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ObjectiveTo observe the effects of Hirudo， Notoginseng Radix et Rhizoma， and drug pair on renal pathological morphology and protein phosphatase 2A （PP2A）/adenylate activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signal pathway in rats with chronic renal failure （CRF）. MethodThe 55 male SD rats were randomly divided into a normal group （n=11） and a modeling group （n=44）. The normal group was fed conventionally， and the modeling group was given 0.25 g·kg-1·d-1 adenine by gavage for 28 days to replicate the CRF model. After successful modeling， rats were randomly divided into model group， Hirudo group （3 g·kg-1·d-1）， Notoginseng Radix et Rhizoma group （3 g·kg-1·d-1）， and Hirudo + Notoginseng Radix et Rhizoma group （3 g·kg-1·d-1）， with 9 rats in each group. The normal group and model group were given a constant volume of normal saline by intragastric administration for 30 days. At the end of the experiment， the levels of serum creatinine （SCr） and urea nitrogen （BUN） in all groups were measured. The renal pathological morphology changes were observed by hematoxylin-eosin （HE） staining， Masson staining， and electron microscopy. The mRNA expressions of PP2A， AMPK， and mTOR were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein expression levels of PP2A， AMPK， phosphorylation（p）-AMPK， mTOR， and p-mTOR in renal tissue were detected by Western blot. ResultCompared with the normal group， the renal pathological structure changes were obvious， and the levels of SCr and BUN were significantly increased. The mRNA expression of PP2A， protein expression of PP2A， and p-mTOR/mTOR expression were significantly increased， and the p-AMPK/AMPK was significantly decreased in the model group （P<0.05）. Compared with the model group， the renal pathological morphology changes were significantly improved， and the levels of SCr and BUN were significantly decreased. The mRNA expression of PP2A， protein expression of PP2A， and p-mTOR/mTOR expression in the renal tissue were significantly decreased， and the p-AMPK/AMPK was significantly increased （P<0.05） in all groups after drug intervention. In addition， the effect in the Hirudo+Notoginseng Radix et Rhizoma group was better. The mRNA expression levels of AMPK and mTOR in the renal tissue were not significantly different among the normal group， model group， and other groups. ConclusionThe efficacy of Hirudo and Notoginseng Radix et Rhizoma pairs in improving renal fibrosis in rats with CRF is significantly better than that of the single drug， and its improvement on renal fibrosis in rats with CRF may be related to the regulation of PP2A/AMPK/mTOR signaling pathway.
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By reviewing the relevant literature of ancient herbal works and modern codices， this paper sorted out the historical evolution and developmental venation of processing of Notoginseng Radix et Rhizoma. On this basis， the modern research of processed products of Notoginseng Radix et Rhizoma was used as the breakthrough point to analyze the literature in terms of processing technology， chemical composition changes and changes in pharmacological effects before and after processing. According to the research status of processing of Notoginseng Radix et Rhizoma， some existing problems were analyzed in this paper， such as not many ancient processing methods used in modern time， lack of standardized research on processing technology. And saponins， polysaccharides， amino acids， flavonoids and other chemical components in Notoginseng Radix et Rhizoma may change to different degrees before and after processing， which was the main reason for the difference of efficacy before and after processing. However， the current research on the pharmacological effects of Notoginseng Radix et Rhizoma mainly focuses on raw products， resulting in a lack of in-depth research on the transformation mechanism of Notoginseng Radix et Rhizoma in processing difference， and the scientific connotation of "Shengxiao Shubu" has not been clearly elaborated， which is not conducive to the standardized clinical use of drugs. Therefore， it is necessary to further analyze the material basis of Notoginseng Radix et Rhizoma and its processed products， and to explore the change rule of chemical components before and after processing and its correlation with pharmacodynamic activity， so as to clarify the processing mechanism for providing scientific basis for its standardized processing， quality control and clinical rational use.
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Objective:To analyze and explore the possible mechanism of anti-tumor metastasis of Notoginseng Radix et Rhizoma using Internet pharmacology. Methods:The active components and targets of Notoginseng Radix et Rhizoma were screened by retrieving Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP). GeneCards database was used to screen the anti-tumor metastasis-related targets, and compounds and disease targets were under mapping analysis. Key targets of Notoginseng Radix et Rhizoma for anti-tumor metastasis were screened through Venn map. With the help of Cytoscape 3.7.2 software, a compound-disease network diagram was constructed. String platform was used to build a PPI network. Bioconductor was used to enrich the target genes for KEGG signaling pathway and GO biological process analysis. Results:Totally 119 active components were selected from Notoginseng Radix et Rhizoma. There were 8 eligible active components, corresponding to 162 related targets, 121 targets related to anti-tumor metastasis, and 30 key targets screened by PPI network, including AKT1, MAPK1, JUN, RELA, IL6, etc. GO enrichment analysis mainly involved biological processes such as cytokine receptor binding, heme binding, RNA polymerase Ⅱ transcription factor binding, ubiquitin protein ligase binding, and steroid hormone receptor activity. 149 signal pathways related to Notoginseng Radix et Rhizoma anti-tumor metastasis were obtained by KEGG enrichment analysis, mainly involving multiple signal pathways, such as AGE-RAGE and PI3K-Akt, and hepatitis B, Kaposi's sarcoma-associated herpes virus infection, human cytomegalovirus infection and other viral infections and various tumors. Conclusion:Notoginseng Radix et Rhizoma can pass multiple active components, such as ginsenoside f2, ginsenoside rh2 β-, sitosterol, stigmasterol and quercetin, and multiple targets, such as AKT1, MAPK1, JUN, RELA and IL6, acting on multiple pathways such as PI3K-Akt, thereby playing the role of anti-tumor metastasis.
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ObjectiveTo evaluate the clinical efficacy and safety of Notoginseng Radix et Rhizoma powder in treating dyslipidemia by regulating the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway. MethodSixty patients with dyslipidemia （syndrome of combined phlegm and stasis） treated in the Third Affiliated Hospital of Henan University of Chinese Medicine from May 2021 to June 2022 were selected in this study and randomized into two groups according to the randomized， double-blind control principle. The control group was treated with Xuezhikang capsules + Notoginseng Radix et Rhizoma powder placebo and the observation group with Notoginseng Radix et Rhizoma powder + Xuezhikang capsules placebo for 6 weeks. The clinical efficacy， traditional Chinese Medicine （TCM） syndrome scores， and liver and kidney function indicators were evaluated at weeks 0， 3， and 6. Enzyme-linked immunosorbent assay （ELISA） was employed to determine the expression of vascular endothelial growth factor （VEGF）， kinase insert domain receptor （KDR）， epidermal growth factor （EGF）， and epidermal growth factor receptor （EGFR） in the peripheral serum. Quantitative Real-time PCR was employed to measure the mRNA levels of KDR， EGFR， PI3K， and Akt in the mononuclear cells of the peripheral blood. ResultThe observation group （83.33%） showed the total effective rate comparable to that of the control group （89.66%） and no adverse reactions. Compared with before treatment， the patients in the observation group showed decreased TCM syndrome score and serum levels total cholesterol （TC）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C） and after being treated for 3 and 6 weeks （P<0.05）， the level of high-density lipoprotein cholesterol （HDL-C） showed an upward trend, but the difference was not statistically significant. After treatment， the two groups showed no significant differences. Compared with that before treatment, the mRNA expression of PI3K, Akt and EGFR in peripheral blood mononuclear cell and the expression of EGF, VEGF and KDR in serum of the observation group showed a downward trend with time, in which the mRNA expression of PI3K, Akt, VEGF and KDR decreased more significantly (P<0.05)，The expression levels of KDR mRNA and serum EGFR show a trend of first increasing and then decreasing.Compared with the control group after treatment, there was no statistically significant difference in mRNA expression of PI3K, Akt, EGFR, and KDR, as well as serum levels of EGF, EGFR, VEGF, and KDR between the two groups of patients at the same time point. ConclusionNotoginseng Radix et Rhizoma powder is safe and effective in correcting dyslipidemia. It may inhibit the PI3K/Akt signaling pathway by down-regulating the expression of VEGF/KDR to lower the blood lipid level.
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ObjectiveTo investigate the effect of Qi-invigorating and blood-activating therapy on the miR216b/Beclin1 pathway in mice with atrophic precancerous lesions of gastric cancer （PLGC） and analyze its mechanism in autophagy of PLGC. MethodSeventy-five healthy male SPF KM mice were randomly divided into a blank group and a model group. Mice in the model group were given 1-methyl-3-nitroso-1-nitrosoguanidine （MNNG） solution （150 mg·L-1） for free drinking and gavage and ranitidine solution （0.03 g·kg-1） daily for 12 weeks. According to the random control table， mice were divided into a model group， a Qi-invigorating group （3.5 g·kg-1 of Astragali Radix）， a blood-activating group （0.7 g·kg-1 of Notoginseng Radix et Rhizoma powder）， a Qi-invigorating and blood-activating group （3.5 g·kg-1 of Astragali Radix + 0.7 g·kg-1 of Notoginseng Radix et Rhizoma powder）， and a folic acid group （2 mg·kg-1）. The corresponding drugs were given to mice in each group for 8 weeks and then the tissues were collected. Hematoxylin-eosin （HE） staining was carried out to observe the changes in gastric mucosa. Western blot was used to detect the protein expression of microtuble-associated protein 1 light chain 3 （LC3）Ⅰ， LC3Ⅱ， and Beclin1. Real-time polymerase chain reaction （Real-time PCR） was used to detect the mRNA expression of Beclin1 and miR-216b. ResultPathological observation showed that as compared with the blank group， the intrinsic glands of gastric mucosa decreased with atrophy and intestinal metaplasia in the model group， which were improved in all treatment groups， and the improvement of the Qi-invigorating and blood-activating group was the most obvious. As compared with the blank group， the content of LC3Ⅰ， LC3Ⅱ， LC3Ⅱ/LC3Ⅰ， and Beclin1 protein in gastric tissues of the model group was significantly decreased （P<0.05）. As compared with the model group， the content of LC3Ⅰ， LC3Ⅱ， LC3Ⅱ/LC3Ⅰ， and Beclin1 protein in gastric tissues of each treatment group was increased （P<0.05， P<0.01）. The increase was most obvious in the Qi-invigorating and blood-activating group. As compared with the blank group， the mRNA expression of Beclin1 in the model group was decreased （P<0.05）， and that of miR216b was increased （P<0.05）. As compared with the model group， the mRNA expression of Beclin1 was increased and that of miR216b was decreased in each treatment group （P<0.05）， and the changes were the most obvious in the Qi-invigorating and blood-activating group. ConclusionThe mechanism of the Qi-invigorating and blood-activating therapy， represented by Astragali Radix and Notoginseng Radix et Rhizoma， in treating PLGC may be through inhibiting the expression of miR216b and activating Beclin1， thus promoting autophagy and repairing gastric mucosa.
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ObjectiveTo study on the correlation between the apparent color， near-infrared spectroscopy and dynamic changes of index constituent content of samples during the processing of steamed Notoginseng Radix et Rhizoma， and to provide reference for the processing process optimization and quality control of this decoction piece. MethodSamples were dried and crushed by setting three steaming temperature gradients of 100-105， 114-118， 130-136 ℃， and sampled at steaming times of 1， 2， 4， 8， 12， 24 h， respectively. The effects of different steaming temperatures and times on the color and absorption of steamed Notoginseng Radix et Rhizoma at different infrared wavelengths were observed， and principal component analysis and cluster analysis were performed on the obtained data by R 4.1.0 and SPSS 21.0 software to observe the changes in color and infrared absorption characteristics of samples. High performance liquid chromatography （HPLC） was employed to determine the content changes of notoginsenoside R1， ginsenoside Rg1， ginsenoside Rb1， ginsenoside Re， 20（S）-ginsenoside Rg3 and 20（R）-ginsenoside Rg3 before and after steaming of Notoginseng Radix et Rhizoma， mobile phase was acetonitrile （A）-water （B） for gradient elution （0-30 min， 19%A； 30-60 min， 19%-44%A； 60-78 min， 44%-74%A； 78-80 min， 74%-100%A； 80-86 min， 100%A； 86-87 min， 100%-19%A； 87-95 min， 19%A） with the detection wavelength of 203 nm. ResultDuring the steaming process of Notoginseng Radix et Rhizoma， with the increase of steaming temperature and time， the b* （yellow-blue value）， L* （brightness）， △E* （comprehensive color difference value） of sample powder showed a decreasing trend， while the a* （red-green value） showed an increasing trend， and the color gradually deepened from gray to brown and dark black. There was no significant difference in the infrared absorption between raw and steamed Notoginseng Radix et Rhizoma sample powder in the low and medium wavelength bands， but significant difference in the infrared absorption of high band， especially in the band of 9 600-10 000 cm-1. HPLC showed that the contents of the original components （notoginsenoside R1， ginsenoside Rg1， Re and Rb1） decreased and 20（S）-ginsenoside Rg3 and 20（R）-ginsenoside Rg3 were newly produced after steaming of Notoginseng Radix et Rhizoma. The content proportion of these six index components reached the best when the steaming temperature at 130-136 ℃ and the steaming time of 1 h. ConclusionThe color and infrared absorption of samples are affected by the dynamic changes of chemical composition during the steaming process， and the composition change is the result of the joint influence of steaming temperature and time. In this paper， through multi-dimensional analysis of the apparent color indexes， the change pattern of near-infrared absorption characteristics and the index components of the samples， the different process parameters of steamed Notoginseng Radix et Rhizoma were evaluated holistically， and the quality transfer pattern of its processing process was initially revealed， which can provide scientific basis for processing optimization and quality evaluation of steamed Notoginseng Radix et Rhizoma.
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ObjectiveTo establish blood stasis models in zebrafish using three inducers and select the optimal model for evaluating the activity of Notoginseng Radix et Rhizoma in promoting blood circulation. MethodArachidonic acid （AA）， ponatinib， and isoprenaline （ISO） were used to induce blood stasis models in zebrafish. A normal group， a model group， a positive drug group， and Notoginseng Radix et Rhizoma water extract freeze-dried powder groups at different concentrations were set up. The staining intensity of cardiac erythrocytes and the fluorescence intensity of cardiac apoptotic cells were calculated， the anti-thrombotic effect and anti-myocardial hypoxia activity of Notoginseng Radix et Rhizoma were evaluated. The activities of water extract and 70% methanol extract of Notoginseng Radix et Rhizoma were compared based on the preferred AA- and ISO-induced blood stasis models in zebrafish and the difference in the chemical composition was analyzed by UHPLC LTQ-Orbitrap MS/MS. ResultAfter induction by AA and ponatinib， the staining intensity of cardiac erythrocytes was reduced （P<0.01）， and the fluorescence intensity of cardiac apoptotic cells increased after the induction by ISO （P<0.01）. The freeze-dried powder of the water extract of Notoginseng Radix et Rhizoma could antagonize the thrombosis in the AA-induced model （P<0.01） and the myocardial apoptosis in the ISO-induced model （P<0.05）， while no significant improvement in the thrombosis was observed in the ponatinib-induced model. The freeze-dried powder of 70% methanol extract of Notoginseng Radix et Rhizoma could inhibit myocardial apoptosis in the ISO-induced blood stasis model （P<0.01）， and the effect was stronger than that of the freeze-dried powder of Notoginseng Radix et Rhizoma water extract. The difference in chemical composition lay in some saponins （such as ginsenoside Re）， amino acids， and acetylenic alcohols. ConclusionAA， ponatinib， and ISO all can serve as inducers for the blood stasis model in zebrafish. AA- and ISO-induced models can be used to evaluate the activity of freeze-dried powder of Notoginseng Radix et Rhizoma water extract in promoting blood circulation. The chemical compositions of the freeze-dried powders of Notoginseng Radix et Rhizoma extracted with water and 70% methanol are quite different. For the ISO-induced blood stasis model， the freeze-dried powder of Notoginseng Radix et Rhizoma extracted with 70% methanol has a stronger ability against myocardial hypoxia. Saponins and acetylenic alcohols may be closely related to the effects of promoting blood circulation and resolving blood stasis.
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This study aims to establish the high-performance liquid chromatography(HPLC) fingerprints of different batches of Notoginseng Radix et Rhizoma, determine their pharmacodynamic indexes of promoting blood circulation, and explore the spectrum-effect relationship between the chemical components of Notoginseng Radix et Rhizoma and the efficacy of promoting blood circulation. Firstly, the HPLC fingerprints of different batches of Notoginseng Radix et Rhizoma were established. Then, the pharmacodynamic indexes were determined after the capillary coagulation experiment and the cerebral ischemia-reperfusion in rats, including capillary coagulation time, percentage of cerebral ischemic area, cerebral water loss rate, and brain-body index. Afterward, the partial least-squares method was used to explore the spectrum-effect relationship between the chemical components of Notoginseng Radix et Rhizoma and the pharmacodynamic indexes. The results showed that this study successfully established the HPLC fingerprints of different batches of Notoginseng Radix et Rhizoma, found 23 common peaks, and identified 12 of them, all of which were saponins. The method was proved stable and reliable. Both the capillary coagulation experiment and the middle cerebral artery occlusion(MCAO)-induced cerebral ischemia-reperfusion experiment on rats revealed that there were obvious differences in the pharmacodynamic indexes of different batches of Notoginseng Radix et Rhizoma. The relationships between 23 common components of Notoginseng Radix et Rhizoma in different batches and the pharmacodynamic indexes were discussed by means of spectrum-effect correlation analysis, of which 17 components had positive effects while 6 components had negative effects on the pharmacodynamic indexes. This study provides a certain reference basis for the clinical rational use and quality control of Notoginseng Radix et Rhizoma.
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Animals , Rats , Blood Coagulation , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Quality Control , Rhizome , SaponinsABSTRACT
This article aims to establish the fingerprints, determine the hemostatic pharmacodynamic indicators, and explore the spectrum-effect relationship of Notoginseng Radix et Rhizoma in 12 different specifications. Firstly, HPLC and liquid chromatography-mass spectrometry(LC-MS) were employed to establish the fingerprints of Notoginseng Radix et Rhizoma. The rat plasma recalcification experiment and the rat gastric bleeding experiment were conducted to determine the pharmacodynamic indicators, including plasma recalcification time(PRT), thrombin time(TT), prothrombin time(PT), and activated partial thromboplastin time(APTT). Afterwards, the partial least squares method was employed to explore the spectrum-effect relationship of Notoginseng Radix et Rhizoma in different specifications. Twenty-six common peaks were detected in the HPLC fingerprints of different specifications of Notoginseng Radix et Rhizoma, and 11 out of the 26 common peaks represented saponins. The content of dencichine was determined by LC-MS. The rat experiments showed that the pharmacodynamic indicators were significantly different among different specifications of Notoginseng Radix et Rhizoma. The spectrum-effect relationship was explored between 27 common components and pharmacodynamic indicators. Among them, 16 components had positive effects on the pharmacodynamic indicators of Notoginseng Radix et Rhizoma, and 11 exerted negative effects. This study provides a basis for the precision medication and quality control of Notoginseng Radix et Rhizoma.
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Animals , Rats , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal/pharmacology , Hemostatics , Quality Control , Rhizome , SaponinsABSTRACT
This study aims to explore the effect of extract of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Chuanxiong Rhizoma(hereinafter referred to as GNS) on the SIRT1-autophagy pathway of endothelial cell senescence induced by hydrogen peroxide(H_2O_2). To be specific, vascular endothelial cells were classified into the blank control group(control), model group(model), model + DMSO group(DMSO), resveratrol group(RESV), and GNS low-dose(GNS-L), medium-dose(GNS-M), and high-dose(GNS-H) groups. They were treated with H_2O_2 for senescence induction except the control. After intervention of cells in each group with corresponding drugs for 24 h, cell growth status was observed under an inverted microscope, and the formation of autophagosome under the transmission electron microscope. In addition, the changes of microtubule-associated protein 1 light chain 3β(LC3 B) were detected by immunofluorescence staining. The autophagy flux was tracked with the autophagy double-labeled adenovirus(mRFP-GFP-LC3) fusion protein. Dansylcadaverine(MDC) staining was employed to determine the autophagic vesicles, and Western blot the expression of sirtuin 1(SIRT1), ubiquitin-binding protein p62, and LC3Ⅱ. After H_2O_2 induction, cells demonstrated slow growth, decreased adhesion ability, raised number of SA-β-gal-stained blue ones, a certain number of autophagosomes with bilayer membrane and secondary lysosomes in the cytoplasm, and slight rise of autophagy flux level. Compared with the model group, GNS groups showed improved morphology, moderate adhesion ability, complete and smooth membrane, decreased SA-β-gal-stained blue cells, many autophagosomes, autophagic vesicles, and secondary lysosomes in the cytoplasm, increased autophagolysosomes, autophagy flux level, and fluorescence intensity of LC3 B and MDC, up-regulated expression of SIRT1 and LC3Ⅱ, and down-regulated expression of p62, suggesting the improvement of autophagy level. GNS can delay the senescence of vascular endothelial cells. After the intervention, the autophagy flux and related proteins SIRT1, LC3Ⅱand p62 changed significantly, and the autophagy level increased significantly. However, EX527 weakened the effect of Chinese medicine in delaying vascular senescence. GNS may delay the senescence of vascular endothelial cells through the SIRT1 autophagy pathway.
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Autophagy , Cells, Cultured , Cellular Senescence , Drugs, Chinese Herbal/pharmacology , Endothelial Cells/drug effects , Hydrogen Peroxide , Panax/chemistry , Sirtuin 1/geneticsABSTRACT
Pharmacology network was used to investigate the common key target and signaling pathway of Notoginseng Radix et Rhizoma in the protection against diabetic nephropathy(DN), diabetic encephalopathy(DE) and diabetic cardiomyopathy(DCM). The chemical components of Notoginseng Radix et Rhizoma were obtained through TCMSP database and literature mining, and SwissTargetPrediction database was used to predict potential targets of Notoginseng Radix et Rhizoma. The disease targets of DN, DE and DCM were obtained through OMIM and GeneCards databases. The overlapped targets of component targets and disease targets of DN, DE and DCM were obtained, and the network of "chemical component-target-disease" was established. The enriched GO and KEGG of the overlapped genes were investigated by using ClueGo plug-in with Cytoscape. At the same time, the PPI network was constructed through STRING database, and the common key targets for the treatment of three diseases by Notoginseng Radix et Rhizoma were obtained through topological parametric mathematical analysis by Cytoscape. A total of 166 chemical components and 835 component targets were screened out from Notoginseng Radix et Rhizoma. Briefly, 216, 194 and 230 disease targets of DN, DE and DCM were collected, respectively. And 54, 45 and 57 overlapped targets were identified when overlapping these disease targets with component targets of Notoginseng Radix et Rhizoma, respectively. Enrichment analysis indicated that the AGE-RAGE signaling pathway and FoxO signaling pathway were the common pathways in the protection of Notoginseng Radix et Rhizoma against DN, DE and DCM. Network analysis of the overlapped targets showed that TNF, STAT3, IL6, VEGFA, MAPK8, CASP3 and SIRT1 were identified as key targets of Notoginseng Radix et Rhizoma against DN, DE and DCM, the selected key targets were verified by literature review, and it was found that TNF, IL6, VEGFA, CASP3 and SIRT1 had been reported in the literature. In addition, there were the most compounds corresponding to the commom core target STAT3, indicating that more compounds in Notoginseng Radix et Rhizoma could regulate STAT3. This study indicated that Notoginseng Radix et Rhizoma potentially protected against DN, DE and DCM through regulating AGE-RAGE signaling pathway and FoxO signaling pathway and 7 common targets including TNF, STAT3, IL6, VEGFA, MAPK8, CASP3 and SIRT1. This study provided a reference for the research of "different diseases with same treatment" and also elucidated the potential mechanism of Notoginseng Radix et Rhizoma against DN, DE and DCM.
Subject(s)
Humans , Brain Diseases , Diabetes Mellitus , Diabetic Cardiomyopathies/genetics , Diabetic Nephropathies/genetics , Research Design , Signal TransductionABSTRACT
Objective:To investigate the effect of Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma extract on endothelial microparticles （EMPs）-induced vascular endothelial cell senescence， and explore the possible mechanism. Method:Human umbilical vein endothelial cells （HUVECs） were used as the research objects， and the aged model was established with 10-12 passages of replicative senescence cells. The experimental cells were divided into young group （2-4 passage cells）， aged group （10-12 passage cells）， only EMPs intervention group （extract EMPs produced by aged cells to intervene young cells） and low dose， middle dose and high dose drug intervention groups （200， 300， 400 mg·L<sup>-1</sup>）. Senescence related <italic>β</italic>-galactosidase （SA-<italic>β</italic>-gal） staining and cell cycle propidium iodide （PI） staining were used to determine cell senescence. Cell counting kit-8 （CCK-8） assay was used to screen the drug concentration. EMPs were extracted by two-step centrifugation， EMPs labeled with phycoerythrin （PE） anti-human CD31 antibody or fluorescein isothiocyanate （FITC） annexin V were detected by flow cytometry， intracellular reactive oxygen species （ROS） were detected by 2'，7'- dichlorofluorescein diacetate （DCFDA） staining. Result:After treatment with the drug， SA-<italic>β</italic>-gal activity of the aged cells significantly decreased （<italic>P</italic><0.01）， the S phase arrest was restored （<italic>P</italic><0.01）， and the number of CD31<sup>+</sup> EMPs and annexin V<sup>+</sup> EMPs secreted by aged cells decreased （<italic>P</italic><0.05）. Compared with the young group， only EMPs intervention group could induce increased SA-<italic>β</italic>-gal activity and S phase arrest in young cells （<italic>P</italic><0.05，<italic>P</italic><0.01）. However， after intervention of EMPs and the drug， EMPs-mediated increase of SA-<italic>β</italic>-gal activity was significantly inhibited and S phase arrest was restored （<italic>P</italic><0.05）. The increase of intracellular ROS induced by EMPs was also significantly inhibited by the drug （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Ginseng Radix et Rhizoma-Notoginseng Radix et Rhizoma-Chuanxiong Rhizoma extract can delay the senescence of vascular endothelial cells by influencing EMPs， and the mechanism may be related to the inhibition of increased intracellular ROS induced by EMPs.
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Objective:To observe the effect of Ginseng Radix et Rhizoma， Notoginseng Radix et Rhizoma， and Chuanxiong Rhizoma extract （GNC） on mitochondrial oxidative stress in hydrogen peroxide （H<sub>2</sub>O<sub>2</sub>）-induced aging of human umbilical vein endothelial cells （HUVECs）， and explore the therapeutic mechanism of GNC on aging HUVECs. Method:The HUVECs were classified into the control group （control）， H<sub>2</sub>O<sub>2</sub> model group （H<sub>2</sub>O<sub>2</sub>）， H<sub>2</sub>O<sub>2</sub> + DMSO group （DMSO， 1 mL·L<sup>-1</sup>）， resveratrol group （Resv， 8 μmol·L<sup>-1</sup>）， and low- （200 mg·L<sup>-1</sup>）， medium- （300 mg·L<sup>-1</sup>）， and high-dose （400 mg·L<sup>-1</sup>） GNC （GNC-L， GNC-M， and GNC-H） groups. Except control group and H<sub>2</sub>O<sub>2</sub> group， the other groups were intervened with corresponding agents. Subsequently， 300 μmol·L<sup>-1</sup> H<sub>2</sub>O<sub>2</sub> was given to other groups except the control group for 4 h to induce aging， and then the cells were cultured in normal media for 24 h. The aging degree， cell cycle， and mitochondrial reactive oxygen species （mtROS） level were determined by SA-<italic>β</italic>-galactosidase （SA-<italic>β</italic>-Gal） staining， flow cytometry， and MitoSox red fluorescence staining， respectively. JC-10 was used as a fluorescent probe to detect the changes in mitochondrial membrane potential， and Western blot was performed to detect the expression of manganese superoxide dismutase （MnSOD） and p-p66 proteins. Result:The SA-<italic>β</italic>-gal staining results showed that H<sub>2</sub>O<sub>2</sub> group had increased blue-stained cells compared with other groups （<italic>P</italic><0.01）. Compared with those in the control group， the ratio of G<sub>0</sub>/G<sub>1</sub> phase cells significantly increased （<italic>P</italic><0.05） and that of G<sub>2</sub>/M phase cells decreased （<italic>P</italic><0.05） in the H<sub>2</sub>O<sub>2</sub> group. Compared with those in the H<sub>2</sub>O<sub>2</sub> group， the proportion of G<sub>0</sub>/G<sub>1</sub> cells decreased （<italic>P</italic><0.05） while that of G<sub>2</sub>/M cells increased （<italic>P</italic><0.05） in GNC-H groups and Resv group. The fluorescence staining for determining mitochondrial ROS level showed that the H<sub>2</sub>O<sub>2</sub> group had weakened fluorescence intensity than the control， GNC-H， and GNC-M groups （<italic>P</italic><0.05）. The mitochondrial membrane potential fluorescence intensity of the H<sub>2</sub>O<sub>2</sub> group was weaker than that of the control， GNC-H， GNC-M， and GNC-L groups （<italic>P</italic><0.01）， as well as the Resv group （<italic>P</italic><0.05）. Western blot showed that the protein level of MnSOD was significantly lower in the H<sub>2</sub>O<sub>2</sub> group than in the control， GNS-H， and GNS-M groups （<italic>P</italic><0.05）， whereas the protein level of p-p66 showed an opposite trend （<italic>P</italic><0.01）， indicating that the medication can alleviate the intracellular mitochondrial oxidative stress. Conclusion:GNC can delay the H<sub>2</sub>O<sub>2</sub>-induced aging of vascular endothelial cells. The GNC intervention significantly regulated the mitochondrial ROS， mitochondrial membrane potential， and related proteins MnSOD and p-p66 to alleviate oxidative stress. Chinese medicinal materials may delay the aging of vascular endothelial cells by inhibiting mitochondrial oxidative stress.
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<b>Objective::To explore the feasibility of Photoshop image processing software in the micro-spherical diameter measurement and identification of medicinal materials, in order to provide a new method for the measurement and mapping of traditional Chinese medicine. <b>Method::Photoshop (CS3 version and above) software was used to measure the diameter of 200 Notoginseng Radix et Rhizoma albumin microspheres in the same batch, digital microscopy and Photoshop image processing software was used to draw schematic diagrams of microstructures of Notoginseng Radix et Rhizoma medicinal powders and its four common pseudo-products and powders, namely Bletillae Radix, Alpiniae Officinarum Rhizoma, Curcumae Longae Rhizoma and <italic>Manihot esculenta,</italic> and make the identification table for classification and identification. <b>Result::The average diameter of the microspheres was (30.62±4.21)μm, and the diameter was mainly distributed in 20-40 μm. The average diameter of the microspheres was verified by laser particle size analyzer (30.18±4.67)μm. The differences between the two methods were not statistically significant. There were many starch granules in Notoginseng Radix et Rhizoma powder, with no calcium oxalate needle, oil cell, brown pigment and stone cell. Microscopic identification could be made for Notoginseng Radix et Rhizoma powder and 4 common counterfeits. <b>Conclusion::Photoshop image processing software measures the diameter of the microspheres with a high speed, high accuracy, simplicity, and low requirements for measuring instruments. It provides a new way to quickly measure the diameter of microspheres. Photoshop image processing software draws a schematic diagram of the microstructure, which is convenient and faster. The original morphology of the ground-reactive microstructure provides a new method for the microstructural drawing.
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The aim of this paper was to observe the changes of intestinal flora in vascular aging mice, in order to explore the relationship between vascular aging and intestinal flora and the effects of extracts of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma on intestinal flora of vascular aging mice. A model of vascular aging in mice was induced through intrape-ritoneal injection with streptozotocin(STZ) combined with high-fat diet. Biochemical detection was performed on serum cholesterol(CHO), triglyceride(TG), high-density liptein cholesterol(HDL-C), low-density liptein cholesterol(LDL-C) and blood glucose(GLU). HE staining was used to detect mice thoracic aorta morphology, and the expressions of cyclin-dependent kinase inhibitor 2 A(p16) and cyclin-dependent kinase inhibitor 1 A(p21) protein in mice thoracic aorta were detected by Western blot. The 16 S rDNA gene of mice intestinal flora was detected by Illumina MiSeq high-throughput sequencing technology to explore the changes of intestinal flora in each group. The results demonstrated that the GLU level in low-dose and high-dose TCM groups decreased, but with unobvious changes in blood lipid indexes. Metformin could significantly decrease the levels of GLU(P<0.01), CHO and LDL-C in mice(P<0.05). Intravascular injury was not obvious in each drug group, and the expressions of p16 and p21 protein were significantly decreased(P<0.05). The intestinal flora of each group was mainly composed of Firmicutes(F) and Bacteroidetes(B) at the level of the phylum, but the B/F ratio was different from that of the youth group and the blank control group. The B/F ratio of the model group was significantly lower(P<0.01), and compared with the model group, the B/F ratio of the high-dose group and the metformin group was signi-ficantly higher(P<0.05). There were dominant and differential floras in the intestine of each group of mice. The results showed that extracts of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma could improve the intestinal flora structure and create a good intestinal environment by increasing the B/F ratio, which provides a new possible pathway for lowering blood glucose and blood lipids and delaying vascular aging.
Subject(s)
Animals , Mice , Aging , Drugs, Chinese Herbal , Gastrointestinal Microbiome , Glucose , Lipids , PanaxABSTRACT
Objective::To explore the protective mechanism of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma (GNC) extracts on cardiac aging in diabetic mice by observing the activation of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, changes of cardiac pathomorphological and related senescent proteins. Method::C57BL/6 male mice, SPF level, were randomly divided into normal control group and high-glucose group. The mice in high-glucose group were intraperitoneally injected with streptozotocin (STZ) and fed with high-fat diet. After successful modeling, they were randomly divided into model group, low-dose GNC group (0.819 g·kg-1), high-dose GNC group (1.638 g·kg-1) and metformin group (150 mg·kg-1). The drug was administered by gavage once a day for a continuous period of 9 weeks. 4-week-old male C57BL/6 mice were normally fed for 1 week as a youth group. General conditions of mice were observed. Hematoxylin-eosin (HE) staining combined with transmission electron microscope (TEM) was used to observe the cardiac pathomorphology in mice. Von Kossa staining was used to determine the degree of calcium salt deposition in cardiac micro vessels. Western blot was used to detect the activation of signaling pathways in myocardial tissue of mice, as well as the expression levels of matrix metalloproteinases-2 (MMP-2), tumor suppressor p53 (p53), and phospho-tumor suppressor p53 (p-p53). Result::As compared with the normal group, the blood glucose in the model group increased (P<0.01), as compared with the model group, the blood glucose in each administration group decreased significantly (P<0.05, P<0.01). The results of three pathological morphology experiments (HE, TEM, and Von Kossa) showed that as compared with the normal control group, the mice in model group showed cardiomyocytes hypertrophy, disordered arrangement of myocardial fibers, focal dissolving and necrosis, mitochondria swelling, degeneration, crest fracture, vacuolar alteration, disordered microvascular structure of the heart, uneven staining, and a large amount of calcium deposition in tunica media and intima. As compared with the model group, the pathomorphological changes of mice in each administration group were improved in varying degrees. Compared with the normal group, the expression levels of MMP-2, p53 and p-p53 protein in the model group were significantly increased (P<0.05, P<0.01), the protein ratios of p-liver kinase B2(LKB1)/LKB1, p-AMPK/AMPK were significantly decreased (P<0.05, P<0.01), and the average gray level of p-mTOR/mTOR and p-p70S6 kinase(p70S6k)/p70S6k protein was significantly increased (P<0.05, P<0.01), while the protein ratios of p-mTOR/mTOR, p-p70S6k/p70S6k were increased (P<0.01). As compared with the model group, the expression levels of MMP-2, p53 and p-p53 protein in each administration group were significantly decreased (P<0.05, P<0.01), the protein ratios of p-LKB1/ LKB1, p-AMPK/AMPK were significantly increased (P<0.05, P<0.01), while the protein ratios of p-mTOR/mTOR and p-p70S6k/p70S6k were decreased (P<0.05, P<0.01). Conclusion::STZ combined with high-fat diet can induce cardiac aging in mice, and GNC can improve cardiac aging in diabetic mice, which may be related to the inhibition of AMPK/mTOR pathway related protein expression.
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Objective::To investigate the protective effect of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma (GNC) extracts on myocardial fibrosis in diabetic mice by observing the degree of myocardial fibrosis and collagen types I (Collagen Ⅰ), collagen types Ⅲ (Collagen Ⅲ) and transforming growth factor-β1 (TGF-β1) protein expression in myocardial tissues. Method::A diabetic mice model was induced by streptozotocin (STZ) and high-fat diet. A normal control group was established. According to random number table method, diabetic mice were divided into model group, GNC low-dose and high-dose groups (0.819, 1.638 g·kg-1), and metformin group (150 mg·kg-1). Intragastrical administration was given in all groups, and the mice in normal control group received an equal dose of deionized water once a day for 9 weeks. The myocardial interstitial fibrosis in mice was observed by Masson trichromatic staining. Image-pro plus 6.0 analysis software was used to calculate the ratio of collagen area to total area. Immunohistochemistry was used to detect Collagen I, Collagen Ⅲ and TGF-β1 protein expression in myocardial tissues. The protein expression electrophoresis and gray value levels of Collagen I, Collagen Ⅲ and TGF-β1 in the myocardial tissues were detected by Western blot. Result::The results of Masson staining showed that as compared with the normal control group, the myocardial cells of diabetic mice were hypertrophic and disordered, and the myocardial stroma, especially the blue-stained collagenous fibers around the blood vessels, were heavily deposited and connected to each other in a network (P<0.01). As compared with the model group, the arrangement of myocardial cells was significantly improved in GNC low-dose and high-dose groups and metformin group, and the collagenous fibers in the myocardial stroma were significantly decreased (P<0.05). Immunohistochemistry and Western blot results showed positive expression of Collagen Ⅰ, Collagen Ⅲ and TGF-β1 in myocardial tissues, with significantly increased content of protein expression in diabetic mice (P<0.05, P<0.01). As compared with the model group, the positive protein expression decreased and the protein content tended to be normal in each administration group (P<0.05, P<0.01). Conclusion::High-fat diet combined with STZ can induce myocardial fibrosis in diabetic mice, and increase Collagen I, Collagen Ⅲ and TGF-β1 protein expression. Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts can improve myocardial fibrosis in diabetic mice by regulating the expression of Collagen I, Collagen Ⅲ and TGF-β1 protein.
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Objective::To investigate the effects of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts (GNC) on the protein expression of α-smooth muscle actin (α-SMA) and runt-related transcription factor2(Runx2) after high glucose-induced vascular aging in mice, and elucidate the protective mechanism of GNC in delaying vascular aging. Method::Totally 130 male C57BL/6 mice were randomly divided into normal control group and high glucose group. The mice in high glucose group were intraperitoneally injected with streptozotocin (STZ). After successful modeling, the mice received high-fat diet for 7 months, and then they were randomly divided into model group, GNC low-dose and high-dose groups (0.819, 1.638 g·kg-1), and metformin group (150 mg·kg-1). The drug was given by intragastric administration once a day for 9 weeks. Seven days before tissues collection, a new batch of 4-week-old male C57BL/6 mice were purchased and fed normally for 1 week as a youth group. The general condition of the mice was observed. Morphological changes of the common carotid artery in mice were determined by hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM). Masson trichromatic staining was used to observe the fibrosis of common carotid artery in mice. The expression levels of matrix metalloproteinases-2 (MMP-2), cyclin-dependent kinase inhibitor 2A (p16), cyclic-dependent kinase inhibitor 1A (p21), α-SMA and Runx2 in the common carotid arteries of mice were detected by immunohistochemistry. Result::The results of HE, TEM and Masson showed that there was almost no change in the inimal and adventitial thickness, ultrastructure and relative contents of collagen and elastic fibers in the common carotid arteries of mice between the youth group and normal control group. As compared with the normal control group, the intima of the common carotid artery in the model group was not smooth, the endothelial cells were almost completely detached, the cytoplasm was lysed, the inner elastic membrane became thinner, fractured, or even detached, and the proliferating collagen fibers sneaked into the tunica media. The hyperplasia of tunica media and tunica adventitia was obvious and disordered (P<0.01). The vascular smooth muscle cells showed deformations, protuberances, bifurcations, and even fragmentation, and focal necrosis was observed. There were significantly more vacuoles, lysosomes, and obvious autophagy vesicles. The relative content of collagen and elastic fibers in vascular walls increased significantly (P<0.01). Compared with the model group, the above situation was relieved in each administration group (P<0.01). The results of immunohistochemistry showed that high glucose induced high expression of MMP-2, p16, p21 and Runx2 in the common carotid arteries(P<0.01), low expression of α-SMA(P<0.01), and the protein expression tended to be normal after drug intervention(P<0.05, P<0.01). Conclusion::High glucose can induce the aging of common carotid artery in mice and change the expression of α-SMA and Runx2 proteins. The Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts can delay vascular aging by regulating the protein expression of α-SMA and Runx2.
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Objective::To investigate the protective effect of Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts on vascular calcification induced by high glucose in mice by observing the expression of osteopontin (OPN) and smooth muscle 22α (SM22α) as well as vascular calcium deposition in the common carotid artery and thoracic aorta of mice. Method::Totally 130 male C57BL/6 mice were randomly divided into normal control group and high glucose group. The mice in high glucose group were intraperitoneally injected with streptozotocin(STZ), and fed on a high-fat diet for 7 months. Then, the mice were randomly divided into model group, low-dose and high-dose Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts groups (0.819, 1.638 g·kg-1), and metformin group (150 mg·kg-1). Each group was intragastrically administered once a day for 9 weeks. The changes in blood glucose were measured. Seven days before the end of the administration, a group of 4-week old male C57BL/6 mice were purchased and fed normally for one week as a youth group. At the end of the administration, the common carotid artery and thoracic aorta tissues of the mice were collected. Von Kossa staining was used to determine the degree of calcium deposition in the common carotid artery and thoracic aorta. The expression levels of OPN and SM22α protein in the common carotid artery and thoracic aorta were detected by immunohistochemistry. The expression of OPN and SM22α protein in the common carotid artery of mice was determined by Western blot. Result::As compared with the young group, the blood glucose of the normal control group was slightly increased without statistical difference, the common carotid artery and thoracic aorta were uniformly stained, and no black granular precipitate was observed. As compared with the normal control group, the blood glucose of the model group was increased (P<0.01), with a large amount of brown-black particles deposited in the intimal elastic fibers, showing obvious calcium salt deposition. As compared with the model group, blood glucose was significantly decreased in each administration group (P<0.05, P<0.01), and the degree of vascular calcium salt deposition was significantly reduced. There were no significant changes in expression levels of OPN protein and SM22α protein in the common carotid artery and thoracic aorta between the youth group and normal control group. As compared with the normal control group, the expression of intimal OPN protein in the common carotid artery and thoracic aorta of the model group was positive, SM22α protein expression was weakly positive, and the gray value of OPN protein expression in the common carotid artery was significantly increased (P<0.01), while the gray value of SM22α protein was decreased significantly (P<0.01). As compared with the model group, the expression levels of intimal OPN protein and SM22α protein in the common carotid artery and thoracic aorta of each administration group were significantly improved, and the gray value of OPN protein expression in the common carotid artery was reduced (P<0.05, P<0.01), while SM22α protein expression was significantly increased (P<0.01). Conclusion::High glucose can induce calcification of common carotid artery and thoracic aorta in mice and accelerate vascular aging. This formation process may be related to the expression of OPN and SM22α. Ginseng Radix et Rhizoma, Notoginseng Radix et Rhizoma and Chuanxiong Rhizoma extracts can reduce vascular calcification and delay vascular aging by regulating the expression of OPN and SM22α.
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Notoginseng Radix et Rhizoma, one of precious and important traditional Chinese medicinal herb, has the functions of dispersing blood stasis and stopping bleeding, detaching swelling and alleviating pain, and invigorating Qi and blood. It is a traditional Chinese medicine for promoting blood circulation and removing blood stasis. After processing, the efficacy of Notoginseng Radix et Rhizoma was obviously different, the raw products mainly dispersed blood stasis and hemostasis, while the main effect of processed products was to replenish blood and Qi. In recent years, more attention has been paid to Notoginseng Radix et Rhizoma research, mainly focusing on the chemical constituent, pharmacological action and clinical application of the raw products. Although the research on processed Notoginseng Radix et Rhizoma has been increasing in these years, the mechanism of processing and the changes of bioactive constituents before and after processing are still unclear. This paper systematically summarized the modern processing methods of Notoginseng Radix et Rhizoma and compared the changes in chemical constituent and pharmacological action before and after processing through literature search. It is proposed that modern technologies should be put forward to study the correlation between the chemical constituent transformation and enriching blood as the breakthrough point, in order to explain the processing mechanism of Notoginseng Radix et Rhizoma, improve the quality evaluation system of this decoction pieces, and provide guarantee for the safety and effectiveness of its clinical medication.