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ObjectiveTo investigate the effect and mechanism of salvianolic acid B combined with puerarin in protecting the SH-SY5Y cells from the damage by oxygen-glucose deprivation/reoxygenation （OGD/R） based on pyroptosis. MethodSH-SY5Y cells were used to establish the model of OGD/R， and cells were classified into the control， OGD/R， 10 μmol·L-1 salvianolic acid B， 100 μmol·L-1 puerarin， 10 μmol·L-1 salvianolic acid B + 100 μmol·L-1 puerarin， and 10 μmol·L-1 NOD-like receptor protein 3 （NLRP3） inhibitor MCC950 groups. Except the control group， other groups were rapidly reoxygenated for 12 h after 6 h OGD for modeling. The cell survival rate was determined by the methyl thiazolyl tetrazolium （MTT） assay. An optical microscope was used to observe the cell morphology. A spectrophotometer was used to determine the content of lactic dehydrogenase （LDH） in culture supernatant. Cell damage was measured by Hoechst/PI staining. The mRNA levels of NLRP3， cysteinyl aspartate specific proteinase-1 （Caspase-1）， gasdermin D （GSDMD）， apoptosis-associated speck-like protein （ASC）， and interleukin-1β （IL-1β） were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein activation of Caspase-1 and NLRP3 was detected by immunofluorescence. Western blot was employed to determine the protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1. ResultCompared with the control group， the OGD/R group showed decreased cell survival rate （P<0.01）， damaged cell morphology， increased leakage rate of LDH （P<0.01）， up-regulated mRNA levels of NLRP3， Caspase-1， GSDMD， ASC， and IL-1β （P<0.01）， and up-regulated protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1 （P<0.01）. Compared with the OGD/R group， salvianolic acid B， puerarin， and salvianolic acid B combined with puerarin improved cell survival rate （P<0.01）， and the combined treatment group outperformed salvianolic acid B and puerarin used alone （P<0.01）. Salvianolic acid B combined with puerarin and MCC950 both improved cell morphology， reduced the leakage of LDH （P<0.01）， alleviated cell damage， and down-regulated the mRNA levels of NLRP3， Caspase-1， GSDMD， ASC， and IL-1β （P<0.05， P<0.01） and also the protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1 （P<0.05， P<0.01）. ConclusionThe results indicated that salvianolic acid B combined with puerarin can alleviate the OGD/R-induced damage of SH-SY5Y cells by inhibiting pyroptosis.
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Salvianolic acid B(Sal B) is the main water-soluble component of Salvia miltiorrhiza Bunge. Studies have found that Sal B has a good protective effect on blood vessels. Sal B can protect endothelial cells by anti-oxidative stress, inducing autophagy, inhibiting endoplasmic reticulum stress(ERS), inhibiting endothelial inflammation and adhesion molecule expression, inhibiting endothelial cell permeability, anti-thrombosis, and other ways. In addition, Sal B can alleviate endothelial cell damage caused by high glucose(HG). For vascular smooth muscle cell(VSMC), Sal B can reduce the synthesis and secretion of inflammatory factors by inhibiting cyclooxygenase. It can also play a vasodilatory role by inhibiting Ca~(2+) influx. In addition, Sal B can inhibit VSMC proliferation and migration, thereby alleviating vascular stenosis. Sal B also inhibits lipid deposition in the subendothelium, inhibits macrophage conversion to foam cells, and reduces macrophage apoptosis, thereby reducing the volume of subendothelial lipid plaques. For some atherosclerosis(AS) complications, such as peripheral artery disease(PAD), Sal B can promote angiogenesis, thereby improving ischemia. It should be pointed out that the conclusions obtained from different experiments are not completely consistent, which needs further research. In addition, previous pharmacokinetics showed that Sal B was poorly absorbed by oral administration, and it was unstable in the stomach, with a large first-pass effect in the liver. Sal B had fast distribution and metabolism in vivo and short drug action time. These affect the bioavailability and biological effects of Sal B, and the development of clinically valuable Sal B non-injectable delivery systems remains a great challenge.
Subject(s)
Endothelial Cells , Oxidative Stress , Benzofurans/pharmacology , LipidsABSTRACT
With the development of small-molecule immunotherapy drugs, its combination with the programmed cell death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) antibodies would provide a new opportunity for cancer treatment. Therefore, targeting PD-L1/PD-1 axis by small-molecule drug is an attractive approach to enhance antitumor immunity and considered as the next generation of tumor immunotherapy. In the present study, we investigated the anti-tumor role of salvianolic acid B (SAB) by regulating the PD-L1 level in tumors. Changes of total PD-L1 and membrane PD-L1 levels were determined by Western blot, flow cytometry and PD-1/PD-L1 interaction assays. The expression of mRNA level of PD-L1 was detected by real-time PCR. The cytotoxicity of activated peripheral blood mononuclear cell (PBMC) cells toward co-cultured tumor cells was measured by cell impedance assay and crystal violet experiment. Surface plasma resonance technique was used to analyze the direct interaction between SAB and ubiquitin carboxyl-terminal hydrolase 2 (USP2). The antitumor effect of SAB in vivo was examined by C57BL/6 mice bearing MC38 xenograft tumor (all animal experiments were conducted in accordance with the Animal Ethics Committee of the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences). Western blot and flow cytometry assay showed that SAB can significantly downregulate the abundance of PD-L1 in RKO and PC3 cells in dose- and time-dependent manner. PD-1/PD-L1 binding assay revealed that SAB reduces the binding of tumor cells to recombinant PD-1 protein. Mechanism studies revealed that SAB can bind directly to USP2 protein and inhibit its activity, thus promote the ubiquitin-proteasome pathway degradation of PD-L1 proteins. In addition, Cell impedance and crystal violet staining indicated that SAB enhances the killing activity of co-cultured PBMC cells toward tumor cells. MC38 tumor transplanted mouse experiments revealed that SAB treatment displayed significant suppression in the growth of MC38 tumor xenografts in C57BL/6 mice with an inhibition rate of 63.2% at 20 mg·kg-1. Our results demonstrate that SAB exerts its anti-tumor activity by direct binding and inhibiting the activity of USP2 and reducing the PD-L1 level. Our study provides an important material basis and scientific basis for the potential application of SAB in tumor immunotherapy drug targeting USP2-PD-L1 axis.
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AIM: To investigate the protective effect of salvianolic acid B on retina and its influence on angiogenesis in retinal vein occlusion(RVO)injured rats.METHODS: SD rats were randomly divided into control group, model group and salvianolic acid B group, with 10 rats in each group. In addition to the control group, rats in model group and salvianolic acid B group were induced RVO by Bengal red combined with laser photodynamic method. The rats in salvianolic acid B group were intraperitoneally injected with salvianolic acid B 50 mg/(kg·d), while the rats in control group and model group were only given the same amount of normal saline for 21 consecutive days. Fundus fluorescein angiography(FFA)was used to observe the retinal vein structure before and after administration. HE staining was used to observe the pathological changes of rat retina. The retinal function of rats was evaluated by electroretinogram(ERG). The fluorescence expression of vascular endothelial growth factor A(VEGFA)in retina of rats in each group was detected by immunofluorescence staining. The relative expression of HIF-1α, STAT3, p-STAT3 and VEGFA proteins in retinal tissue were detected by Western blotting.RESULTS: Compared with the control group, the blood flow at the retinal obstruction in the model group was recanalized, and the effective collateral circulation was abundant, but the shape was irregular, and there was fluorescence leakage. In salvianolic acid B group, the retinal vein circulation recovered, the shape became regular gradually, and the collateral vessels decreased. The retina of the model group and salvianolic acid B group showed different degrees of pathological damage. At the same time, the amplitude of ERG a wave and b wave, the thickness of retinal total layer(RTL), inner nuclear layer(INL)and outer nuclear layer(ONL)decreased, the fluorescence intensity of VEGFA enhanced, and the relative expression of HIF-1α, p-STAT3 and VEGFA protein increased(P&#x003C;0.05). Compared with the model group, the retinal histopathological damage of salvianolic acid B rats was alleviated, the amplitude of ERG a-wave and b-wave, the thickness of RTL, INL and ONL were increased, the fluorescence intensity of VEGFA was weakened, and the relative expression of HIF-1α, p-STAT3 and VEGFA proteins was decreased(P&#x003C;0.05).CONCLUSION: Salvianolic acid B can alleviate the retinal histopathological injury and improve retinal function in RVO rats, which may be related to inhibiting the activation of HIF-1α/STAT3/VEGFA pathway and reducing angiogenesis.
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OBJECTIVE@#To determine whether salvianolic acid B (Sal B) exerts protective effects on diabetic peripheral neuropathy by attenuating apoptosis and pyroptosis.@*METHODS@#RSC96 cells were primarily cultured with DMEM (5.6 mmol/L glucose), hyperglycemia (HG, 125 mmol/L glucose) and Sal B (0.1, 1, and 10 µ mol/L). Cells proliferation was measured by 3-(4, 5-cimethylthiazol-2-yl)-2, 5-dilphenyltetrazolium bromide assay. Reactive oxygen species (ROS) generation and apoptosis rate were detected by flow cytometry analysis. Western blot was performed to analyze the expressions of poly ADP-ribose polymerase (PARP), cleaved-caspase 3, cleaved-caspase 9, Bcl-2, Bax, NLRP3, ASC, and interleukin (IL)-1β.@*RESULTS@#Treatment with HG at a concentration of 125 mmol/L attenuated cellular proliferation, while Sal B alleviated this injury (P<0.05). In addition, Sal B inhibited HG-induced ROS production and apoptosis rate (P<0.05). Furthermore, treatment with Sal B down-regulated HG-induced PARP, cleaved-caspase 3, cleaved-caspase 9, Bax, NLRP3, ASC, and IL-1β expression, but mitigated HG-mediated down-regulation of Bcl-2 expression (P<0.05).@*CONCLUSION@#Sal B may protect RSC96 cells against HG-induced cellular injury via the inhibition of apoptosis and pyroptosis activated by ROS.
Subject(s)
Apoptosis , Benzofurans/pharmacology , Oxidative Stress , Pyroptosis , Reactive Oxygen Species/metabolismABSTRACT
Objective:To evaluate the effect of Salvianolic acid B on the radiosensitivity of human non-small cell lung cancer cells and investigate its possible mechanism.Methods:Non-small cell lung cancer cells A549 and H1299 were cultured in vitro. The toxicity of Salvianolic acid B on non-small cell lung cancer cells was detected by MTT assay. The effect of Salvianolic acid B on the radiosensitivity was assessed by clone formation assay. Transwell chamber assay was used to evaluate the effect of Salvianolic acid B on the migration of tumor cells. Western blot was employed to detect the expression levels of OTUD7B, MMP-2, MMP-9, E-cadherin, Akt and p-Akt regulated by Salvianolic acid B. Results:Salvianolic acid B (5 μmol/L) could inhibit the proliferation of A549 and H1299 cells. Clone formation assay showed that Salvianolic acid B increased the radiosensitivity of A549 and H1299 cells, with a radiosensitization ratio of 1.45 and 1.38, respectively. Transwell chamber assay indicated that the ability of cell migration was significantly inhibited by Salvianolic acid B ( P<0.05). Western blot revealed that the expression levels of OTUD7B in A549 and H1299 cells were induced by irradiation in a time-dependent manner. Salvianolic acid B could down-regulate the expression levels of MMP-2, MMP-9 and p-Akt, whereas up-regulate the expression level of E-cadherin by down-regulating the expression level of OTUD7B. Conclusions:Salvianolic acid B can enhance the radiosensitivity of A549 and H1299 cells. The possible mechanism is that Salvianolic acid B down-regulates the expression level of OTUD7B induced by irradiation and inhibits the epithelial-mesenchymal transition process of tumor cells.
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In recent years, there has been an increase in the incidence of herbal-induced liver injury due to the accidental ingestion of herbal medicines containing pyrrolizidine alkaloids (PAs) in domestic. Salvianolic acid B (Sal B), a hydrophilic component in Salvia miltiorrhiza Bge., shows activities of anticoagulation, antioxidation, and other pharmacological activities. This research aims to investigate the protective effect of Sal B on hepatotoxicity induced by senecionine (SEN) and its potential mechanism. The animal experiment was approved by the Experimental Animal Ethical Committee of Shanghai University of Traditional Chinese Medicine, and all mice have received humane care in compliance with the institutional animal care guidelines. Mice were treated with Sal B (10 mg·kg-1) 3 days before and 1 day after SEN (50 mg·kg-1) treatment. The animals were sacrificed 48 h after SEN administration. As a result, Sal B effectively ameliorated SEN-induced liver injury. The mice in the group treated with Sal B showed lower serum activities of alanine aminotransferase and aspartate aminotransferase, less hepatic sinusoidal hemorrhage, and reduced hepatocyte necrosis. Besides, contents of pyrrole-protein adducts, the marker for PA-induced toxicity, were also decreased in serum. The key factors related to coagulation, oxidative stress, and liver fibrosis were further analyzed. It was found that Sal B inhibited the coagulant system by reducing the expression of plasminogen activator inhibitor-1. Sal B also modulated glutathione and superoxide dismutase levels and improved the anti-oxidative defense system. In addition, Sal B decreased the excessive deposition of extracellular matrix and inhibited the progression of liver fibrosis via down-regulating several key factors related to liver fibrosis, including matrix metalloproteinase 9, transforming growth factor-β1, signal transducer and activator of transcription 3, and chemokine 1. In conclusion, Sal B ameliorated SEN-induced liver injury in mice by regulating the blood coagulation system, improving oxidative stress, and modulating liver fibrosis-related factors. Our present study pointed to the possibility of utilizing salvianolic acid B for protection against PA-induced liver injury clinically.
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Salvianolic acids are the main water-soluble active compounds of Salvia miltiorrhiza and have been widely used for the treatment of cardiovascular diseases. Based on the latest studies in China and abroad, we summarize the pharmacological effects and mechanism of salvianolic acids on ischemic heart disease by describing how salvianolic acid A and salvianolic acid B protect the vascular endothelium, relax coronary arteries, promote angiogenesis and anti-platelet aggregation, inhibit the inflammatory response, anti-cell apoptosis, and scavenge free radicals. This review provides a theoretical basis for further research on the effects of salvianolic acids on ischemic heart disease and their potential for drug development.
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The metabolites of salvianolic acid A and salvianolic acid B in rats were analyzed and compared by ultra-high-perfor-mance liquid chromatography with linear ion trap-orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS). After the rats were administrated by gavage, plasma at different time points and urine within 24 hours were collected to be treated by solid phase extraction(SPE), then they were gradient eluted by Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) and 0.1% formic acid solution(A)-acetonitrile(B) mobile phase system, and finally all biological samples of rats were analyzed under negative ion scanning mode. By obtaining the accurate relative molecular mass and multi-level mass spectrometry information of metabolites, combined with the characteristic cleavage law of the reference standard and literature reports, a total of 30 metabolites, including salvianolic acid A and B, were identified. Among them, there were 24 metabolites derived from salvianolic acid A, with the main metabolic pathways including ester bond cleavage, dehydroxylation, decarboxylation, hydrogenation, methylation, hydroxylation, sulfonation, glucuronidation, and their multiple reactions. There were 15 metabolites of salvianolic acid B, and the main biotransformation pathways were five-membered ring cracking, ester bond cleavage, decarboxylation, dehydroxylation, hydrogenation, methylation, sulfonation, glucuronidation, and their compound reactions. In this study, the cross-metabolic profile of salvianolic acid A and B was elucidated completely, which would provide reference for further studies on the basis of pharmacodynamic substances and the exploration of pharmacological mechanism.
Subject(s)
Animals , Rats , Benzofurans , Caffeic Acids , Chromatography, High Pressure Liquid , Lactates , Mass Spectrometry , TechnologyABSTRACT
Objective:To establish the grade evaluation standard of Salviae Miltiorrhizae Radix et Rhizoma decoction pieces combining traditional character evaluation and modern intrinsic quality analysis. Method:The appearance character parameters （thickness and weight） and contents of internal index components （tanshinones and salvianolic acid B） of 18 batches of Salviae Miltiorrhizae Radix et Rhizoma decoction pieces were determined， and the relative quality constant was calculated. The maximum value of the percentage quality constants of the tested samples was assumed to be 100%， the value ≥80% was classified as the first-class， ≥50% and <80% as the second-class， <50% as the third-class. Result:The relative quality constants of 18 batches of Salviae Miltiorrhizae Radix et Rhizoma decoction pieces ranged from 349 to 884. According to the percentage quality constant， 18 batches of samples were successfully divided into three grades. The relative quality constant of the first-class product was ≥707， including samples ds5， ds8 and ds14， accounting for about 17% of the total number of samples. The relative quality constant of the second-class product was ≥442 and <707， accounting for about 61% of the total number of samples. the other samples were of the third-class， and their relative quality constants were all <442. Conclusion:The method of relative quality constant overcomes the one-sidedness of the single method in the grade evaluation of Salviae Miltiorrhizae Radix et Rhizoma decoction pieces， and the evaluation results can objectively， reasonably and scientifically classify the grade of the decoction pieces， which can provide reference for the establishment of the grade standard of other decoction pieces.
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OBJECTIVE：To e valuate the correlation between processing time ，color and chemical composition content in the wine-fried process of Salvia miltiorrhiza . METHODS ：Wine-fried S. miltiorrhiza with different processing time （7-15 min）was prepared by yellow rice wine. The contents of salvianolic acid B ，tanshinone ⅡA and 5-HMF in raw and wine-fried S. miltiorrhiza were determined by HPLC. The colorimeter was used to determine their chromatic values [red-green axis component （a*）， yellow-blue axis component （b*），lightness（L*）] and calculate the total color difference value （ΔE）. Spearman ’s rho and Kendall ’s Tau-b test were adopted to validate the correlation between processing time ，chromatic value and chemical composition content. RESULTS：The contents of salvianolic acid B ，tanshinone ⅡA and 5-HMF were 17.9-70.6，2.3-3.1，0 mg/g in S. miltiorrhiza decoction pieces ；the contents of them were 14.8-68.4，1.1-3.9，0.7-34.4 mg/g in wine-fried S. miltiorrhiza . The content of salvianolic acid B at first decreased and then increased ，reaching the peak at about 9，11 min，and then gradually decreased ；the content of tanshinone ⅡA increased at first ，reached its peak about 7 min，and then gradually decreased；the content of 5-HMF increased sharply after frying 13 min. The measurement results of chromaticity values were ΔL* －5.369-2.553，Δa* －1.098-0.321， Δb* －1.471- 2.355，ΔE 0.217-5.397. Results of Spearman ’s rho and Kendall ’s Tau-b test showed that ΔE was positively correlated with processing time （the correlation coefficient were 0.517，0.389 respectively）and 5-HMF content （the correlation coefficient were 0.549，0.405 respectively）（both P＜0.01）. The content of tanshinone ⅡA was negatively correlated with Δb（* the correlation coefficient were －0.509，－0.391 respectively），processing time （the corr elation coefficient were －0.556，－0.420 respectively） and 5-HMF content （the correlation coefficient were －0.545，－0.392 respectively）（both P＜0.01）. The content of 5-HMF was positively correlated with the processing time （the correlation coefficient were 0.957，0.870 respectively）（both P＜0.01）. CONCLUSIONS ：The contents of tanshinone ⅡA and 5-HMF in the process of wine-fried process are significantly related to the time and color. With the increase of processing time and temperature ，its color changes from red 话：0553-3844333。E-mail：liulj1@126.com yellow to yellow green ，and tends to be black in black and white；the content of tanshinone ⅡA is decreased and the content of 5-HMF is increased.
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Fufang Danshen preparation (FDP) is consisted of Salviae Miltiorrhizar Radix et Rhizoma (Danshen), Notoginseng Radix et Rhizoma (Sanqi) and Borneolum Syntheticum (borneol). FDP is usually used to treat myocardial ischemia hypoxia, cerebral ischemia and alzheimer's disease, etc. In the treatment of cerebrovascular diseases, borneol is usually used to promote the absorption and distribution of the bioactive components to proper organs, especially to the brain. The purpose of this study is investigating the effects of borneol on the pharmacokinetics and brain distribution of tanshinone IIA (TS IIA), salvianolic acid B (SAB) and ginsenoside Rg1 in FDP. Male healthy Sprague-Dawley (SD) rats were given Danshen extracts, Sanqi extracts (Panax notoginsengsaponins) or simultaneously administered Danshenextracts, Sanqi extracts and borneol. Plasma and brain samples were collected at different points in time. The concentration of TS IIA, SAB and Rg1 was determined by UPLC-MS/MS method. The main pharmacokinetics parameters of plasma and brain tissue were calculated by using Phoenix WinNolin 6.1 software. In comparison with Danshen and Sanqi alone, there were significant differences in pharmacokinetic parameters of TS IIA, SAB and Rg1, and the brain distribution of SAB and TS IIA when Danshen, Sanqi and borneol were administrated together. Borneol statistically significant shortened t
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BACKGROUND: In recent years, bone marrow mesenchymal stem cells have been used in clinical treatment of heart injury caused by permanent cardiomyocytopenia. However, the best inducer to promote the proliferation and differentiation of bone marrow mesenchymal stem cells into cardiomyocytes is still in the research and selection. OBJECTIVE: To investigate the protective effect of salvianolic acid B on oxidative stress injury and cardiomyocyte differentiation of bone marrow mesenchymal stem cells. METHODS: Bone marrow mesenchymal stem cells were used as study objects to establish oxidative stress injury model by 300 μmol/L H2O2 for 24 hours. Simultaneously, bone marrow mesenchymal stem cells were treated with salvianolic acid B for 24 hours. The contents of lactate dehydrogenase and creatine kinase in the supernatant and malondialdehyde and superoxide dismutase in the cell lysate were determined in each group using the kit. Western blot assay was used to detect the expression of nuclear factor E2 related factor 2 (Nrf2), Keap1, Bcl-2 and Bax in bone marrow mesenchymal stem cells. RT-PCT was used to detect cardiomyocyte differentiation-related markers 14 days after intervention. RESULTS AND CONCLUSION: (1) Compared with the model group, the levels of lactate dehydrogenase and creatine kinase in the supernatant were significantly decreased in the salvianolic acid B group (P < 0.05). (2) Compared with the model group, the level of malondialdehyde in cell lysate was decreased, and the level of superoxide dismutase was increased in the salvianolic acid B group (P < 0.05). (3) Compared with the model group, expression of Nrf2 protein in cells was significantly increased, and the expression of Keap1 protein was significantly decreased in the salvianolic acid B group. (4) The expression of Bcl-2 in salvianolic acid B group was significantly higher than that in model group, and the expression of Bax in salvianolic acid B group was significantly lower than that in model group. (5) Compared with the model group, the expression of GATA4 and cTnT mRNA was significantly increased in the salvianolic acid B group. (6) Salvianolic acid B can induce the expression of proteins that activate Nrf2-ARE signaling pathway in bone marrow mesenchymal stem cells for anti-oxidation, and salvianolic acid B can inhibit apoptosis and improve the ability of bone marrow mesenchymal stem cells to differentiate into cardiomyocytes.
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To investigate the protective effect of salvianolic acid B(SAB)on the intestinal tract of rats after intestinal ischemia-reperfusion injury(IIRI). Forty-eight healthy male SD rats were equally randomized into IIRI group,SAB+IIRI group,sham control group,and SAB+sham control group. The malonyldialdehyde(MDA)level and superoxide dismutase(SOD)activity in the ileum were measured in each group according to the kit instructions,the transcription levels of inflammatory factors in the ileum of rats were detected by real-time fluorescence quantitative RT-PCR,the secretion level of inflammatory factors was detected by ELISA,and the effects of intestinal ischemia-reperfusion on intestinal permeability and histological lesions were measured by histopathology. The MDA level in IIRI group was significantly higher than those in negative control group(=0.005)and SAB+IIRI group(=0.012). SOD activity of IIRI group was significantly lower than those of negative control group(=0.006)and SAB+IIRI group(=0.017). The optical densities of tumor necrosis factor-α(TNF-α)(=0.003,=0.009),interleukin(IL)-1β(=0.026,=0.005),IL-6(=0.015,=0.003),and nuclear factor kappa-B(NF-κB)(=0.007,=0.015)in IIRI group were significantly higher than those in sham control group and SAB+IIRI group. The TNF-α(=0.002,=0.006),IL-1β(=0.002,=0.006),IL-6(=0.008,=0.002),and NF-κB(=0.026,=0.005)levels in IIRI group were significantly higher than those in sham control group and SAB+IIRI group. The inulin level in IIRI group was significantly lower than that in negative control group(=0.015)and significantly higher than that in SAB+IIRI group(=0.011). The dextran level in IIRI group was significantly lower than those in sham control group(=0.011)and SAB+IIRI group(=0.012). The dextran gel level in IIRI group was significantly higher than those in sham control group(=0.031)and SAB+IIRI group(=0.020). SAB pretreatment remarkably improved the edema,necrosis,and villus stripping of the intestinal mucosa in the ileum of rats. The Chiu score was significantly higher in SAB+sham control group than in sham control group(=0.001)and was significantly lower in SAB+IIRI group than in IIRI group(=0.001). SAB pretreatment can alleviate IIRI in rat models,and this protective effect may be achieved by alleviating oxidative stress and inflammation in the intestinal tract.
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The aim of this paper was to observe the effect of salvianolic acid B(Sal B) on high-glucose induced renal tubular epithelial-mesenchymal transition(EMT) in rats, and to explore its possible mechanisms of prevention and treatment of diabetic nephropathy. The rat renal tubular epithelial NRK-52 E cells were cultured in vitro. The cells were divided into control group, high glucose group, high glucose+10 μmol·L~(-1)Sal B group(Sal B), the above 3 groups were set at 6, 12, 24 and 48 h for dynamic observation; high glucose+Sal B different concentration(1, 5, 10 μmol·L~(-1)) groups, high glucose+5.0 μmol·L~(-1) pioglitazone group, high glucose+10 μmol·L~(-1)Sal B+5 μmol·L~(-1)GW9662 group. The protein expression levels of PPARγ, PTEN, α-SMA, E-cadherin and PI3 K/Akt signaling molecules were determined by Western blot. The mRNA expression of PPARγ and PTEN were detected by Real-time PCR. The viabi-lity of NRK52 E cells was determined by MTT assay. The results showed that as compared with control group, the mRNA and protein expression levels of PPARγ and PTEN in high glucose group gradually reduced, the protein expression levels of α-SMA and p-Akt~((Thr308))gradually increased, and the protein expression of E-cadherin gradually reduced(P<0.05). As compared with high glucose group, when increases in Sal B doses, the mRNA and protein expression levels of PPARγ, PTEN in high glucose + different concentrations of Sal B groups gradually increased, the protein expression levels of α-SMA and p-Akt~((Thr308)) gradually reduced, and the protein expression of E-cadherin gradually increased(P<0.05), however, the effect of 1 μmol·L~(-1)concentration of Sal B on the expression of PPARγ mRNA and protein and PTEN mRNA was not significantly different. As compared with high glucose group, the mRNA and protein expression levels of PPARγ mRNA(except 6 h) and protein(except 6 h), PTEN mRNA(except 6 h) and protein(except 6, 12 h) kept increasing, the protein expression levels of α-SMA and p-Akt~((Thr308))(except 6 h) continued to reduce, the protein expression of E-cadherin kept increasing in high glucose+10 μmol·L~(-1) Sal B dynamic observation group(P<0.05). As compared with high glucose group, Sal B and the pioglitazone(PIO) can greatly enhance the expression of PPARγ, PTEN at mRNA and protein levels, enhance the expression of E-cadherin at protein levels, and reduce the expression of α-SMA, p-Akt~((Thr308))protein level(P<0.05), there was no significant difference between the two groups. However, the expression levels of PPARγ and PTEN mRNA and protein, E-cadherin, α-SMA and p-Akt(Thr308) protein in the Sal B+GW9662 control group were not statistically significant compared with the high glucose group. The effect of Sal B was blocked by the PPARγ antagonist GW9662. It can be concluded that Sal B can suppress the NRK52 E cells induced by high-glucose EMT. The mechanism may be related to the activation of PPARγ with Sal B, and the up-regulation of PTEN expression, and thereby inhibiting the fibrosis effect of PI3 K/Akt signaling pathway.
Subject(s)
Animals , Rats , Benzofurans , Epithelial Cells , Epithelial-Mesenchymal Transition , Glucose , Transforming Growth Factor beta1ABSTRACT
The aim of this paper was to investigate whether the mechanism of salvianolic acid B in protecting H9 c2 cardiomyocytes from hypoxia/reoxygenation injury is related to the regulation of mitochondrial autophagy mediated by NIX. H9 c2 cardiomyocytes were cultured in vitro and divided into normal group, model group and salvianolic acid B group(50 μmol·L~(-1)). Hypoxia/reoxygenation injury model was established by hypoxia for 4 h and reoxygenation for 2 h. In normal group, high glucose DMEM medium was used for culture. Those in model group were cultured with DMEM medium without glucose and oxygen, and no drugs for hypoxia and reoxyge-nation. In salvianolic acid B group, salvianolic acid B prepared by glucose-free DMEM medium was added during hypoxia, and the other process was as same as the model group. The cell viability was evaluated by CCK-8 assay. The leakage of lactate dehydrogenase(LDH) was detected by microplate method. The levels of intracellular reactive oxygen species(ROS) and mitochondrial membrane potential(ΔΨm) were measured by chemical fluorescence method. The level of intracellular adenosine triphosphate(ATP) was mea-sured by fluorescein enzyme method. The autophagy related proteins LC3-Ⅰ, LC3-Ⅱ, apoptosis related protein cleaved caspase-3 and mitochondrial autophagy receptor protein NIX were detected by Western blot. As compared with the normal group, the activity of H9 c2 cardiomyocytes and ATP level were decreased(P<0.05); LDH leakage and ROS production were increased(P<0.01); ΔΨm was decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio, cleaved caspase-3 and NIX protein expression levels were increased(all P<0.05) in the model group. As compared with the model group, the activity of cells and ΔΨm were significantly increased(P<0.01); ATP level was increased(P<0.05); LDH leakage and ROS generation were decreased(P<0.01); LC3-Ⅱ/LC3-Ⅰ ratio was decreased(P<0.01); cleaved caspase-3 and NIX expression levels were decreased(P<0.05) in the salvianolic acid B group. The protective effect of salvianolic acid B on hypoxia/reoxygenation injury of H9 c2 cardiomyocytes may be associated with inhibiting mitochondrial auto-phagy. The specific mechanism may be related to inhibiting the activation of mitochondrial autophagy mediated by NIX, increasing ΔΨm, reducing ROS production, reducing the expression of cleaved caspase-3, LC3-Ⅱ, and increasing cell viability.
Subject(s)
Humans , Apoptosis , Autophagy , Benzofurans , Cell Hypoxia , Cell Survival , Hypoxia , Myocytes, CardiacABSTRACT
Objective::To investigate the protective effect of salvianolic acid B on HepaRG hepatocyte injury induced by arsenic trioxide (As2O3 ) and its mechanism. Method::HepaRG cells were incubated with 5μmol·L-1 As2O3 for 24 h to induce hepatocyte injury. The cells were divided into control group, model group, salvianolic acid B 10 μmol·L-1 group, salvianolic acid B 10 μmol·L-1+ As2O3 group, salvianolic acid B 5 μmol·L-1+ As2O3 group, and salvianolic acid B 2.5 μmol·L-1+ As2O3 group. HepaRG cells were preincubated with salvianolic acid B for 2 h and then incubated with As2O3 for 24 h. At the end of the incubation, cell viability was detected by thiazolyl blue tetrazolium bromide assay, apoptosis was observed by Hoechst33342 fluorescence staining, apoptosis rate was detected by annexin V-FITC/propidium iodide double staining flow cytometry, and mitochondrial membrane was observed by JC-1 fluorescence staining. Western blot was used to detect the protective effect of expressions of relevant proteins Bcl-2, Bax, Akt, p-Akt on salvianolic acid B in the liver. Result::As2O3 concentration-dependently reduced the survival rate of HepaRG cells(P<0.01), salvianolic acid B had no effect on normal cell viability for 2 h, pre-incubation with salvianolic acid B(5, 10 μmol·L-1) for 2 h significantly increased the decreased cell survival rate caused by As2O3 (P<0.01). As2O3 significantly increased hepatocytes apoptosis rate(P<0.01), while pre-incubation with salvianolic acid B(10 μmol·L-1) deceased apoptosis rate(P<0.01). Incubation with As2O3 for 24 h caused decrease of mitochondrial membrane potential, pre-incubation with salvianolic acid B maintained mitochondrial membrane potential, indicating that the anti-apoptotic effect of salvianolic acid B were related to the mitochondrial pathway modulation. Western blot analysis showed that salvianolic acid B promoted the ratio of Bcl-2/Bax and promoted p-Akt/Akt compared with As2O3 group(P<0.01). Conclusion::Salvianolic acid B has a protective effect on hepatocyte injury induced by As2O3, and its mechanism is related to maintenance of mitochondrial function and inhibition of hepatocyte apoptosis.
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Objective::To establish the quality control method for multi-index content determination and fingerprint of salvianolic acids. Method::Agilent ZORBAX SB-C18 (4.6 mm×250 mm, 5μm) column was adopted, with 0.1%formic acid-water as mobile phase A and 0.1%formic acid-acetonitrile as mobile phase B for gradient elution (0-30 min, 20%-21.5%B; 30-35 min, 21.5%-25%B; 35-45 min, 25%-40%B; 45-50 min, 40%-95%B). The column temperature was set at 30 ℃, the flow rate was set at 1 mL·min-1, and the detection wavelength was set at 288 nm. Relative correction factors of caffeic acid, salvianolic acid E, rosmarinic acid, lithosperic acid, salvianolic acid B and salvianolic acid Y were determined by the concentration method. The content of each indicator component of the reference extract of salvianolic acid polyphenolic acid was determined and compared with the results of the monomer reference substance by the external standard method. At the same time, the fingerprint method was established. and the similarity evaluation was carried out on 10 batches of extracts. Result::Caffeic acid, salvianolic acid E, rosmarinic acid, lithospermic acid, salvianolic acid B, and salvianolic acid Y had a good linear relationship within the respective detection mass concentration ranges (r>0.999 9). The injection precision RSD was 0.1%-1.2%, the reproducible RSD was 1.2%-1.6%, and the recovery of the six components was 82.03%-98.68%. The stability of each component in the sample solution was good within 36 h. The relative correction factors for each indicator component were determined to be caffeic acid (2.92), salvianolic acid E (1.10), rosmarinic acid (1.61), lithosperic acid (1.07), salvianolic acid B (1.00), salvianolic acid Y (0.83). The effects of different methods, concentrations, instruments, columns, wavelengths were investigated, and the measured relative correction factors were found to be suitable. The results of the calibration factor method and the monomer standard reference substance method were less different. The HPLC fingerprints of the reference extract of salvianolic acids were established, and five common characteristic peaks were determined. The chromatographic peaks were confirmed according to the reference substance. The similarity of the fingerprints of the 10 batches of extracts was higher, and the quality difference was smaller. Conclusion::The multi-index content determination method and the fingerprint method established in this study are simple, rapid, accurate and reproducible, and can be used for quality control of Salviae miltiorrhizae Radix et Rhizoma polyphenolic acid reference extract.
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Objective::To screen out the effective components of Salvia miltiorrhiza by establishing an in vitro model of pulmonary epithelial mesenchymal transformation. Method::Different concentrations of salvianolic acid A (10, 20, 40, 80, 160 μmol·L-1), salvianolic acid B (10, 20, 40, 80, 160 μmol·L-1), tanshinol (10, 20, 40, 80, 160 μmol·L-1), tanshinoneⅡA (10, 20, 40, 80, 160 μmol·L-1) and the blank group were applied to A549 cell, cell proliferation and cytotoxicity assay (MTS) were used to detect the proliferation effect of menthol on A549 cells.After screening the safe concentration of the active ingredients of salvia miltiorrhiza by MTS, cells were divided into blank group, model group, salvianolic acid A group, salvianolic acid B group, tanshinol group and tanshinoneⅡA.Then, the inhibitory effect of the active ingredients of salvia miltiorrhiza on the proliferation of A549 cells induced by TGF-β1 was detected by MTS. Enzyme linked immunosorbent assay (ELISA) method to detect salvia miltiorrhiza effective component of fiber protein(FN), collagen type I (COL-Ⅰ) expression. Based on the above results, the active components of salvia miltiorrhiza, which have best inhibition were screened out, and their effects on the expression of E-calcium-viscosity (E-Cad) protein were detected by Western blot. Result::Compared with blank group, salvianolic acid A 40 μmol·L-1, salvianolic acid B 160 μmol·L-1, tanshinol 160 μmol·L-1 had toxic effects on A549 cells (P<0.05). In the non-toxic concentration range, compared with the model group, salvianolic acid A 10, 20 μmol·L-1, salvianolic acid B 80 μmol·L-1 showed inhibition effect after 24 h culture (P<0.05). After 72 h culture, salvianolic acid A 5, 10, 20 μmol·L-1, salvianolic acid B 40, 80 μmol·L-1inhibition effect was very significant (P<0.01). ELISA results showed that with the blank group, model group cells the expression of FN and COL-Ⅰ increased significantly (P < 0.01). Compare with model group, salvianolic acid A 20 μmol·L-1, salvianolic acid B 80 μmol·L-1 inhibited FN and COL-Ⅰ(P<0.05). Western blot results showed that salicylic acid A and salicylic acid B had protective effects on E-Cad (P<0.01). Conclusion::Salvianolic acid A and salvianolic acid B have inhibitory effects on epithelial mesenchymal transformation by TGF-β1, which may be the main effective components of salvianolic acid in the treatment of pulmonary fibrosis.
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Aim To investigate the effect of salvianolic acid B ( SalB) on non-alcoholic fatty liver disease in high fat-induced ApoE knockout mice by regulating AMPK-induced autophagy. Methods Healthy, 8 weeks old, homologous C57BL/6J mice and ApoE_/" mice were randomly divided into control group, model group and Sal B group, fed with high fat diet, intraperitoneal injection of Sal B ( 15 mg • kg"1 • d "') in group Sal B on week 8, and control group and model group were given an equal dose of normal saline. Pathological changes of livers in each group were assessed by HE staining, oil red 0 staining and Masson staining. Biochemical indexes in serum were analyzed commercially available kits. The levels of inflammation and oxidative stress in liver were detected by immuno-histochemistry. Autophagy and AMPK levels were determined by Western blot immunofluorescence. Results In model group, AST and ALT were significantly elevated in serum, and a large number of fat vacuoles and severe lipid deposits and collagen fibrosis were de-tected in liver. The liver lesions were significantly improved after SalB intervention compared with the ApoE~/_group. The levels of hepatic inflammation and oxidative stress significantly increased in model group but significantly decreased in SalB group. The levels of autophagy and AMPK were significantly, reduced in model group and markedly elevated in SalB group. Conclusions Sal B can improve non-alcoholic fatty liver disease in ApoE_/" mice, and the mechanism may involve mediating AMPK level and enhancing autophagy levels in liver.