Procatechuic acid and protocatechuic aldehyde increase survival of Caenorhabditis elegans after fungal infection and inhibit fungal virulence.

Protocatechuic acid (PCA) and protocatechuic aldehyde (PAL) are important phenolic compounds in plants. We here investigated their possible beneficial effect against fungal infection and the underlying mechanism. The model animal of Caenorhabditis elegans was used as host, and Candida albicans was used as fungal pathogen. The nematodes were first infected with C. albicans, and the PCA and PAL treatment were then performed. Post-treatment with 10-100 µM PCA and PAL suppressed toxicity of C. albicans infection in reducing lifespan. Accompanied with this beneficial effect, treatment with 10-100 µM PCA and PAL inhibited C. albicans accumulation in intestinal lumen. In addition, treatment with 10-100 µM PCA and PAL suppressed the increase in expressions of antimicrobial genes caused by C. albicans infection. The beneficial effect of PCA and PAL against C. albicans infection depended on p38 MAPK and insulin signals. Moreover, although treatment with 10-100 µM PCA and PAL could not exhibit noticeable antifungal activity, PCA and PAL treatment obviously suppressed biofilm formation, inhibited hyphal growth, and reduced expressions of virulence genes (ALS3, CaVps34, Vma7, Vac1, and/or HWP1) related to biofilm formation and hyphal growth in C. albicans. Therefore, our data demonstrated the potential of PCA and PAL post-treatment against fungal infection and fungal virulence.

Protocatechuic aldehyde increases pericyte coverage and mitigates pericyte damage to enhance the atherosclerotic plaque stability.

Pericyte dysfunction and loss contribute substantially to the destabilization and rupture of atherosclerotic plaques. Protocatechuic aldehyde (PCAD), a natural polyphenol, exerts anti-atherosclerotic effects. However, the effects and mechanisms of this polyphenol on pericyte recruitment, coverage, and pericyte function remain unknown. We here treated apolipoprotein E-deficient mice having high-fat diet-induced atherosclerosis with PCAD. PCAD achieved therapeutic effects similar to rosuvastatin in lowering lipid levels and thus preventing atherosclerosis progression. With PCAD administration, plaque phenotype exhibited higher stability with markedly reduced lesion vulnerability, which is characterized by reduced lipid content and macrophage accumulation, and a consequent increase in collagen deposition. PCAD therapy increased pericyte coverage in the plaques, reduced VEGF-A production, and inhibited intraplaque neovascularization. PCAD promoted pericyte proliferation, adhesion, and migration to mitigate ox-LDL-induced pericyte dysfunction, which thus maintained the capillary network structure and stability. Furthermore, TGFBR1 silencing partially reversed the protective effect exerted by PCAD on human microvascular pericytes. PCAD increased pericyte coverage and impeded ox-LDL-induced damages through TGF-ß1/TGFBR1/Smad2/3 signaling. All these novel findings indicated that PCAD increases pericyte coverage and alleviates pericyte damage to improve the stability of atherosclerotic plaques, which is accomplished by regulating TGF-ß1/TGFBR1/Smad2/3 signaling in pericytes.

Protocatechuic aldehyde acts synergistically with dacarbazine to augment DNA double-strand breaks and promote apoptosis in cutaneous melanoma cells.

BACKGROUND: Despite rapid developments in immunotherapy and targeted therapy, dacarbazine (DTIC)-based chemotherapy still has been placed at the first-line for advanced melanoma patients who are after failure of immunotherapy or targeted therapy. However, the limited response rate and survival benefit challenge the DTIC-based chemotherapy for advanced melanoma patients. METHODS: Two melanoma cell lines, A375 and SK-MEL-28 were cultured with PA and DTIC over a range of concentrations for 72 h and the cell viabilities were detected by CCK8 assay. The Bliss model and ZIP model were used for calculating the synergistic effect of PA and DTIC. DNA double-strand breaks in the two cell lines were examined by the Comet assay, and cell apoptosis was analyzed by flow cytometry. The short hairpin RNA (shRNA)-mediated knockdown, Real-time polymerase chain reaction (RT-PCR) and Western blot were performed for molecular analysis. RESULTS: In the present study, we report that Protocatechuic aldehyde (PA) synergistically enhances the cytotoxicity of DTIC to two melanoma cell lines, A375 and SK-MEL-28. The combination of PA and DTIC augments DNA double-strand breaks and increases cell apoptosis. Further mechanism study reveals that PA destabilizes MGMT protein (O-6-Methylguanine-DNA Methyltransferase) through the ubiquitin-proteasome process and directly repairs DTIC-induced genetic lesions. Knockdown of MGMT compromises the synergistic effect between PA and DTIC. CONCLUSION: Our study demonstrates that the bioactive compound, Protocatechuic aldehyde, synergistically promotes the cytotoxicity of DTIC to melanoma cells through destabilization of MGMT protein. It could be a potential candidate for melanoma chemotherapy.

Protocatechuic aldehyde promotes the functional recovery of spinal cord injury by activating the Wnt/ß-catenin signaling pathway.

CONTEXT/OBJECTIVE: This study aimed to explore the anti-inflammatory and neuroprotective effects of protocatechuic aldehyde (PCA) in rats with spinal cord injury (SCI) and to clarify the molecular mechanisms underlying its pharmacological effects. DESIGN: Male Sprague Dawley rat model of moderate spinal cord contusion were established. SETTING: Third-class first-class hospital. OUTCOME MEASURES: The Basso, Beattie, and Bresnahan scores and performance on the inclined plane test were evaluated. Histological analyses were performed via hematoxylin and eosin staining. Apoptosis in the spinal cord and neurons was detected by 5 terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining. Apoptotic factors (Bax, Bcl-2, and cleaved caspase-3) were also evaluated. INOS, IL-1ß, IL-10, TNF-α, Wnt-3α, ß-catenin, iBA-1, and NeuN were assessed by real-time reverse transcription-polymerase chain reaction (RT-PCR), western blotting (WB), and enzyme-linked immunosorbent assay. Cell viability and the immunofluorescence of IL-1ß were measured in PC-12 cells. RESULTS: Using WB and quantitative reverse transcription-PCR, we confirmed that PCA treatment activated the Wnt/ß-catenin signaling axis in vivo and in vitro. Hematoxylin and eosin staining and hindlimb motor functional evaluation revealed that treatment with PCA improved tissue protection and functional recovery via the Wnt/ß-catenin axis. The upregulation of TUNEL-positive cells, downregulation of neurons, elevated apoptosis-associated factors in rats, and increased apoptotic rates were observed in microglia and PC-12 after PCA application. Finally, PCA mitigated SCI-induced inflammation by targeting the Wnt/ß-catenin axis. CONCLUSION: This study provided preliminary evidence that PCA inhibits neuroinflammation and apoptosis through the Wnt/ß-catenin pathway, thereby attenuating the secondary injury after SCI and promoting the regeneration of injured spinal tissues.

Transdermal delivery of Protocatechuic aldehyde using hyaluronic acid/gelatin-based microneedles for the prevention and treatment of hypertrophic scars.

The formation of hypertrophic scar (HS) involves many pathological processes, such as reduced apoptosis in fibroblasts, excessive collagen deposition by fibroblasts, over-abundant angiogenesis, etc. The therapeutic effects of current treatments targeting one single pathological process are limited. Due to their diverse biological activities, natural products offer a potential solution to this issue. In this study reported herein, we investigated the effects of Protocatechuic aldehyde (PA) on both hypertrophic scar-derived fibroblasts (HSF) and vascular endothelial growth factor (VEGF)-stimulated human umbilical vein endothelial cells (HUVECs). Microneedles (MN) containing PA and hyaluronic acid (HA) or containing PA, HA, and gelatin were prepared by mixing PA stock solution with HA or HA/gelatin at a ratio of 1:10. The HS prevention and treatment outcomes of these HA-PA-MN and HA/gelatin-PA-MN were tested using a rabbit ear HS model. Our data indicate that PA induces apoptosis and reduces collagen deposition in HSF. In addition, PA attenuates VEGF-stimulated angiogenesis of HUVECs. Furthermore, HA-PA-MN or HA/gelatin-PA-MN are able to effectively penetrate the epidermis of the HS tissues and then quickly dissolve, enabling the fast release of PA directly into the dermis of the HS tissues. HA-PA-MN or HA/Gelatin-PA-MN have also been found to effectively prevent or alleviate HS in a rabbit ear HS model. In conclusion, this study demonstrates that PA can be used to prevent and treat HS by simultaneously regulating HSF and HUVECs, which offers a potential novel reagent for HS management.

Antibiofilm effect and mechanism of protocatechuic aldehyde against Vibrio parahaemolyticus.

This study investigated the effect of protocatechuic aldehyde (PCA) on Vibrio parahaemolyticus biofilm formation and its effects on gene expression. Crystal violet assay, metabolic activity assay, and fluorescence experiments were used to evaluate the antibiofilm activities of PCA and to reveal its possible antibiofilm mechanisms using transcriptomic analysis. The results indicated that the minimum antibacterial concentration of PCA against V. parahaemolyticus was 300 µg/mL. PCA (9.375 µg/mL) inhibited biofilm generation and adhesion of the mature biofilm. PCA (75 µg/mL) significantly reduced the metabolic viability of V. parahaemolyticus, reduced polysaccharide production, and inhibited cell surface flagella-mediated swimming and aggregation phenotypes. Meanwhile, transcriptome analysis showed that the key genes of V. parahaemolyticus expressed under PCA (75 µg/mL) inhibition were mainly related to biofilm formation (pfkA, galE, narL, and oppA), polysaccharide production and adhesion (IF, fbpA, and yxeM), and motility (cheY, flrC, and fliA). By regulating these key genes, PCA reduced biofilm formation, suppressed polysaccharide production and transport, and prevented the adhesion of V. parahaemolyticus, thereby reducing the virulence of V. parahaemolyticus. This study demonstrated that protocatechuic aldehyde can be used to control V. parahaemolyticus biofilm to ensure food safety.

Protocatechuic aldehyde prevents ischemic injury by attenuating brain microvascular endothelial cell pyroptosis via lncRNA Xist.

BACKGROUND: Pyroptosis is a pro-inflammatory cell death characterized by the formation of inflammasomes. Abnormal inflammation in brain microvascular endothelial cells (BMECs) has been correlated with ischemic stroke. Protocatechuic aldehyde (PCA) is a hydrophilic phenolic acid derived from the traditional Chinese herb Salvia miltiorrhiza with significant anti-inflammatory effects. However, the mechanism of PCA on BMEC pyroptosis under ischemic injury has been largely unexplored. PURPOSE: We aimed to study the effects and mechanism of PCA on BMEC pyroptosis under ischemic injury. METHODS: Sprague-Dawley (SD) rats were injected through the tail vein with different concentrations of PCA after transient middle cerebral artery occlusion (MCAO) was performed. The protective effects of PCA in SD rats were examined via neurological scores, infarct volume evaluation, and anti-pyroptosis effects using immunofluorescence staining and western blot. Rat BMECs (rBMECs) were treated with different concentrations of PCA after oxygen and glucose deprivation (OGD). The ability of PCA to protect rBMECs was examined via cell viability, anti-oxidative activity, and anti-pyroptosis effects as determined by qRT-PCR and western blot. Additionally, the role of lncRNA Xist in anti-pyroptosis responses of PCA-treated rBMECs was validated with lncRNA Xist siRNA. RESULTS: We found that treatment with MCAO and OGD increased the expression of NOD-like receptor protein 3, gasdermin D, Caspase-1, interleukin-1ß, and NIMA-related kinase 7, which was reversed by treatment with PCA or MCC950 (a pyroptosis inhibitor). In addition, PCA reduced the cerebral infarct volume in MCAO rats and promoted cell survival and proliferation in OGD/reperfusion-treated rBMECs. PCA enhanced the antioxidant activity and mitochondrial membrane potential in rBMECs. PCA also enhanced lncRNA Xist expression, and when the expression of lncRNA Xist was silenced, PCA could not alleviate pyroptosis well in rBMECs. CONCLUSION: Protocatechuic aldehyde prevents ischemic injury by attenuating rBMEC pyroptosis via lncRNA Xist.

Protocatechuic aldehyde protects cardiomycoytes against ischemic injury via regulation of nuclear pyruvate kinase M2.

Rescuing cells from stress damage emerges a potential therapeutic strategy to combat myocardial infarction. Protocatechuic aldehyde (PCA) is a major phenolic acid in Chinese herb Danshen (Salvia miltiorrhiza root). This study investigated whether PCA regulated nuclear pyruvate kinase isoform M2 (PKM2) function to protect cardiomyocytes. In rats subjected to isoprenaline, PCA attenuated heart injury and protected cardiomyocytes from apoptosis. Through DARTS and CETSA assays, we identified that PCA bound and promoted PKM2 nuclear translocation in cardiomyocytes exposed to oxygen/glucose deprivation (OGD). In the nucleus, PCA increased the binding of PKM2 to ß-catenin via preserving PKM2 acetylation, and the complex, in cooperation with T-cell factor 4 (TCF4), was required for transcriptional induction of genes encoding anti-apoptotic proteins, contributing to rescuing cardiomyocyte survival. In addition, PCA ameliorated mitochondrial dysfunction and prevented mitochondrial apoptosis dependent on PKM2. Consistently, PCA increased the binding of PKM2 to ß-catenin, improved heart contractive function, normalized heart structure and attenuated oxidative damage in mice subjected to artery ligation, but the protective effects were lost in Pkm2-deficient heart. Together, we showed that PCA regulated nuclear PKM2 function to rescue cardiomyocyte survival via ß-catenin/TCF4 signaling cascade, suggesting the potential of pharmacological intervention of PKM2 shuttle to protect the heart.

Evaluation of the Therapeutic Effects of Protocatechuic Aldehyde in Diabetic Nephropathy.

Diabetic nephropathy (DN) is one of the most severe chronic kidney diseases in diabetes and is the main cause of end-stage renal disease (ESRD). Protocatechuic aldehyde (PCA) is a natural product with a variety of effects on pulmonary fibrosis. In this study, we examined the effects of PCA in C57BL/KS db/db male mice. Kidney morphology, renal function indicators, and Western blot, immunohistochemistry, and hematoxylin and eosin (H&E) staining data were analyzed. The results revealed that treatment with PCA could reduce diabetic-induced renal dysfunction, as indicated by the urine albumin-to-creatinine ratio (db/m: 120.1 ± 46.1µg/mg, db/db: 453.8 ± 78.7 µg/mg, db/db + 30 mg/kg PCA: 196.6 ± 52.9 µg/mg, db/db + 60 mg/kg PCA: 163.3 ± 24.6 µg/mg, p < 0.001). However, PCA did not decrease body weight, fasting plasma glucose, or food and water intake in db/db mice. H&E staining data revealed that PCA reduced glomerular size in db/db mice (db/m: 3506.3 ± 789.3 µm2, db/db: 6538.5 ± 1818.6 µm2, db/db + 30 mg/kg PCA: 4916.9 ± 1149.6 µm2, db/db + 60 mg/kg PCA: 4160.4 ± 1186.5 µm2p < 0.001). Western blot and immunohistochemistry staining indicated that PCA restored the normal levels of diabetes-induced fibrosis markers, such as transforming growth factor-beta (TGF-ß) and type IV collagen. Similar results were observed for epithelial-mesenchymal transition-related markers, including fibronectin, E-cadherin, and α-smooth muscle actin (α-SMA). PCA also decreased oxidative stress and inflammation in the kidney of db/db mice. This research provides a foundation for using PCA as an alternative therapy for DN in the future.

ß-Cyclodextrin Inclusion Complexes with Catechol-Containing Antioxidants Protocatechuic Aldehyde and Protocatechuic Acid-An Atomistic Perspective on Structural and Thermodynamic Stabilities.

Protocatechuic aldehyde (PCAL) and protocatechuic acid (PCAC) are catechol derivatives and have broad therapeutic effects associated with their antiradical activity. Their pharmacological and physicochemical properties have been improved via the cyclodextrin (CD) encapsulation. Because the characteristics of ß-CD inclusion complexes with PCAL (1) and PCAC (2) are still equivocal, we get to the bottom of the inclusion complexation by an integrated study of single-crystal X-ray diffraction and DFT full-geometry optimization. X-ray analysis unveiled that PCAL and PCAC are nearly totally shielded in the ß-CD wall. Their aromatic rings are vertically aligned in the ß-CD cavity such that the functional groups on the opposite side of the ring (3,4-di(OH) and 1-CHO/1-COOH groups) are placed nearby the O6-H and O2-H/O3-H rims, respectively. The preferred inclusion modes in 1 and 2 help to establish crystal contacts of OH⋅⋅⋅O H-bonds with the adjacent ß-CD OH groups and water molecules. By contrast, the DFT-optimized structures of both complexes in the gas phase are thermodynamically stable via the four newly formed host-guest OH⋯O H-bonds. The intermolecular OH⋅⋅⋅O H-bonds between PCAL/PCAC 3,4-di(OH) and ß-CD O6-H groups, and the shielding of OH groups in the ß-CD wall help to stabilize these antioxidants in the ß-CD cavity, as observed in our earlier studies. Moreover, PCAL and PCAC in distinct lattice environments are compared for insights into their structural flexibility.

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.

Protocatechuic Aldehyde Inhibits α-MSH-Induced Melanogenesis in B16F10 Melanoma Cells via PKA/CREB-Associated MITF Downregulation.

Protocatechuic aldehyde (PA) is a naturally occurring phenolic compound that is a potent inhibitor of mushroom tyrosinase. However, the molecular mechanisms of the anti-melanogenesis activity of PA have not yet been reported. The aim of the current study was to clarify the melanogenesis inhibitory effects of PA and its molecular mechanisms in murine melanoma cells (B16F10). We first predicted the 3D structure of tyrosinase and used a molecular docking algorithm to simulate binding between tyrosinase and PA. These molecular modeling studies calculated a binding energy of -527.42 kcal/mol and indicated that PA interacts with Cu400 and 401, Val283, and His263. Furthermore, PA significantly decreased α-MSH-induced intracellular tyrosinase activity and melanin content in a dose-dependent manner. PA also inhibited key melanogenic proteins such as tyrosinase, tyrosinase-related protein 1 (TRP-1), and TRP-2 in α-MSH-stimulated B16F10 cells. In addition, PA decreased MITF expression levels by inhibiting phosphorylation of cAMP response element-binding protein (CREB) and cAMP-dependent protein kinase A (PKA). These results demonstrate that PA can effectively suppress melanin synthesis in melanoma cells. Taken together, our results show that PA could serve as a potential inhibitor of melanogenesis, and hence could be explored as a possible skin-lightening agent.

Qingxue jiedu formulation ameliorated DNFB-induced atopic dermatitis by inhibiting STAT3/MAPK/NF-κB signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Qingxue jiedu Formulation (QF) is composed of two classic prescriptions which have been clinically used for more than 5 centuries and appropriately modified through basic theory of traditional Chinese medicine for treating various skin inflammation such as atopic dermatitis (AD), acute dermatitis and rash. Although QF possesses a prominent clinical therapeutic effect, seldom pharmacological studies on its anti-AD activity are conducted. AIM OF THE STUDY: We used AD mice model to investigate the anti-AD activities of QF, as well as its underlying molecular mechanisms which involved signal transducer and activator of transcription 3 (STAT3), nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways. MATERIALS AND METHODS: 2,4-dinitrofluorobenzene (DNFB)-induced AD mice were used to collect serum and skin tissues for consequential determination. The levels of various inflammatory factors [interleukin (IL)-12, Interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL-4, IL-6 and immunoglobulin E (IgE)] were determined by enzyme-linked immunosorbent assay (ELISA). Real-time polymerase chain reaction (RT-PCR) was contributed to detect the effects of relevant inflammatory factors on mRNA. The roles of STAT3, NF-κB and MAPK signaling pathways in AD response were analyzed by Western blotting (WB), and the thickening of mice dorsal skin and inflammatory cell infiltration were observed by hematoxylin and eosin (H&E) staining. RESULTS: QF significantly reduced the skin thickening, inflammatory cell infiltration and other symptoms in AD mice. The levels of IL-12, TNF-α, IL-4, IL-6 and IgE were decreased, while IFN-γ was increased by QF in the ELISA analysis. QF lessened the levels of lL-6 and elevated IFN-γ on the mRNA level. In addition, WB analysis showed QF thoroughly inhibited the activation of NF-κB, STAT3 and phosphorylation of JAK1, JAK2, JAK3, while partially suppressed MAPK signaling pathways. CONCLUSIONS: QF inhibited the activations of STAT3, MAPK and NF-κB signaling pathways and possessed a significant therapeutic effect on AD. Therefore, QF deserves our continuous attention and research as a prominent medicine for AD.

Protective Effect of Protocatechuic Aldehyde on Neurovascular Unit Homeostasis Damage in Rats after Cerebral Ischemia-reperfusion Injury / 中国药房

OBJECTIVE：To obs erve the protective effect of protoca techuic aldehyde（PAL）on neurovascular unit （NVU） homeostasis damage in rats after cerebral ischemia-reperfusion injury （CIRI）. METHODS ：SD rats were randomly divided into sham operation group ，model group ，PAL high-dose and low-dose groups （10，20 mg/kg），with 11 rats in each group. Administration groups were given relevant medicine intragastrically. Sham operation group and model group were given the same volume of normal saline intragastrically ，10 mL/kg once a day ，for 5 days. After last administration ，CIRI model was induced by suture method ；the ultrastructural changes of NVU were observed by transmission electron microscope. Western blot assay was used to detect the expression of NUV related proteins （MAP-2，GFAP，AQP-4）in cerebral tissue. Immunofluorescence staining was used to observe the positive expression of above proteins in cerebral cortex. RESULTS ：Compared with sham operation group ， blood-brain barrier （BBB）structure of model group was destroyed severely ，the vascular lumen became narrower ，lateral edema of endothelial cells was severe ，and the thickness of basement membrane varied ；the nuclei of neurons were pyknosis and there was a large area of edema in the surrounding tissues ；the structure of glial cells was seriously damaged ，the cell body was shrunk and organelles were lost ；protein expression （or positive expression ）of MAP- 2 in brain tissue （or cerebral cortex ）were significantly decreased （P＜0.05 or P＜0.01），while protein expression （or positive expression ） of GFAP and AQP- 4 were increased significantly（P＜0.01）. After PAL intervention ，the rats had less BBB damage ，and the morphology of vascular lumen and basement membrane were not completely destroyed ；the damage of neurons was alleviated ，the pyknosis of neurons was decreased ， the chromatin was homogeneous and the heterochromatin was decreased；the damage of glial cell structure was alleviated protein expression of GFAP and AQP- 4（except for low-dose group） in cerebral tissue and positive expression of MAP- 2 and GFAP protein in cerebral cortex were reversed @qq.com significantly （P＜0.05 or P＜0.01）. CONCLUSIONS ：PAL can protect the stability of NVU from damage in CIRI model rats； the mechanism may be related to up-regulating the expression of MAP- 2 protein in cerebral cortex and down-regulating the expression of GFAP and AQP- 4 protein in brain tissue.

Protocatechuic aldehyde from Salvia miltiorrhiza exhibits an anti-inflammatory effect through inhibiting MAPK signalling pathway.

BACKGROUND: The aerial parts of Salvia miltiorrhiza, which was considered to be the waste part and discarded during the root harvest, is rich in protocatechuic aldehyde (PAI). This study investigated the health-promoting effects of extracts and PAI from the aerial parts of Salvia miltiorrhiza, including its anti-inflammatory effects and the underlying mechanisms of action in vitro and in vivo. METHOD: Purification of the sample paste of Salvia miltiorrhiza was accomplished using HPLC analysis. TheMTT (Methylthiazolyldiphenyl-tetrazolium bromide) assay was employed to determine the cell viability. The production of inflammatory factors was detected by ELISA assays. The histopathological analysis was used to analyse the lungs and livers of mice treated with PAI. Western blot was performed to reveal the mechanism of PAI in anti-inflammatory. RESULTS: The extracts and PAI from the aerial parts of Salvia miltiorrhiza inhibited TNF-α, IL-6 production and promoted the production of IL-10 in vivo in mice and in vitro in the macrophage cell line RAW264.7. NF-κB and MAPKs kinase phosphorylation were also suppressed by PAI in vivo and in vitro, indicating that PAI exhibited an anti-inflammatory effect. CONCLUSION: These findings suggest that the aerial parts of Salvia miltiorrhiza extract may serve as potential protective agents for inflammatory.

Protocatechuic Aldehyde Attenuates UVA-Induced Photoaging in Human Dermal Fibroblast Cells by Suppressing MAPKs/AP-1 and NF-κB Signaling Pathways.

Ultraviolet radiation (UV) is a major causative factor of DNA damage, inflammatory responses, reactive oxygen species (ROS) generation and a turnover of various cutaneous lesions resulting in skin photoaging. The purpose of this study is to investigate the protective effect of protocatechuic aldehyde (PA), which is a nature-derived compound, against UVA-induced photoaging by using human dermal fibroblast (HDF) cells. In this study, our results indicated that PA significantly reduced the levels of intracellular ROS, nitric oxide (NO), and prostaglandins-E2 (PGE2) in UVA-irradiated HDF cells. It also inhibited the levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression. Besides, PA significantly suppressed the expression of matrix metalloproteinases-1 (MMP-1) and pro-inflammatory cytokines and promoted collagen synthesis in the UVA-irradiated HDF cells. These events occurred through the regulation of activator protein 1 (AP-1), nuclear factor-κB (NF-κB), and p38 signaling pathways in UVA-irradiated HDF cells. Our findings suggest that PA enhances the protective effect of UVA-irradiated photoaging, which is associated with ROS scavenging, anti-wrinkle, and anti-inflammatory activities. Therefore, PA can be a potential candidate for the provision of a protective effect against UVA-stimulated photoaging in the pharmaceutical and cosmeceutical industries.

Protocatechuic aldehyde mitigates hydrogen peroxide-triggered PC12 cell damage by down-regulating MEG3.

Background: Protocatechuic aldehyde (PA) extracts from S. miltiorrhiza, which anti-oxidative and anti-inflammatory functions have been certified in diverse diseases. Nonetheless, the influence of PA in spinal cord injury (SCI) is still hazy. The research probed the function of PA in hydrogen peroxide (H2O2)-damaged PC12 cells.Methods: The disparate dosages of H2O2 (0-400 µM) or PA (0-2 µM) were applied for stimulating PC12 cells, and subsequently cell viability, apoptosis, apoptosis- and autophagy-correlative factors were evaluated. After pc-MEG3 transfection, functions of MEG3 overexpression in H2O2 and/or PA-managed PC12 cells were reassessed. Western blot was conducted to determine Wnt/ß-catenin and PTEN/PI3K/AKT pathways.Results: H2O2 stimulation clearly triggered PC12 cell damage via prohibiting cell viability and accelerating apoptosis and autophagy. But, PA management mitigated H2O2-triggered PC12 cells damage. Down-regulated MEG3 triggered by PA was presented in H2O2-managed cells. What's more, overexpressed MEG3 dramatically overturned the influences of PA in H2O2-damaged PC12 cells. Beyond that, PA activated Wnt/ß-catenin and PTEN/PI3K/AKT via repression of MEG3 in H2O2-managed PC12 cells.Conclusions: The results disclosed the protective impacts of PA on PC12 cells to resist H2O2-provoked damage. MEG3, Wnt/ß-catenin and PTEN/PI3K/AKT pathways joined in adjusting the activity of PA in H2O2-damaged PC12 cells.

Ginsenoside Rg1-Notoginsenoside R1-Protocatechuic Aldehyde Reduces Atherosclerosis and Attenuates Low-Shear Stress-Induced Vascular Endothelial Cell Dysfunction.

Background: The Fufang Danshen formula is a clinically important anti-atherosclerotic preparation in traditional Chinese medicine. However, its anti-atherosclerotic effect is not well recognized, and the mechanisms of its combined active ingredients, namely Ginsenoside Rg1-Notoginsenoside R1-Protocatechuic aldehyde (RRP), remain unclear. The purpose of this study was to investigate the anti-atherosclerotic effects and potential mechanism of RRP in ApoE-/- mice and in low-shear stress-injured vascular endothelial cells. Methods: ApoE-/- mice were randomly divided into three groups: model group, rosuvastatin group, and RRP group, with C57BL/6J mice as the control group. Oil-red O, hematoxylin and eosin, Masson, and Movat staining were utilized for the observation of aortic plaque. Changes in the blood lipid indexes were observed with an automatic biochemistry analyzer. ET-1, eNOS, TXA2, and PGI2 levels were analyzed by enzyme-linked immunosorbent assay. In vitro, a fluid shear stress system was used to induce cell injury. Piezo1 expression in HUVECs was silenced using siRNA. Changes in morphology, proliferation, migration, and tube formation activity of cells were observed after RRP treatment. Quantitative Real-Time PCR and western blot analysis were employed to monitor mRNA and protein expression. Results: RRP treatment reduced the atherosclerotic area and lipid levels and improved endothelial function in ApoE-/- mice. RRP significantly repaired cell morphology, reduced excessive cell proliferation, and ameliorated migration and tube formation activity. In addition, RRP affected the FAK-PI3K/Akt signaling pathway. Importantly, Piezo1 silencing abolished the protective effects of RRP. Conclusion: RRP has anti-atherosclerotic effects and antagonizes endothelial cell damage via modulating the FAK-PI3K/Akt signaling pathway. Piezo1 is a possible target of RRP in the treatment of atherosclerosis. Thus, RRP has promising therapeutic potential and broad application prospect for atherosclerosis.

Optimization of water-precipitation process in pretreatment process of Salviae Miltiorrhizae (Danshen) and Ligustrazine Hydrochloride Injection based on sequential central composite design / 中草药

Objective: To optimize the water-precipitation in the pretreatment manufacture process of Salviae Miltiorrhizae (Danshen) and Ligustrazine Hydrochloride Injection (SMLHI) based on the quality by design (QbD) concept and the sequential design. Methods: A design space was obtained by the global-type sequential design methodology combined with the fishbone-diagram risk analysis and central composite experiment based on global type sequential design method. The preliminary optimized zone of critical process parameters was extended in axial direction on the basis of the statistical result gained from the first central composite design. Then an updated multivariate linear regression model was fitted and a design space was delineated and verified. Results: According to the design space, the recommended operation space of the water-precipitation was as follows: when the pH value of the material was between 3.1 and 3.4, 3.25-5.00 times of water adding into the solution was recommened, the standing time was 7-17 h and the standing temperature was 7 ℃. Conclusion: This study introduces the sequential design into the process optimization of traditional Chinese medicine pharmaceuticals. Compared with the experimental design methods such as uniform space grid design and orthogonal design with a large number of samples, while samples cover the same feasible zone in a continuous operation form, sequential design methodology can effectively reduce the workload of the optimization experiment, avoid the waste of manpower and resources as well as guarantee the prediction ability of the fitting model.

Neuroprotective effects of protocatechuic aldehyde through PLK2/p-GSK3ß/Nrf2 signaling pathway in both in vivo and in vitro models of Parkinson's disease.

Mitochondrial dysfunction and oxidative damage are closely related to the pathogenesis of Parkinson's disease (PD). The pharmacological mechanism of protocatechuic aldehyde (PCA) for PD treatment have retained unclear. The purposes of the present study were to clarify the neuroprotective effects of post-treatment of PCA for PD treatment by mitigating mitochondrial dysfunction and oxidative damage, and to further determine whether its effects were mediated by the polo-like kinase 2/phosphorylated glycogen synthase kinase 3 ß/nuclear factor erythroid-2-related factor 2 (PLK2/p-GSK3ß/Nrf2) pathways. We found that PCA improved 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced behavioral deficits and dopaminergic cell loss. Moreover, PCA increased the expressions of PLK2, p-GSK3ß and Nrf2, following the decrease of α-synuclein (α-Syn) in MPTP-intoxicated mice. Cell viability was increased and the apoptosis rate was reduced by PCA in 1-methyl-4-phenylpyridinium iodide (MPP+)-incubated cells. Mitochondrial membrane potential (MMP), mitochondrial complex I activity and reactive oxygen species (ROS) levels in MPP+-incubated cells were also ameliorated by treatment with PCA. The neuroprotective effects of PCA were abolished by inhibition or knockdown of PLK2, whereas overexpression of PLK2 strengthened the protection of PCA. Furthermore, GSK3ß and Nrf2 were involved in PCA-induced protection. These results indicated that PCA has therapeutic effects on PD by the PLK2/p-GSK3ß/Nrf2 pathway.