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OBJECTIVE To investigate the mechanism of catalpol affecting the differentiation of helper T cell 17 （Th17） by interfering the expressions of pyruvate kinase M2 （PKM2） and lactate dehydrogenase A （LDHA）. METHODS The naive CD4+ T cells were selected from the spleen of C57BL/6 mice， and were differentiated into Th17 cells by adding directional differentiation stimulants for 72 hours. At the same time， the cells were treated with 0 （directed control）， 20， 40 and 80 μg/mL catalpol. The flow cytometry was used to detect the proportion of Th17 cell differentiation in cells； the colorimetric method was adopted to detect the levels of pyruvate and lactate in cell culture supernatant； mRNA expressions of retinoid-related orphan nuclear receptor gamma t （RORγt）， PKM2 and LDHA were detected by qRT-PCR method； Western blot was used to detect the expression levels of PKM2， LDHA， signal transducer and activator of transcription 3 （STAT3）， and phosphorylated STAT3 （p-STAT3） proteins in cells. RESULTS Compared with the directed control group， after 72 hours of treatment with 20， 40， 80 μg/mL catalpol， the differentiation ratio of Th17 cells were decreased by 6.74%， 8.41%， 9.24%， and the levels of pyruvate and lactate in the cell culture supernatant， the mRNA expressions of PKM2， LDHA and RORγt as well as the protein expressions of PKM2 and LDHA and the phosphorylation of STAT3 were significantly reduced （P＜0.05）. CONCLUSIONS Catalpol can reduce the glycolysis level by down-regulating the expressions of PKM2 and LDHA， thereby inhibiting the differentiation of Th17 cells.
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OBJECTIVE To study the improvement effects and its mechanism of catalpol on testicular lesions in KK-Ay spontaneous diabetic mice on the basis of glycolysis process mediated by advanced glycation end products （AGEs） and their receptors （RAGE）. METHODS KK-Ay spontaneous diabetic mice fed with high-fat diet were used as diabetic model， and then randomly divided into model group， catalpol group （100 mg/kg）， aminoguanidine group （AGEs inhibitor， 100 mg/kg） and FPS- ZM1 group （RAGE inhibitor， 1 mg/kg）， and C57BL/6J mice fed in the same period were set as normal group， with 6 mice in each group. The catalpol group and aminoguanidine group mice were given relevant medicine intragastrically， normal group and model group mice were given constant volume of normal saline intragastrically， and FPS-ZM1 group mice were given relevant medicine 1 mL/g intraperitoneally， for consecutive 8 weeks. After the last administration， the body mass， fasting blood glucose， 24-hour food intake， water consumption， urine volume， testicular organ coefficient， and sperm motility of the mice were measured； pathological morphology and ultrastructural structure of testicular tissue were observed； the levels of reduced glutathione （GSH）， superoxide dismutase （SOD）， lactate dehydrogenase （LDH） and sugar metabolites in testicular tissue of mice were detected； pathway enrichment analysis was performed； the level of AGEs in serum and testicular tissue， protein expressions of RAGE， B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）， and mRNA expressions of key rate-limiting enzymes [hexokinase （HK）， phosphofructose kinase （PFK）， pyruvate kinase （PK）， LDH] in testicular tissue were alldetected. RESULT S Catalpol could significantly improve the general symptoms， testicular organ coefficients and motility ofsperm in KK-Ay spontaneous diabetic mice （P＜0.05 or P＜0.01）. The morphology and ultrastructure of spermatogenic cells in each layer of the seminiferous tubules were all improved. The levels of GSH， SOD and LDH in testicular tissue，the levels of the metabolic product glucose fructose-1，6-diphosphate， 3-phosphate glycerate， 3-phosphate glyceraldehyde， lactic acid and pyruvate， the expressions of HK， PFK， PK and LDH mRNA were all significantly increased（P＜0.05 or P＜0.01）； the levels of AGEs in serum and testicular tissue， the expression of RAGE protein and the ratio of Bax to Bcl-2 in testicular tissue were significantly decreased（P＜0.05 or P＜0.01）. Aminoguanidine and FPS-ZM1 could significantly improve the levels of most of above indicators in mice（P＜0.05 or P＜0.01）. CONCLUSIONS Catalpol shows significant improvement effects on testicular lesions of KK-Ay spontaneous diabetic mice， and its mechanism of action was associated with upregulation of AGEs/RAGE signaling pathway- mediated glycolysis.
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Objective:To investigate effect and underlying lipid-lowering mechanisms of catalpol in non-alcoholic fatty liver disease(NAFLD).Methods:In vivo model of NAFLD was established with high-fat diet-fed ICR mice for 8 weeks. Low(50 mg/kg), medium(150 mg/kg), and high(300 mg/kg) doses of catalpol were administered, and the body weight, liver weight, hepatic index, and biochemical parameters of the mice were analyzed. Free fatty acid-induced LO2 in human hepatocytes to establish NAFLD cell model. Quantitative realtime PCR reaction to detect fatty acid synthesis-related gene levels. Western blotting assay was adopted to analyze proteins in the endoplasmic reticulum stress(ERS)-mediated protein kinase RNA-like endoplasmic reticulum kinase(PERK)-eukaryotic translation initiation factor 2α(eIF2α) signaling pathway. Results:Compared with model mice, body weight [(39.43±1.84)g, (34.01±1.83)g, (32.28±1.11)g vs(42.17±1.37)g, all P<0.001], liver weight [(1.03±0.06)g, (0.79±0.05)g, (0.64±0.04)g vs(1.30±0.13)g, P<0.01 or P<0.001], and liver index [(2.60±0.09)%, (2.32±0.09)%, (1.99±0.11)% vs(3.07±0.30)%, P<0.05 or P<0.001] were reduced in low, medium, and high doses of catapol model. Medium and high doses of catalpol diminished total cholesterol, triglyceride, low density lipoprotein-cholesterol, aspartate aminotransferase, and alanine aminotransferase( P<0.01 or P<0.001), increased high density lipoprotein-cholesterol( P<0.01 or P<0.001). In the cell model, elevated levels of both fatty acid synthesis genes and PERK-eIF2α pathway proteins were attenuated by catalase, and this attenuation was reversed by signaling pathway agonists. Conclusion:The Chinese herb catalpol may play a role in improving NALFD by regulating the ERS-mediated PERK-eIF2α signaling pathway.
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Objective:To establish high performance liquid chromatography （HPLC） fingerprint and multi-component determination for the substance benchmark of Yiweitang， and to evaluate its quality in combination with chemical pattern recognition method. Method:Fifteen batches of substance benchmark of Yiweitang were prepared， the "Chinese medicine chromatographic fingerprint similarity evaluation system" （2012 edition） was used to calculate similarity. Cluster analysis， principal component analysis and orthogonal partial least squares-discriminant analysis were employed to handle the common peaks for evaluating the quality difference among 15 batches of the substance benchmark. The contents of catalpol， verbascoside and methylophiopogonanone A were determined with mobile phase system of acetonitrile-phosphoric acid solution at detection wavelengths of 210 nm and 334 nm. Result:There were 22 common peaks in HPLC fingerprint of the substance benchmark， among them， peaks 1， 9， 12， 14-17， 19 and 20 belonged to Rehmanniae Radix， peaks 3， 4， 6， 7 and 21 belonged to Glehniae Radix， peaks 5 and 22 belonged to Ophiopogonis Radix， peaks 2 and 18 belonged to Polygonati Odorati Rhiaoma， peak 8 was the common peak of Ophiopogonis Radix and Rehmanniae Radix， peak 10 was shared by Ophiopogonis Radix， Polygonati Odorati Rhiaoma<italic> </italic>and<italic> </italic>Rehmanniae Radix， peak 11 was the common peak of these four herbs， and peak 13 was shared by Polygonati Odorati Rhiaoma and Rehmanniae Radix. The similarities between HPLC fingerprints of 15 batches of the substance benchmark and the control fingerprint were all >0.90， the samples could be divided into four categories by three chemical pattern recognition methods. Quantitative analysis showed that the contents of catalpol， verbascoside and methylophiopogonanone A among 15 batches of samples ranged from 0.37% to 1.14%， 0.002% to 0.054% and 0.016% to 0.079%， respectively. Conclusion:The established fingerprint and determination for the substance benchmark of Yiweitang have good separation and high accuracy， which reflect the overall chemical composition characteristics of Yiweitang， and can provide experimental basis for the further development and quality control of the compound preparations of this famous classical formula.
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This study was designed to explore the protective effect and underlying mechanism of catalpol on hepatocyte apoptosis in nonalcoholic fatty liver disease (NAFLD). High fat diet (HFD) was used to establish NAFLD model in the in vivo experiment, and the procedures of the experiments and animal care protocol were approved by the Animal Care and Use Committee of Jianghan University. Human liver cancer cell line HepG2 was treated with palmitate (PA) to establish a lipid toxicity model in the in vitro experiments. The results showed that catalpol significantly decreased the contents of serum total glyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate transaminase (AST) in HFD-fed mice. Results of TUNEL staining and flow cytometry analyses revealed that catalpol significantly inhibited hepatocytes apoptosis in HFD-fed mice and PA-treated HepG2 cells. Moreover, catalpol treatment significantly reduced the endoplasmic reticulum stress-related protein expression levels of binding immunoglobulin protein (BiP), phosphorylated PKR-like endoplasmic reticulum kinase (p-PERK), inositol-requiring kinase 1α (IRE1α), and transcriptional factor activating transcription factor 6 (ATF6), and apoptosis-related protein expression levels of C/EBP homology protein (CHOP), phosphorylated c-Jun N-terminal kinase (p-JNK), and cleaved cysteinyl aspartate specific proteinases (caspases)-12, -9, and -3 in HFD-fed mice and PA-treated HepG2 cells. Furthermore, endoplasmic reticulum stress agonist tunicamycin (TM) significantly reversed the inhibitory effect of catalpol on protein expression levels of BiP, p-PERK, IRE1α, and ATF6, subsequently the inhibitory effect of catalpol on expression levels of CHOP, p-JNK, Bcl-2, Bax, and cleaved caspases (-12, -9, and -3) was also attenuated in PA-treated HepG2 cells. Taken together, these findings demonstrated that catalpol could inhibit hepatocytes apoptosis and had a significant protective effect on liver injury, and its mechanism might be related to the relief of endoplasmic reticulum stress.
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BACKGROUND: Inflammatory transmitters secreted from the synovium may be one of the important factors inducing the onset of knee osteoarthritis, and further exacerbate knee osteoarthritis. OBJECTIVE: To observe the effect of different concentrations of catalpol from the root of Rehmannia glutinosa on the expression of interleukin-1β, galectin 3 and S100A12 in the synovium of the knee joint in rats with early knee osteoarthritis, and to explore the mechanism of catalpol from the root of Rehmannia glutinosa in the treatment of early knee osteoarthritis. METHODS: The 14 of 48 adult male Wistar rats were randomly selected as a normal control group, and the remaining 34 rats were injected with 4% papain and 0.03 mol/L cysteine solution (0.2 mL) into the right knee joint cavity at 1, 4, 7 days after the initialization of the experiment to duplicate the model of early knee osteoarthritis. At 10 days after the initialization of the experiment, 4 rats in each group were randomly selected for histological observation using hematoxylin-eosin staining and validation of the model using Mankin and OARSI grading evaluation. After the model was successfully verified, the remaining 30 rats in the model group were randomly divided into model control group, low-dose group and high-dose group. At 2 days after successful modeling, each treatment group was infused with corresponding drugs at a dose of 0.2 mL/kg, and normal control group and model control group were infused with normal saline, once a day for 28 consecutive days. The synovium of the right knee joint was then taken from rats in each group, and the expression of interleukin-1β, galectin 3 and S100A12 in the rat synovium was detected by ELISA. RESULTS AND CONCLUSION: Mankin score and OARSI grading were significantly higher in the model control group than the normal control group (P < 0.05). The levels of S100A12, interleukin-1β and galectin 3 in the synovium were significantly higher in the model control group than the normal control group (P < 0.01). Compared with the model control group, the levels of S100A12, interleukin-1β and galectin 3 in the synovium was decreased somewhat in the low-dose group (P < 0.05), but significantly decreased in the high-dose group (P < 0.01). Therefore, catalpol from the root of Rehmannia glutinosa can delay the progression of knee osteoarthritis by reducing the level of inflammatory factors in the synovium.
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BACKGROUND: Studies have shown that abnormal osteoblast metabolism may cause abnormal subchondral bone mineralization in knee osteoarthritis. OBJECTIVE: To verify the effects of catalpol from the root of Rehmannia glutinosa on osteoblast proliferation and drug toxicity, and the anti-inflammatory protective effect on inflammatory osteoblasts induced by lipopolysaccharide. METHODS: (1) Osteoblasts from newborn Sprague-Dawley rats were primarily extracted, cultured, and passaged. The activity and proliferation of osteoblasts were determined by observing the morphology of osteoblasts alkaline phosphatase staining and staining of mineralized nodules. (2) Cell counting kit-8 (CCK-8) method was used to observe the effect of different concentrations of catalpol from the root of Rehmannia glutinosa on osteoblast activity. (3) Different concentrations of lipopolysaccharide (0, 10, 20, 40, 80, 160 mg/L) were used to induce inflammatory osteoblasts in fetal rats. CCK-8 method was used to verify the best concentration. (4) The experiment was divided into three groups: blank group, model group, low-, medium- and high-concentration catalpol groups. Inflammatory osteoblasts were induced by lipopolysaccharide at 80 mg/L, and then treated for 8 hours. CCK-8 method was used to observe the effect of different concentrations of catalpol on inflammatory osteoblasts. The study protocol was approved by the Medical Ethics Committee of Liuhe District People’s Hospital in Nanjing. RESULTS AND CONCLUSION: When osteoblasts were cultured to the fourth generation, followed by alkaline phosphatase staining, black grey granules were found in the cytoplasm of the positive cells, and the morphology of the cells was irregular. When the concentration of catalpol was lower than 1 mg/L, it had no significant effect on osteoblasts; when it was higher than 10 mg/L, it had a significant effect on the proliferation of osteoblasts but no drug toxicity (P < 0.05). When the concentration of lipopolysaccharide was higher than 80 mg/L, there was a significant trend of inflammatory damage to osteoblasts (P < 0.01). Therefore, 80 mg/L was selected as the best injury concentration in this experiment. When the concentration of catalpol from the root of Rehmannia glutinosa was lower than 1 mg/L, it had no significant protective effect on the inflammatory response of osteoblasts (P < 0.05); when it was higher than 10 mg/L, it had significant protective effect on the inflammatory response of osteoblasts (P < 0.05). Therefore, when the concentration of catalpol from the root of Rehmannia glutinosa reaches a certain level, it has no toxic effect on osteoblasts, promotes proliferation, and has anti-inflammatory protective effect on inflammatory osteoblasts induced by lipopolysaccharide.
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Osteoporosis fracture with high disability and mortality is a difficult problem that seriously affects the life quality of individuals. At present, there is still a lack of anti-osteoporosis drugs with clear target and significant efficacy in the clinical practice. Rehmanniae Radix and its prescriptions have significant clinical effects. In this regard, more and more studies have reported the effects and mechanisms of Rehmanniae Radix and its active components, and the certain research outputs have been achieved. In this article, the PubMed, Web of science, China National Knowledge Infrastructure, and Wanfang database were searched to collect and organize the latest research progress of Rehmanniae Radix treatment of osteoporosis in the recent 10 years. We summarized the research dynamics as well as the function indexes and mechanisms of the raw and processed Rehmanniae Radix, active ingredients such as catalpol, aucubin, acteoside and Rehmanniae Radix polysaccharide, and their formulating prescriptions, and then excavated the potential active ingredients, targets and signaling pathways, including the effect on bone marrow mesenchymal stem cells, promoting the osteoblast proliferation and promoting osteogenesis differentiation(increasing alkaline phosphatase, typeⅠ collagen, osteoprotegerin, and osteocalcin and promoting calcium deposits), increasing the bone density, inhibiting the osteoclast quantity and differentiation, promoting the osteoclast apoptosis, and reducing tartrate resistant acid phosphatase and bone resorption pit area to provide the reference and develop new ideas for developing Rehmanniae Radix prescriptions for treatment of osteoporosis and exploring its mechanism.
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Humans , China , Drugs, Chinese Herbal , Osteogenesis , Osteoporosis , RehmanniaABSTRACT
Aim To investigate the effect of catalpol on the stimulation of macrophage-mediated mouse mesang-ial cells ( MMCs) injury by advanced glycation end products (AGEs). Methods MMCs and RAW264.7 macrophages were co-cultured in vitro and divided into control group, model group,catalpol group (0. 1, 1.0, 10. 0 jjimol • L"1 ) , and aminoguanidine group ( 10. 0 fimol • L"1) , which was setas the positive control. After drug administration for 1 h, RAW264. 7 was stimulated by AGEs (100 mg • L_l) for 24 h. MTT assay was employed to detect the proliferation rate of MMCs . Immunofluorescence was used to investigate the expres-sion levels of COL-IV in MMCs. Western blot was applied to assess the expression of FN,COL-IV and TGF-P in MMCs. ELISA was utilized to determine the levels of IL-6, IL-12 and TNF-ot in supernatant liquid of RAW264. 7 macrophages. Results Catalpol could inhibit RAW264.7 macrophage-mediated MMC-induced proliferation stimulated by AGEs ( P < 0. 05, P < 0.01), down-regulate the levels of FN, COL-IV and TGF-0 proteins in MMCs (P < 0. 05, P < 0. 01) , and decrease IL-6, IL-12 and TNF-a level (P < 0. 05, P < 0. 01). Conclusions Catalpol has apparent protective effect on AGEs-stimulated macrophage-mediated me-sangial cell injury. It down-regulates FN, COL-IV and TGF-p protein expression in MMCs, down-regulates inflammatory factor levels and reduces inflammation, thereby alleviating diabetic kidney damage.
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Catalpol is an iridoid glycoside extracted from the root of Rehmannia glutinosa. It has been reported to have antioxidant stress effects. Adenosine 5' monophosphate-activated protein kinase( AMPK) plays an important role in inhibiting oxidative stress. This study was designed to investigate the protective effects of catalpol on TNF-α-exposed human aorta epithelial cells( HAECs) via inhibit oxidative stress,and the relationship between catalpol and AMPK was detected by RNA interference technique. Levels of superoxide dismutase( SOD),malonaldehyde( MDA),glutathione( GSH) and lactate dehydrogenase( LDH) were measured with a colorimetric assay kit. The level of ROS was measured with FACS calibur. Western blot was employed to detect the protein expression of AMPK,phosphorylated-AMPK and NOX4. Finally,RNA interference technique was used to investigate the role of AMPK in catalpol-induced protective effects. TNF-α treatment decreased the expression of phosphorylated-AMPK protein level,however,catalpol could reverse the decreased phosphorylated-AMPK level. Catalpol could inhibit NOX4 protein expression and decrease ROS overproduction. After using AMPK siRNA that effects of catalpol on ROS overproduction and NOX4 protein expression inhibition were attenuated. The above results suggest that catalpol inhibits oxidative stress in TNF-α-exposed HAECs by activating AMPK.
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Humans , Iridoid Glucosides , Pharmacology , Iridoids , Oxidative Stress , Reactive Oxygen Species , Tumor Necrosis Factor-alphaABSTRACT
Objective: Rehmanniae Radix has been traditionally used to treat diabetes. Catalpol (CAT) and stachyose (STA) are two of the main bioactive compounds in Rehmannia Radix and found to have similar therapeutic effects on diabetes and its complications. In this paper, we aimed to investigate whether there were synergistic therapeutic effects of CAT and STA on diabetes. Methods: Streptozotocin (STZ) with the feeding of high-sugar-high-fat diet (HFD) was applied to induce diabetic C57BL/6 mice. STZ-HFD induced diabetic mice were then divided into model and six medical-treated groups: metformin (MET), STA, CAT, and three combinations of CAT:STA (1:1, 1:2, 2:1). Blood, liver, and kidney samples were isolated after six-week oral administration for biochemical assays of serum lipids, the indicators of kidney and liver functions and HE staining for liver tissues. Results: It turned out that CAT, STA and their three combinations (1:1, 1:2, 2:1) could effectively control body weight, blood glucose, kidney weight and liver weight index, and well regulate levels of TC, HDL-c, TG, ALT, and TBA. In addition, CAT and its combination with STA at the ratio of 2:1 could significantly improve albumin content, compared to that in model group. STA and CAT and their combinations showed the improvements on kidney function in terms of urinary creatinine (Ucr). However, there were no such consistent observations on serum creatinine (Scr) and creatinine clearance rate (Ccr). The combination of CAT and STA at the ratio of 1:1 exhibited the better adjusting effects on kidney weight and liver weight indexes and the levels of ALT, Ucr, Scr, and Ccr. Our results demonstrated that the combinations of CAT and STA especially 1:1 showed similar or better improvements on diabetes-associated complications, compared to the sole CAT or STA treatment. Conclusion: Thus, we concluded that there were synergistic therapeutic effects between CAT and STA on STZ/HFD-induced type 2 diabetes. This project provided insights and technical supports for the innovation of discovering bioactive constituents in Rehmannia Radix and studying its integrative mechanism in curing diabetes.
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Iridoid glycosides are widely distributed in Chinese materia medica (CMM) with various biological activities such as anti-inflammation. They are also used as quality control constituents in some Chinese medicines. Iridoid glycosides are usually divided into nine-carbon skeleton iridoid glycoside type, ten-carbon skeleton iridoid glycoside type, and secoiridoid glycoside type. In this paper, 15 representative iridoid glycosides from three types which are received extensive attention (including geniposide, catalpol, gentiopicroside, etc) have been selected. Their anti-inflammatory effects and possible related mechanisms are summarized to find out the acting feature of different types. Through comparing the structures and function characteristics, it was concluded that the anti-inflammatory effects of iridoid glycosides were mostly related to NF-κB pathway and MAPK pathway. They have obvious inhibitory effects on TNF-α, IL-6, and IL-1β inflammatory factors, some of which could play a role by reducing the expression of iNOS and COX-2 in NLRP3, Nrf2/HO-1, PI3K, and other pathways. From the structure-activity relationship, the double bond on the cyclopentane, the C-11 substituent and the bond formation after ring opening in iridoid glycosides all have important effects on its anti-inflammatory activities.
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Objective: To prepare substance benchmarks of Baihe Dihuang Decoction (BDD), and evaluate the scientificity and rationality of preparation process by analyzing the process quality. Methods: Fifteen batches of substance benchmarks were prepared according to the ancient method, the content of catalpol and acteoside in the preparation process was determined, and the transfer rate and extractum rate were calculated. Fingerprints of 15 batches of decoction pieces, decoction, concentrate and substance benchmarks were detected by HPLC, and the common peaks of fingerprints were attributed and identified; In addition, the similarity of fingerprints were evaluated. Results: In 15 batches of substance benchmarks, the transfer rates of catalpol and acteoside were 81.40%—92.88% and 28.90%—41.41% respectively, the extractum rate was 36.06%—41.71%, and without discrete data. During the process of decoction, concentration and freeze-drying, the transfer rates of effective components were stable. In the fingerprints of substance benchmarks, 16 common peaks were determined, of which six peaks belong to Lilii Bulbus, nine peaks belong to Rehmanniae Radix. Three peaks were identified. The similarity of fingerprints of decoction pieces, decoction, concentrate, and substance benchmarks were all over 0.9. The similarity of reference fingerprints of decoction, concentrate, and substance benchmarks were over 0.99. Conclusion: The fingerprint method is reasonable and feasible, which can be used for simultaneously determining the fingerprint of decoction pieces, intermediates and substance benchmarks. The preparation process is scientific and reasonable, and will not changes substance basis significantly. The paper establishes a foundation for the development of preparation of BDD, and provides a new idea for the evaluation of process and quality of the substance benchmarks of classical famous prescriptions.
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Objective: The fingerprint of Rehmannia glutinosa was established by HPLC to provide scientific basis for improving the quality of R. glutinosa. Methods: The characteristic silicagel C18 column-Atlantis T3 was used, and the mobile phase was 0.1% phosphate water and acetonitrile by gradient elution. Absorption wavelength was 203 nm, and flow rate was 1.0 mL/mL with column temperature of 35 ℃. Setting catalpol as the control peak, 20 batches of R. glutinosa fingerprint was established. Fingerprint similarity evaluation software was used for data evaluation and determinating the catalpol content of 20 batches of R. glutinosa. Results: The standard contrast chromatographic fingerprint similarity of 20 batches R. glutinosa reached above 0.917. The 20 batches catalpol content was above 0.2%. Conclusion: The established fingerprint chromatography was proved that it had good precision, stability, and repeatability. The catalpol content determination met requirement which can avoid interference of saccharide compared with China Pharmacopoeia method and provide scientific reference for improving the quality of R. glutinosa.
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Catalpol is the main active ingredient of an extract from Radix rehmanniae, which in a previous study showed a protective effect against various types of tissue injury. However, a protective effect of catalpol on uterine inflammation has not been reported. In this study, to investigate the protective mechanism of catalpol on lipopolysaccharide (LPS)-induced bovine endometrial epithelial cells (bEECs) and mouse endometritis, in vitro and in vivo inflammation models were established. The Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway and its downstream inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), western blot (WB), and immunofluorescence techniques. The results from ELISA and qRT-PCR showed that catalpol dose-dependently reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6, and chemokines such as C-X-C motif chemokine ligand 8 (CXCL8) and CXCL5, both in bEECs and in uterine tissue. From the experimental results of WB, qRT-PCR, and immunofluorescence, the expression of TLR4 and the phosphorylation of NF-κB p65 were markedly inhibited by catalpol compared with the LPS group. The inflammatory damage to the mouse uterus caused by LPS was greatly reduced and was accompanied by a decline in myeloperoxidase (MPO) activity. The results of this study suggest that catalpol can exert an anti-inflammatory impact on LPS-induced bEECs and mouse endometritis by inhibiting inflammation and activation of the TLR4/NF-κB signaling pathway.
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OBJECTIVE： To determine and compare the contents of catalpol and aucubin in different parts （root， stem， leaf and flower） of wild Centranthera grandiflora， and to provide reference for the selection of medicinal parts and source development. METHODS： HPLC method was used to determine the contents of catalpol and aucubin in root， stem， leaf and flower of wild C. grandiflora， and the contents of different parts were analyzed comparatively. The determination of catalpol was performed on Agilent TC-C18 column with mobile phase consisted of methanol-0.1％ phosphoric acid （1 ∶ 99， V/V） at the flow rate of 1 mL/min； the detection wavelength was set at 210 nm， and sample size was 20 μL. The column temperature was 35 ℃； the determination of aucubin was performed on SPHERI-5RP-C18 column with mobile phase consisted of acetonitrile-water （3 ∶ 97， V/V） at the flow rate of 1 mL/min； the detection wavelength was set at 205 nm， and sample size was 20 μL； the column temperature was 25 ℃. RESULTS： The linear range of catalpol and aucubin were 0.061 5-3.321 and 0.000 36-0.216 mg/mL （all r＝0.999 9）. The limits of detection were 0.016 and 0.007 μg/mL. The limits of quantitation were 0.052 and 0.023 μg/mL. RSDs of precision， stability （24 h） and reproducibility tests were all lower than 2.00％ （n＝6）. The average recoveries were 99.34％ and 99.61％， and RSDs were 1.06％ and 1.12％， respectively （n＝6）. The average content of catalpol in root， stem， leaf and flower wild C. grandiflora were 1.609， 3.030， 11.095 and 1.921 mg/g， respectively. The contents of aucubin in different parts were 0.441， 0.020， 0.005 and 0.006 mg/g，respectively. CONCLUSIONS：The established HPLC method meets the requirements of quantitative analysis. Catalpol is mainly distributed in the leaves of wild C. grandiflora， and aucubin is mainly distributed in the roots of wild C. grandiflora. The experimental conclusion provides a reference for the reasonable selection of different medicinal parts as raw materials to develop medicine with different efficacy.
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Aim: To investigate the effect of catalpol-mediated autophagy on oxidative damage of cardiomyocytes induced by glucose deprivation based on estrogen receptor(ER) and the related mechanism. Methods: The cardiomyocytes (H9c2) injury model in rat was induced by glucose deprivation for 6 h. The protective effect of catalpol on H9c2 injury and its mechanism were observed. The cells were divided into five groups: control group, model group, catalpol group (0. 28, 2. 8, 28 μmol · L-1). The effects of catalpol on reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) in cells were detected, and the effect of catalpol on autophagy was assessed by electron microscopy. ER blockers was used to detect whether the protective effect of catalpol on oxidative damage was related to ER. Lentivirus transfection was used to lower the expression of ER alpha, and the oxidative damage and autophagy of H9c2 treated with catapol or not were observed. Results: Compared with control group, the levels of ROS and MDA increased and SOD decreased in model group, and there was no significant difference in the expression of autophagy related proteins. Compared with model group, catalpol could reduce oxidative damage and increase autophagy level. After transfection of ER alpha with lentivirus, catalpol inhibited the level of oxidative damage and promoted the effect of autophagy compared with the empty virus catalpol group. Conclusions: Catalpol can activate autophagy by up-regulating the expression of ER alpha and inhibiting the oxidative damage of cardiomyocytes.
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Objective To study the protective effects ofcatalpol on cardiomyocytes in type 2 diabetic rats.Methods Based on the method of random number table,8-wee k-old SD rats were randomly divided into normal group (10 rats) and experimental group (90 rats).The rats in the experimental group that were fed with high-fat and high-sugar diet for 8 weeks were injected intraperitoneally with STZ (15 mg/kg),with fasting blood glucose≥16.7mmol/L as a model for type 2 diabetes.Then based on fasting blood glucose and body weight,the rats meeting the criteria of type 2 diabete mellitus were randomy divided into five group,namely,the model group,the control group,the low or medium and high doses of the catalpol group,and there were 12 rats in each group.The normal group and the model group were filled with distilled water (5 ml/kg·d,the control group were treated with metformin (90 mg/kg·d),and Low,medium and high dose group were intragastric administration of Catalpol(2.5,5,10 mg/kg·d).Then,the level of glucose,myocardial oxidative stress related protein,cardiac strcture and function change were measured after 12 weeks of intervention in rats.Results Compared with the model group,the level of fasting blood glucose in catalpol low,medium and high dose groups was significantly lower (P＜0.05).The levels of SOD (197.43 ± 8.85 U/ml,186.54 ± 5.89 U/ml,175.62 ± 7.67 U/ml vs.157.75 ±11.29 U/ml) in myocardial tissue of those groups significantly increased (P＜0.05),the levels of MDA (7.26 ±0.72 nmol/mg,8.58 ± 0.93 nmol/mg,10.62 ± 0.59 nmol/mg vs.14.80 ± 0.71 nmol/mg) in myocardial tissue of those groups significantly decreased (P＜0.05),the E/A value (1.25 ± 0.18,1.09 ± 0.14,0.97 ± 0.11 vs.0.51 ±0.11) in those groups significantly increased (P＜0.05),the E/E'values (12.33 ± 0.73,13.26 ± 1.07,14.73 ± 1.23 vs.20.54 ± 1.64) and the IVRT (21.90 ± 2.60 ms,22.05 ± 2.84 ms,24.42 ± 2.22 ms vs.27.40 ± 2.81 ms) in those groups significantly decreased (P＜0.05).Conclusions The catalpol can reduce oxidative stress,improve cardiac function and protect cardiomyocytes.
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To investigate the effect of catalpol on high-fat diet(HFD)-induced nonalcoholic fatty liver disease(NAFLD)and its underlying molecular mechanisms. Sixty C57BL/6J male mice were randomly divided into six groups:control group;HFD group;HFD+catalpol(100 mg/kg)group;HFD+catalpol(200 mg/kg)group;HFD+catalpol(400 mg/kg)group;and HFD+atorvastatin calcium(ATC)(30 mg/kg)group.The control group was fed a normal diet containing 4.4 kJ/g fat,whereas the other five groups were fed a high-fat diet containing 19.8 kJ/g fat.Mice in the catalpol or ATC treatment groups were administered by gavage for different doses of catalpol or ATC,whereas other mice were treated with saline.Body weight was measured once a week.Experiments were terminated after 18 weeks,and blood and liver samples were collected after an overnight fast(12 hours)for analysis.The body weight and liver weight were measured and the levels of serum total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),and aspartate transaminase(AST)as well as inflammatory factors tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,and IL-6 were determined by commercially available kits.Liver sections were stained with Oil Red O and HE to investigate the lipid accumulation and histopathological changes.The protein expressions of nuclear factor kappa-B(NF-κB)p65,inhibitor of nuclear factor kappa-B α(IκBα),B-cell lymphoma-2(Bcl-2),Bcl-2 associated x protein(Bax),and Caspase-3 were determined by Western blot. Compared to the model group,the body weight gains(all =0.001),liver index(=0.008,=0.001,=0.001),ALT(=0.004,=0.001,=0.001),and AST(=0.008,=0.001,=0.001)were significantly decreased in catalpol treatment groups,and the serum levels of TC(=0.005,=0.001),TG (all =0.001),and LDL-C(all =0.001)were also significantly decreased in middle and high dose groups,and the serum level of HDL-C was significantly increased in high group(=0.009).Moreover,compared to the model group,the degree of liver injury and lipid accumulation were obviously decreased in the catalpol treatment groups according to the pathology.Similarly,the release of inflammatory factors was significantly inhibited by the treatment with catalpol.The results of Western blot showed that the protein levels of NF-κB p65(=0.014,=0.001,=0.001)and Caspase-3(all =0.001)in the livers of HFD-fed mice were significantly reduced by catalpol treatment.In addition,the protein level of IκBα(=0.028,=0.001,=0.001)and the ratio of Bcl-2/Bax in high dose group(=0.003)was increased by treatment with catalpol. Catalpol can effectively improve the body weight gains,liver index,dyslipidemia,and lipid accumulation in HFD-fed mice and inhibit the release of inflammatory factors and hepatocyte apoptosis,thereby preventing the development of NAFLD induced by HFD.
Subject(s)
Animals , Male , Mice , Diet, High-Fat , Iridoid Glucosides , Mice, Inbred C57BL , Non-alcoholic Fatty Liver DiseaseABSTRACT
This paper aimed to observe the protective effect of catalpol on the high glucose induced destruction of tight junctions of rat primary brain microvascular endothelial cells (BMECs). Catalpol co-administrated with high glucose increased BMECs survival, decreased its ET-1 secretion, and improved transmembrane electrical resistance in a time-dependent manner. Furthermore, transmission electron microscopy was used to observe catalpol's protective effect on tight junction. Fluorescence staining displayed that catalpol reversed the rearrangement of the cytoskeleton protein F-actin and up-regulated the tight junction proteins claudin-5 and ZO-1, which were further demonstrated by the mRNA expression levels of claudin-5, occludin, ZO-1, ZO-2, ZO-3, -actintin, vinculin and cateinins. This study indicated that catalpol reverses the disaggregation of cytoskeleton actin in BMECs and up-regulates the expression of tight junction proteins, such as claudin-5, occludin, and ZO-1, and finally alleviates the increase in high glucose-induced BMECs injury.