Catalpol promotes articular cartilage repair by enhancing the recruitment of endogenous mesenchymal stem cells.

Articular cartilage defect is challenged by insufficient regenerative ability of cartilage. Catalpol (CA), the primary active component of Rehmanniae Radix, could exert protective effects against various diseases. However, the impact of CA on the treatment of articular cartilage injuries is still unclear. In this study, full-thickness articular cartilage defect was induced in a mouse model via surgery. The animals were intraperitoneally injected with CA for 4 or 8 weeks. According to the results of macroscopic observation, micro-computed tomography CT (µCT), histological and immunohistochemistry staining, CA treatment could promote mouse cartilage repair, resulting in cartilage regeneration, bone structure improvement and matrix anabolism. Specifically, an increase in the expression of CD90, the marker of mesenchymal stem cells (MSCs), in the cartilage was observed. In addition, we evaluated the migratory and chondrogenic effects of CA on MSCs. Different concentration of CA was added to C3H10 T1/2 cells. The results showed that CA enhanced cell migration and chondrogenesis without affecting proliferation. Collectively, our findings indicate that CA may be effective for the treatment of cartilage defects via stimulation of endogenous MSCs.

A study on the mechanism of Beclin-1 m6A modification mediated by catalpol in protection against neuronal injury and autophagy following cerebral ischemia.

OBJECTIVE: Catalpol (CAT) has various pharmacological activities and plays a protective role in cerebral ischemia. It has been reported that CAT played a protective role in cerebral ischemia by upregulaing NRF1 expression. Bioinformatics analysis reveals that NRF1 can be used as a transcription factor to bind to the histone acetyltransferase KAT2A. However, the role of KAT2A in cerebral ischemia remains to be studied. Therefore, we aimed to investigate the role of CAT in cerebral ischemia and its related mechanism. METHODS: In vitro, a cell model of oxygen and glucose deprivation/reperfusion (OGD/R) was constructed, followed by evaluation of neuronal injury and the expression of METTL3, Beclin-1, NRF1, and KAT2A. In vivo, a MCAO rat model was prepared by means of focal cerebral ischemia, followed by assessment of neurological deficit and brain injury in MCAO rats. Neuronal autophagy was evaluated by observation of autophagosomes in neurons or brain tissues by TEM and detection of the expression of LC3 and p62. RESULTS: In vivo, CAT reduced the neurological function deficit and infarct volume, inhibited neuronal apoptosis in the cerebral cortex, and significantly improved neuronal injury and excessive autophagy in MCAO rats. In vitro, CAT restored OGD/R-inhibited cell viability, inhibited cell apoptosis, LDH release, and neuronal autophagy. Mechanistically, CAT upregulated NRF1, NRF1 activated METTL3 via KAT2A transcription, and METTL3 inhibited Beclin-1 via m6A modification. CONCLUSION: CAT activated the NRF1/KAT2A/METTL3 axis and downregulated Beclin-1 expression, thus relieving neuronal injury and excessive autophagy after cerebral ischemia.

Exploration of the Dynamic Variations of the Characteristic Constituents and the Degradation Products of Catalpol during the Process of Radix Rehmanniae.

Radix Rehmanniae (RR), a famous traditional Chinese medicine (TCM) widely employed in nourishing Yin and invigorating the kidney, has three common processing forms in clinical practice, including fresh Radix Rehmanniae (FRR), raw Radix Rehmanniae (RRR), and processed Radix Rehmanniae (PRR). However, until now, there has been less exploration of the dynamic variations in the characteristic constituents and degradation products of catalpol as a representative iridoid glycoside with the highest content in RR during the process from FRR to PRR. In this study, an ultra-performance liquid chromatography coupled with photodiode array detector (UPLC-PDA) method was successfully established for the simultaneous determination of ten characteristic components to explore their dynamic variations in different processed products of RR. Among them, iridoid glycosides, especially catalpol, exhibited a sharp decrease from RRR to PRR. Then, three degradation products of catalpol were detected under simulated processing conditions (100 °C, pH 4.8 acetate buffer solution), which were isolated and identified as jiofuraldehyde, cataldehyde, and norviburtinal, respectively. Cataldehyde was first reported as a new compound. Moreover, the specificity of norviburtinal in self-made PRR samples was discovered and validated, which was further confirmed by testing in commercially available PRR samples. In conclusion, our study revealed the decrease in iridoid glycosides and the production of new degradation substances during the process from FRR to PRR, which is critical for unveiling the processing mechanism of RR.

Omics analyses of Rehmannia glutinosa dedifferentiated and cambial meristematic cells reveal mechanisms of catalpol and indole alkaloid biosynthesis.

BACKGROUND: Rehmannia glutinosa is a rich source of terpenoids with a high medicinal reputation. The present study compared dedifferentiated cells (DDCs) and cambial meristematic cells (CMCs) cell cultures of R. glutinosa for terpenoid (catalpol) and indole alkaloid (IA) biosynthesis. In this regard, we used widely targeted metabolomics and transcriptome sequencing approaches together with the comparison of cell morphology, cell death (%), and catalpol production at different time points. RESULTS: We were able to identify CMCs based on their morphology and hypersensitivity to zeocin. CMCs showed higher dry weight content and better catalpol production compared to DDCs. The metabolome analysis revealed higher concentrations of IA, terpenoids, and catalpol in CMCs compared to DDCs. The transcriptome sequencing analysis showed that a total of 27,201 genes enriched in 139 pathways were differentially expressed. The higher catalpol concentration in CMCs is related to the expression changes in genes involved in acetyl-CoA and geranyl-PP biosynthesis, which are precursors for monoterpenoid biosynthesis. Moreover, the expressions of the four primary genes involved in monoterpenoid biosynthesis (NMD, CYP76A26, UGT6, and CYP76F14), along with a squalene monooxygenase, exhibit a strong association with the distinct catalpol biosynthesis. Contrarily, expression changes in AADC, STR, and RBG genes were consistent with the IA biosynthesis. Finally, we discussed the phytohormone signaling and transcription factors in relation to observed changes in metabolome. CONCLUSIONS: Overall, our study provides novel data for improving the catalpol and IA biosynthesis in R. glutinosa.

Catalpol Ameliorates Oxidative Stress and Neuroinflammation after Traumatic Brain Injury in Rats.

Oxidative stress and neuroinflammation are deemed the prime causes of neurological damage after traumatic brain injury (TBI). Catalpol, an active ingredient of Rehmannia glutinosa, has been suggested to possess antioxidant and anti-inflammatory properties. This study was designed to investigate the protective effects of catalpol against TBI and the underlying mechanisms of action of catalpol. A rat model of TBI was induced by controlled cortical impact. Catalpol (10 mg/kg) or vehicle was administered via intravenous injection 1 h post trauma and then once daily for 3 consecutive days. Following behavioural tests performed 72 h after TBI, the animals were sacrificed and pericontusional areas of the brain were collected for neuropathological experiments and analysis. Treatment with catalpol significantly ameliorated neurological impairment, blood-brain barrier disruption, cerebral oedema, and neuronal apoptosis after TBI (P < 0.05). Catalpol also attenuated TBI-induced oxidative insults, as evidenced by reduced reactive oxygen species generation; decreased malondialdehyde levels; and enhanced superoxide dismutase, catalase and glutathione peroxidase activity (P < 0.05). Catalpol promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2 and the expression of its downstream antioxidant enzyme HO-1 following TBI (P < 0.05). Moreover, catalpol treatment markedly inhibited posttraumatic microglial activation and neutrophil infiltration, suppressed NLRP3 inflammasome activation and reduced the production of the proinflammatory cytokine IL-1ß (P < 0.05). Taken together, these findings reveal that catalpol provides neuroprotection against oxidative stress and neuroinflammation after TBI in rats. Therefore, catalpol may be a novel treatment strategy for TBI patients.

Catalpol ameliorates dexamethasone-induced osteoporosis by promoting osteogenic differentiation of bone marrow mesenchymal stem cells via the activation of PKD1 promoter.

OBJECTIVE: To investigate the effects of CA on glucocorticoid-induced osteoporosis (GIOP) and lucubrate the underlying mechanism of CA via the activation of polycystic kidney disease-1(PKD1) in bone marrow mesenchymal stem cells (BMSCs). METHODS: In vivo, a GIOP model in mice treated with dexamethasone (Dex) was established. Biomechanical, micro-CT, immunofluorescence staining of OCN, ALP and PKD1 and others were severally determined. qRT-PCR and Western blot methods were adopted to elucidate the particular mechanisms of CA on GIOP. In addition, BMSCs cultured in vitro were also induced by Dex to verify the effects of CA. Finally, siRNA and luciferase activity assays were performed to confirm the mechanisms. RESULTS: We found that CA could restore the destroyed bone microarchitecture and increase the bone mass in GIOP mice. CA could also upregulate PKD1 protein expression, reduce oxidative stress, and promote mRNA expression of bone formation-associated markers in GIOP mice. Furthermore, it was also observed that CA reduced oxidative stress and promoted osteogenic differentiation in Dex-induced BMSCs. Mechanically, CA could promote protein expression via increasing the activity of PKD1 promoter. CONCLUSION: This study provides important evidences for CA in the further clinical treatment of GIOP, reveals the activation of PKD1 promoter as the underlying mechanism.

Catalpol modulating the crosstalking between mesenchymal stromal cells and macrophages via paracrine to enhance angiogenesis and osteogenesis.

Due to the inflammatory responses associated with defect occurrence and materials implantation, immunoregulation has emerged as a promising strategy to enhance bone regeneration. It has been widely reported that a material could facilitate osteogenesis if it can guide macrophages to anti-inflammatory M2 phenotype, vice versa, a substrate will influence macrophage phenotype if it is osteoinductive. However, few studies have looked into the intercellular crosstalking directly. Herein, the compound catalpol was selected for its multiple functions to study the interactions between bone marrow mesenchymal stromal cells (BMSCs) and macrophages. This iridoid glucoside exhibits excellent anti-inflammatory and osteoinductive activities. The effects of catalpol on mediating M1/M2 polarization of macrophages, inhibiting osteoclast differentiation, promoting osteogenesis and angiogenesis were systematically investigated to correlate the biological responses of BMSCs and macrophages. To extend its in vivo application, the catalpol was then loaded onto an electrospun polylactide/gelatin composite fibrous mesh and subcutaneously implanted to evaluate the local inflammation and ectopic osteogenesis. The results revealed that the functions of catalpol displayed in modulating cellular behaviors are via cell paracrine to strengthen intercellular crosstalking, hence demonstrating that catalpol itself could serve as a promising bioactive stimulator for bone tissue engineering.

Catalpol ameliorates inflammation and oxidative stress via regulating Sirt1 and activating Nrf2/HO-1 signaling against acute kidney injury.

BACKGROUND: Septic acute kidney injury (SAKI) is usually caused by sepsis. It has been shown that catalpol (Cat) impairs sepsis-evoked organ dysfunction to a certain degree. The current work aims to evaluate the protective effects of Cat on SAKI and potential mechanisms in vivo and in vitro. METHODS: SAKI cellular and murine model were set up using lipopolysaccharide (LPS) in vitro and in vivo. Cell apoptosis in cells was determined by TUNEL assay. Levels of inflammatory cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The levels of the markers of oxidative injury were evaluated by corresponding commercial kits. Protein levels were assayed via western blotting and immunohistochemistry (IHC) staining. RESULTS: The results demonstrated that LPS upregulated TNF-α, IL-6, and malondialdehyde levels, and downregulated superoxide dismutase, whereas Cat treated cells have the opposite results. Functional assays displayed that Cat remarkably reversed the LPS-challenged damage as the impairment of TNF-α and IL-6 levels, oxidative stress, and the apoptosis in HK-2 cells. Moreover, knockdown of Sirtuin 1 (Sirt1) counteracted the suppressive impact of Cat on LPS-triggered inflammatory response, oxidative stress, and renal damage. Further, Cat elevated Sirt1 expression and activated the Nrf2/HO-1 signaling in LPS-engendered SAKI in vivo and in vitro. CONCLUSION: Our study clearly proved that Cat protected against LPS-induced SAKI via synergic antioxidant and anti-inflammatory actions by regulating Sirt1 and Nrf2/HO-1 signaling pathways.

Anti-inflammatory effect and mechanism of catalpol in various inflammatory diseases.

Catalpol is a kind of iridoid glucoside, widely found in a variety of plants, mostly extracted from the rhizome of the traditional medicinal herb rehmanniae. It has various biological activities such as anti-inflammatory, antioxidant, and antitumor. The anti-inflammatory effects of catalpol have been demonstrated in a variety of diseases, such as neurological diseases, atherosclerosis, renal diseases, respiratory diseases, digestive diseases, bone and joint diseases, eye diseases, and periodontitis. The purpose of this review is to summarize the existing literature on the anti-inflammatory effects of catalpol in a variety of inflammatory diseases over the last decade and to focus on the anti-inflammatory mechanisms of catalpol.

Analyses of Transcriptomics upon IL-1ß-Stimulated Mouse Chondrocytes and the Protective Effect of Catalpol through the NOD2/NF-κB/MAPK Signaling Pathway.

The overall objective of this study was to investigate the mechanism of inflammation on chondrocyte injury and the protective effect of catalpol on chondrocytes in an inflammatory environment. Chondrocytes were isolated and cultured from the knee joints of three-day-old newborn mice. Alcian Blue staining and the immunocytochemistry staining of type II collagen were used to identify the purity of chondrocytes. Primary chondrocytes were stimulated by IL-1ß (10 ng/mL) and subjected to transcriptome analysis. Differentially expressed genes (DEGs) were further analyzed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. In this experimental study, we performed the viability assay to determine the effects of different concentrations of catalpol on the cell viability of chondrocytes. Chondrocytes were seeded in six-well plates and exposed to 10 µM catalpol 2 h prior to treatment with IL-1ß (10 ng/mL). Quantitative real-time (qPCR) and Western blotting were performed to evaluate the RNA and protein expression, respectively. Based on the results of transcriptomics analysis, we found the NOD2 signaling pathway, the NF-kappa B signaling pathway, and the MAPK signaling pathway showed significant changes in chondrocyte damage caused by inflammation. Catalpol (10 µM and 100 µM) could significantly reduce NO, IL-6, IL-1ß, and TNF-α in supernatant of chondrocytes. Catalpol significantly inhibited the mRNA expression of IL-1, IL-6, and IL-12 in chondrocytes induced by IL-1ß. Catalpol markedly inhibited MMP3, MMP13 mRNA, and protein levels. Catalpol could significantly reduce TNF-α mRNA levels in inflammatory chondrocytes. Inflammation causes significant increases in mRNA levels and protein levels of NOD2, mRNA levels, and protein levels were markedly suppressed by catalpol. In addition, catalpol could significantly increase IKBα protein levels and significantly lower intranuclear P65 levels. Catalpol significantly lowered the phosphorylation protein levels of ERK, p38, and JNK. Our transcriptomic analysis demonstrated that the activation of NOD2 and its downstream pathways, NF-κB and MAPK, is an important cause of the inflammatory injury to chondrocytes induced by IL-1ß. Catalpol inhibited the activation of the NOD2 signaling pathway, which reduced the phosphorylation of ERK, p38, and JNK, inhibited the degradation of IκBα, inhibited p65 translocation into the nucleus, reduced the release of inflammatory cytokines, and attenuated the inflammatory damage to chondrocytes.

Catalpol alleviates myocardial ischemia reperfusion injury by activating the Nrf2/HO-1 signaling pathway.

PURPOSE: Myocardial ischemia/reperfusion injury (MI/RI) is a major problem in the clinical treatment of ischemic cardiomyopathy, and its specific underlying mechanisms are complicated and still unclear. A number of studies have indicated that the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxidase-1(HO-1) signaling pathway might serve as an important target for the management of MI/RI. Catalpol is a kind of iridoid glucoside that has been found to exhibit diverse anti-inflammatory and antioxidant properties. This study was aimed at investigating the role of Catalpol in targeting MI/RI and its related mechanisms in an oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro and a preclinical ischemia/reperfusion (I/R) model. METHODS: This study using both in vitro and in vivo models investigated the possible role and underlying mechanisms used by Catalpol for modulating of MI/RI. The potential effects of Catalpol on the viability of cardiomyocytes were measured by cell counting kit-8 (CCK-8) assays. The phenotypes of myocardial injury, oxidative stress and inflammation markers were measured by western blot, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) etc. Nrf2/HO-1 signaling pathway was detected by immunofluorescence and western blot analysis. RESULTS: We found that Catalpol significantly suppressed the process of MI/RI and protected OGD/R-treated cardiomyocytes by inhibiting the various markers of inflammation and suppressing oxidative stress. Additionally, mechanistically it was also demonstrated that Catalpol could effectively activate Nrf2/HO-1 signaling pathway to suppress the damage caused by inflammation and oxidative stress in MI/RI. CONCLUSION: In summary, the findings suggest that Catalpol exerted significant cardioprotective effects following myocardial ischemia, possibly through the activation of the Nrf2/HO-1 signaling pathway.

Catalpol induces apoptosis in breast cancer in vitro and in vivo: Involvement of mitochondria apoptosis pathway and post-translational modifications.

Breast cancer is a fatal cancer with the highest mortality in female. New strategies for anti-breast cancer are still urgently needed. Catalpol, an iridoid glycoside extracted from the traditional Chinese medicinal plant Rehmannia glutinosa, has shown anticancer efficacy in various cancer cells. However, its effect on breast cancer remains unclear. In this study, we aim to investigate the anti-breast cancer activity of catalpol and elucidate its underlying mechanism. Cell counting kit-8 (CCK-8) and morphology change showed that catalpol could inhibit the proliferation and viability of MCF-7 cells. Catalpol administration reduced the tumor volume in xenograft model. Catalpol induced apoptosis in MCF-7 cells confirmed by Hoechst 33342 staining and Annexin V-FITC/PI double staining. In vivo, catalpol also induced apoptosis as seen from the increased level of terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) in tumor. According to JC-1 and Dichlorodi-hydrofluorescein Diacetate (DCFH-DA) staining, loss of mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation was found in MCF-7 cells treated with catalpol. Furthermore, catalpol also increased the level of cytoplasmic cytochrome c and activity of caspase-3 in MCF-7 cells. Likewise, histopathological and immunohistochemical (IHC) assay also found that catalpol enhanced the levels of cytochrome c and caspase-3 in breast cancer tissues. Ultimately, acetylation, 2-hydroxyisobutyrylation and lactylation were dramatically increased, whereas succinylation, malonylation and phosphorylation were markedly decreased in the breast cancer tumor treated with catalpol. Taken together, catalpol inhibited breast cancer in vitro and in vivo through induction of apoptosis via mitochondria apoptosis pathway and regulation of protein post-translational modifications (PTMs). Thus, it can be considered as an excellent candidate compound for treatment of breast cancer.

Catalpol Alleviates Isoflurane-Induced Hippocampal Learning and Memory Dysfunction and Neuropathological Changes in Aged Mice.

INTRODUCTION: Isoflurane-associated perioperative neurocognitive disorders (PNDs) is a common complication that occurs commonly in elderly patients characterized by deterioration of hippocampus-dependent cognitive function. Mounting evidence has shown that hippocampal impairment and inflammatory processes are implicated in the pathogenesis of PNDs. Catalpol has been suggested to play a role in the modulation of neuroprotection and neurotransmission. Therefore, we surmised that catalpol may play a similar role during isoflurane-induced PNDs. METHODS: In our current study, aged mice were exposed to isoflurane to develop a mouse model of PNDs and preconditioned with catalpol for 2 weeks before modeling. Three weeks after isoflurane exposure, behavioral, histological, biochemical, electrophysiological, and immunofluorescent assays were performed. RESULTS: Our results showed that catalpol preadministration significantly alleviated cognitive impairment in the Morris water maze, novel object recognition, and Y-maze behavioral tests. Neuropathological analyses showed that catalpol preadministration reduced the loss of neurons and synapses; in line with this, it is revealed that hippocampal synaptic plasticity was restored. Mechanistically, catalpol preadministration suppressed the activation of microglia and decreased the expression of NLRP3 inflammasome. CONCLUSION: Our results indicate that catalpol preadministration could effectively alleviate cognitive impairment and neuropathological damage in isoflurane-exposed aged mice with its neuroprotective effects via modulation of the NLRP3 inflammatory pathway. Furthermore, the NLRP3 inflammatory pathway was revealed to be involved in these effects.

Catalpol protects AC16 cells from hypoxia/reoxygenation injury by regulating the miR-22-3p/DPP4 axis.

Catalpol (CA) is widely used in the protection of cardiomyocytes. Nevertheless, the mechanism of CA in alleviating ischemia-reperfusion-induced injury of cardiomyocytes remains unclear. Human cardiomyocyte AC16 cells were subjected to hypoxia/reoxygenation (H/R) injury. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis were applied to detect tumor necrosis factor-alpha (TNF-α) mRNA, interleukin-6 (IL-6) mRNA, interleukin-1beta (IL-1ß) mRNA, microRNA-22-3p (miR-22-3p), dipeptidyl peptidase 4 (DPP4) mRNA, and DPP4 protein expressions. The cell viability and apoptosis were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Lactate dehydrogenase (LDH) and creatine kinase (CK-MB) were examined by enzyme-linked immunosorbent assay (ELISA) kits. A dual-luciferase reporter gene assay was performed to confirm the binding sequence between miR-22-3p and DPP4 mRNA 3'-untranslated region (3'UTR). CA promoted the viability and reduced cell apoptosis of AC16 cells and repressed the release of inflammatory cytokines TNF-α, IL-6, and IL-1ß, and inhibited the leakage of myocardial injury markers LDH and CK-MB. Furthermore, CA enhanced the expression of miR-22-3p in cardiomyocytes, and DPP4 was validated to be the target gene of miR-22-3p. The inhibition of miR-22-3p and augmentation of DPP4 reversed the above effects of CA. CA protects A16 cells from H/R injury by regulating the miR-22-3p/DPP4 axis.

Catalpol improves impaired neurovascular unit in ischemic stroke rats via enhancing VEGF-PI3K/AKT and VEGF-MEK1/2/ERK1/2 signaling.

Neurovascular unit (NVU) is organized multi-cellular and multi-component networks that are essential for brain health and brain homeostasis maintaining. Neurovascular unit dysfunction is the central pathogenesis process of ischemic stroke. Thus integrated protection of NVU holds great therapeutic potential for ischemic stroke. Catalpol, classified into the iridoid monosaccharide glycoside, is the main active ingredient of the radix from traditional Chinese medicine, Rehmannia glutinosa Libosch, that exhibits protective effects in several brain-related diseases. In the present study, we investigated whether catalpol exerted protective effects for NVU in ischemic stroke and the underlying mechanisms. MCAO rats were administered catalpol (2.5, 5.0, 10.0 mg·kg-1·d-1, i.v.) for 14 days. We showed that catalpol treatment dose-dependently reduced the infarction volume and significantly attenuated neurological deficits score in MCAO rats. Furthermore, catalpol treatment significantly ameliorated impaired NVU in ischemic region by protecting vessel-neuron-astrocyte structures and morphology, and promoting angiogenesis and neurogenesis to replenish lost vessels and neurons. Moreover, catalpol treatment significantly increased the expression of vascular endothelial growth factor (VEGF) through up-regulating PI3K/AKT signaling, followed by increasing FAK and Paxillin and activating PI3K/AKT and MEK1/2/ERK1/2 pathways. The protective mechanisms of catalpol were confirmed in an in vitro three-dimensional NVU model subjected to oxygen-glucose deprivation. In conclusion, catalpol protects NVU in ischemic region via activation of PI3K/AKT signaling and increased VEGF production; VEGF further enhances PI3K/AKT and MEK1/2/ERK1/2 signaling, which may trigger a partly feed-forward loop to protect NVU from ischemic stroke.

Study on Catalpol Content in Rehmannia glutinosa Root, an Important Ingredient in Kampo Prescriptions.

Rehmannia glutinosa is an important medicinal plant in Asia, and its roots are used as an ingredient in herbal medicine. However, the roots exhibit different medicinal effects depending on the processing conditions. Since the catalpol content differs greatly during the process, the catalpol content is an essential index for quality evaluation. R. glutinosa roots have various weights, diameters, and lengths, and there are differences between individuals and within an individual immediately after harvest. We found that, catalpol content in the roots tended to increase as root diameter increased. Furthermore, it has been reported that catalpol content decreased with drying, and our results also supported this phenomenon. To clarify the reason for the decrease in catalpol content, we investigated the effect of ß-glucosidase in R. glutinosa cells. An in situ assay for ß-glucosidase activity revealed that the activity in the tissue inside the cambium disappeared one month after drying under natural conditions, and the activity in the tissue outside the cambium completely disappeared after two months. Because catalpol content remained almost unchanged even after drying for two months, it was clarified that ß-glucosidase activity had minimal involvement in the decrease in catalpol content in R. glutinosa roots. Based on the above results, we proposed that slicing the roots and rapidly removing water by natural drying is best to obtain dry root with little loss of catalpol content.

[Anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol against H_2O_2-induced oxidative damage in pancreatic ß cells (INS-1 cells)].

The present study investigated the anti-oxidative and anti-apoptotic effects and molecular mechanisms of catalpol on the H_2O_2-induced pancreatic ß-cells(INS-1 cells).The oxidative damage model of INS-1 cells was induced and optimized by the stimulation of H_2O_2 of different concentrations for different time.CCK-8 assay was used to detect cell viability after catalpol intervention(1, 5, 10, 20, 40, 80, and 160 µmol·L~(-1)) for 24 h.Intracellular reactive oxygen species(ROS), superoxide dismutase(SOD), and lipid peroxide malondialdehyde(MDA) were measured by DCFH-DA fluorescent probe, WST-1, and TBA respectively.Moreover, the apo-ptotic effect was detected by AO-EB and Annexin V-FITC/PI staining.In addition, the protein expression levels were detected by Wes-tern blot, and intracellular insulin concentration was measured by ELISA.The results showed that the oxidative damage model of INS-1 cells was stably induced by 50 µmol·L~(-1) H_2O_2 treatment for 2 h, and catalpol at 1-80 µmol·L~(-1) did not affect cell viability of INS-1 cells.Compared with the conditions in the model group, 1, 5, and 10 µmol·L~(-1) catalpol intervention for 2 h could protect INS-1 cells from oxidative damage(P<0.001), reduce ROS and MDA, increase SOD, and inhibit excessive cell apoptosis.Moreover, 1, 5, and 10 µmol·L~(-1) catalpol could also up-regulate the phosphorylation of nuclear transcription factor NF-E2 related factors, negatively regulate Kelch-like ECH-associated protein 1(Keap1), phosphorylation of extracellular signal-regulated kinase(ERK), and heme oxyge-nase 1(HO-1), and promote the protein expression of pancreatic-duodenal homeobox factor-1(PDX-1) and glucose transporter 2(GLUT2).In addition, 1, 5, and 10 µmol·L~(-1) catalpol increased insulin secretion of INS-1 cells under oxidative damage in the high-glucose culture medium, indicating function recovery of pancreatic ß cells.PDX-1 is a key nuclear transcription factor of pancreatic ß cell function that directly regulates GLUT2 and insulin synthesis, and affects glucose homeostasis.In conclusion, catalpol can reduce the oxidative damage and apoptosis of INS-1 cells, activate antioxidant pathway, protect the function of pancreatic ß cells, and improve insulin synthesis and secretion.

Suppression of apoptosis in vascular endothelial cell, the promising way for natural medicines to treat atherosclerosis.

Atherosclerosis, a chronic multifactorial disease, is closely related to the development of cardiovascular diseases and is one of the predominant causes of death worldwide. Normal vascular endothelial cells play an important role in maintaining vascular homeostasis and inhibiting atherosclerosis by regulating vascular tension, preventing thrombosis and regulating inflammation. Currently, accumulating evidence has revealed that endothelial cell apoptosis is the first step of atherosclerosis. Excess apoptosis of endothelial cells induced by risk factors for atherosclerosis is a preliminary event in atherosclerosis development and might be a target for preventing and treating atherosclerosis. Interestingly, accumulating evidence shows that natural medicines have great potential to treat atherosclerosis by inhibiting endothelial cell apoptosis. Therefore, this paper reviewed current studies on the inhibitory effect of natural medicines on endothelial cell apoptosis and summarized the risk factors that may induce endothelial cell apoptosis, including oxidized low-density lipoprotein (ox-LDL), reactive oxygen species (ROS), angiotensin II (Ang II), tumor necrosis factor-α (TNF-α), homocysteine (Hcy) and lipopolysaccharide (LPS). We expect this review to highlight the importance of natural medicines, including extracts and monomers, in the treatment of atherosclerosis by inhibiting endothelial cell apoptosis and provide a foundation for the development of potential antiatherosclerotic drugs from natural medicines.

Catalpol synergistically potentiates the anti-tumour effects of regorafenib against hepatocellular carcinoma via dual inhibition of PI3K/Akt/mTOR/NF-κB and VEGF/VEGFR2 signaling pathways.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver cancer characterized by dysregulation of several crucial cellular signaling pathways such as PI3K/p-Akt/mTOR/NF-κB and VEGF/VEGFR2 pathways. Novel therapies targeting these pathways have been discovered such as regorafenib which is small molecular multi-kinase inhibitor mainly targets VEGF/VEGFR2. Catalpol is an iridoid glycoside richly found in rehmannia glutinosa which is a fundamental herb used extensively in traditional Chinese medicine. It is evidenced that catalpol has many pharmacological effects on nervous and cardiovascular systems, in addition to exhibiting hypoglycemic, anti-inflammatory, anti-proliferative and anti-tumour activities. However, its effect on HCC isn't clear enough. So, this study aimed to investigate the anti-tumour effects of catalpol either alone or in combination with regorafenib on HCC. METHODS AND RESULTS: In vitro experiments were performed using HepG2 and HUH-7 hepatocellular carcinoma cell lines. MTT assays evaluated anti-proliferative effects of catalpol and/or regorafenib. Combination index was calculated via compusyn software to detect synergism. Tumour biomarkers were measured using ELISA technique. Results showed that catalpol has anti-tumour effects against HCC via targeting PI3K/p-Akt/mTOR/NF-κB and VEGF/VEGFR2 pathways. In addition, results revealed that our novel combination of catalpol and regorafenib showed potent synergistic anti-tumour effect via suppressing both of PI3K/p-Akt/mTOR/NF-κB and VEGF/VEGFR2 signaling pathways and their downstreams. CONCLUSION: Catalpol and/or regorafenib markedly suppressed PI3K/p-Akt/mTOR/NF-κB and VEGF/VEGFR2 signaling pathways and consequently showed potent anti-tumour effects against HCC. Results encourage further pre-clinical and clinical studies of this novel combination as a promising targeted therapy for HCC management.

Catalpol counteracts the pathology in a mouse model of Duchenne muscular dystrophy by inhibiting the TGF-ß1/TAK1 signaling pathway.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease caused by a mutation in the gene encoding the dystrophin protein. Catalpol is an iridoid glycoside found in Chinese herbs with anti-inflammatory, anti-oxidant, anti-apoptotic, and hypoglycemic activities that can protect against muscle wasting. In the present study we investigated the effects of catalpol on DMD. Aged Dystrophin-deficient (mdx) mice (12 months old) were treated with catalpol (100, 200 mg·kg-1·d-1, ig) for 6 weeks. At the end of the experiment, the mice were sacrificed, and gastrocnemius (GAS), tibialis anterior (TA), extensor digitorum longus (EDL), soleus (SOL) muscles were collected. We found that catalpol administration dose-dependently increased stride length and decreased stride width in Gait test. Wire grip test showed that the time of wire grip and grip strength were increased. We found that catalpol administration dose-dependently alleviated skeletal muscle damage, evidenced by reduced plasma CK and LDH activity as well as increased the weight of skeletal muscles. Catalpol administration had no effect on dystrophin expression, but exerted anti-inflammatory effects. Furthermore, catalpol administration dose-dependently decreased tibialis anterior (TA) muscle fibrosis, and inhibited the expression of TGF-ß1, TAK1 and α-SMA. In primary myoblasts from mdx mice, knockdown of TAK1 abolished the inhibitory effects of catalpol on the expression levels of TGF-ß1 and α-SMA. In conclusion, catalpol can restore skeletal muscle strength and alleviate skeletal muscle damage in aged mdx mice, thus may provide a novel therapy for DMD. Catalpol attenuates muscle fibrosis by inhibiting the TGF-ß1/TAK1 signaling pathway.