificed and the ovarian visceral index was calculated. The pathological changes in ovarian tissues were observed by hematoxylin eosin (HE) staining. The protein expression levels of TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1 </sub>receptor (TGF-<italic>β</italic><sub>1</sub>R) in the ovarian tissues of rats were measured by immunohistochemistry (IHC), and the mRNA expression levels of Smad2, Smad3, and Smad7 in the ovarian tissues by real-time polymerase chain reaction (Real-time PCR). Result:Compared with the normal group, the model group exhibited disordered estrus cycle (<italic>P</italic><0.05), reduced visceral index (<italic>P</italic><0.01), and down-regulated TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1</sub>R protein and Smad2 and Smad3 mRNA expression in the ovarian tissues (<italic>P</italic><0.01), and up-regulated Smad7 mRNA expression (<italic>P</italic><0.01). Compared with the model group, Danggui Shaoyaosan at the low, medium, and high doses and estradiol valerate improved the estrus cycle of rats to varying degrees (<italic>P</italic><0.05) and increased the visceral index, with better effects observed in the medium-group and high-dose Danggui Shaoyaosan groups (<italic>P</italic><0.05,<italic>P</italic><0.01). Besides, the protein expression levels of TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1</sub>R and the mRNA expression levels of Smad2 and Smad3 in the ovarian tissues were elevated to varying degrees (<italic>P</italic><0.01), and the Smad7 mRNA expression declined (<italic>P</italic><0.01). The improvements in TGF-<italic>β</italic><sub>1</sub> and TGF-<italic>β</italic><sub>1</sub>R protein expression of the medium-dose Danggui Shaoyaosan group and estradiol valerate group were more obvious. Conclusion:Danggui Shaoyaosan significantly improves ovarian reserve in DOR rats, which is closely related to the regulation of TGF-<italic>β</italic><sub>1</sub>/Smads signaling pathway.

6.
Article in Chinese | WPRIM | ID: wpr-873241

ABSTRACT

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.

7.
Article in Chinese | WPRIM | ID: wpr-802029

ABSTRACT

Objective:To investigate the effect of copper ion(Cu2+) on the aggregation and neurotoxicity of Aβ, and affirm the role of Danggui Shaoyaosan in vitro,the Neuroblastoma (SH-SY5Y) cells treated with β-amyloid 1-42 (Aβ1-42) and Cu2+ were used as a vitro models of Alzheimer's disease(AD). Method:Aβ 1-42 (20 μmol·L-1) was reacted with different concentrations of copper sulfate (CuSO4,20,40 μmol·L-1), and then the thioflavine T (ThT) staining method was used to detect the Aβ aggregation state. The Aβ aggregation status was also detected by ThT staining in the Aβ1-42-Cu2+ group(20+20 μmol·L-1), and Danggui Shaoyaosan groups(1.6,3.2,6.4 mg·L-1).The SH-SY5Y cells were cultured and incubated with different concentrations of Aβ1-42(1.25,2.5,5,10,20,40 μmol·L-1) and Danggui Shaoyaosan(1.6, 3.2, 6.4,12.8 mg·L-1) for 24 h. Subsequently, SH-SY5Y cells were incubated with Aβ1-42 (20 μmol·L-1) and CuSO4(20 μmol·L-1) in the Aβ1-42-Cu2+ group, and incubated with Aβ1-42 (20 μmol·L-1), Danggui Shaoyaosan (1.6 mg·L-1) and CuSO4 (20 μmol·L-1) in Danggui Shaoyaosan group. Control group was added with the medium. After 24 h of co-action, the cell viability was detected by the methylthiazolyl tetrazolium (MTT) assay. The morphology of the cells was photographed by microscopy. The intracellular extracellular Aβ1-42 aggregation was detected by Western blot. Result:Cu2+ and Aβ1-42 bound to more and larger Aβ aggregates compared with the Aβ1-42 group. Compared with the normal group, cell viability was significantly reduced (PPβ1-42 aggregation was increased(PPβ-Cu2+ (PPβ1-42 protein (PPConclusion:Cu2+ can increase the aggregation and toxicity of Aβ; Danggui Shaoyaosan can significantly reduce the damage of SH-SY5Y cells induced by Cu2+-mediated Aβ aggregation, promote Aβ endocytosis, reduce extracellular Aβ aggregation and increase cell viability.

8.
Article in Chinese | WPRIM | ID: wpr-801995

ABSTRACT

Objective: To discuss the effect of Juantong decoction on phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway proteins in rats with endometriosis (EMS). Method: The EMS Rat model was established by autologous endometrial transplantation. And 48 rats were randomly divided into 6 group, namely the sham operation group, the model group, the low-dose Juantong decoction group, the middle-dose Juantong decoction group, high-dose Juantong decoction group, and the PI3K pathway blocker group (LY294002). Then, the high-dose Juantong decoction group, the middle-dose Juantong decoction group and the low-dose Juantong decoction group were given different doses (42.9,14.3,4.8 g·kg-1). The pathway blocker group (LY294002) was given LY294002 (0.04 g·kg-1) through peritoneal injection weekly. The sham operation group and the model group were given saline irrigation (10 mL·kg-1). The administration lasted for 28 days. At last, the ectopic endometrial tissues in rats were observed by transmission electron microscope, the proteins of PI3K, Akt and mTOR in the tissue were detected by immunohistochemical method, and the p70 ribosomal S6 kinase (p70S6K) mRNA expression of ectopic endometrial tissue was tested by Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) method. Result: Compared with the normal group, the protein expressions of PI3K, Akt and mTOR and the mRNA expression of p70S6K of ectopic endometrium in the model group increased significantly (PPConclusion: The proteins of PI3K/Akt/mTOR signaling pathway participate in the occurrence of endometriosis. Juantong decoction can inhibit the activity of epithelial mesenchymal cells and promote apoptosis by reducing the protein expressions of PI3K, Akt and mTOR and the mRNA expression of P70S6K in endometriotic tissues of model rats, thus inhibiting the PI3K/Akt/mTOR signaling pathway in the treatment of endometriosis.

9.
Article in Chinese | WPRIM | ID: wpr-801869

ABSTRACT

Objective:To observe the effects of addition and subtraction therapy of Danggui Shaoyaosan combined with Huaihuasan on ulcerative colitis (UC) with syndrome of dampness-heat in large intestine during active stage, and the effects on brain-gut petide neurotransmitter and inflammatory cytokines. Method:A total of 130 cases were included and randomly divided into control group and observation group, 65 cases in each group. In the control group, the patients received oral administration of mesalazine enteric-coated tablets, 1.0 g/time and 3 times/days. Severe patients received prednisone acetate tablets (0.75 mg·kg-1·d-1) in several times by oral administration. Based on the treatment in control group, patients in observation group also received addition and subtraction therapy of Danggui Shaoyaosan combined with Huaihuasan, 1 dose/day. Both groups were treated for 4 weeks. Symptom scores, Mayo scores, colonic mucosa scores and Inflammatory Bowel Disease Questionnaire (IBDQ) scores were assessed before and after treatment. Patients in remission stage were followed up for 6 months to record the recurrence. Before and after treatment, vasoactive intestinal peptide (VIP), substance P (SP), somatostatin (SS), interleukin-1 (IL-1), IL-6, IL-4 and IL-10 were detected. Result:After 4 weeks of treatment, the clinical remission rate was 93.22%in the observation group, better than 80.7%in the control group (χ2=4.035,PPPPχ2=4.509,PPPPPPConclusion:On the basis of conventional western medicine treatment, addition and subtraction therapy of Danggui Shaoyaosan combined with Huaihuasan in the treatment of UC (dampness-heat in large intestine) during active stage can control the disease activity in a short term, promote restoration of the colonic mucosa. And delay the recurrence in a long term, reduce the recurrence rate, regulate ghrelin neurotransmitters and pro-and anti-inflammatory cytokines levels.

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