Functional analysis of the protocatechuate branch of the ß-ketoadipate pathway in Aspergillus niger.

Bacteria and fungi catabolize plant-derived aromatic compounds by funneling into one of seven dihydroxylated aromatic intermediates, which then undergo ring fission and conversion to TCA cycle intermediates. Two of these intermediates, protocatechuic acid and catechol, converge on ß-ketoadipate which is further cleaved to succinyl-CoA and acetyl-CoA. These ß-ketoadipate pathways have been well characterized in bacteria. The corresponding knowledge of these pathways in fungi is incomplete. Characterization of these pathways in fungi would expand our knowledge and improve the valorization of lignin-derived compounds. Here, we used homology to characterize bacterial or fungal genes to predict the genes involved in the ß-ketoadipate pathway for protocatechuate utilization in the filamentous fungus Aspergillus niger. We further used the following approaches to refine the assignment of the pathway genes: whole transcriptome sequencing to reveal genes upregulated in the presence of protocatechuic acid; deletion of candidate genes to observe their ability to grow on protocatechuic acid; determination by mass spectrometry of metabolites accumulated by deletion mutants; and enzyme assays of the recombinant proteins encoded by candidate genes. Based on the aggregate experimental evidence, we assigned the genes for the five pathway enzymes as follows: NRRL3_01405 (prcA) encodes protocatechuate 3,4-dioxygenase; NRRL3_02586 (cmcA) encodes 3-carboxy-cis,cis-muconate cyclase; NRRL3_01409 (chdA) encodes 3-carboxymuconolactone hydrolase/decarboxylase; NRRL3_01886 (kstA) encodes ß-ketoadipate:succinyl-CoA transferase; and NRRL3_01526 (kctA) encodes ß-ketoadipyl-CoA thiolase. Strain carrying ΔNRRL3_00837 could not grow on protocatechuic acid, suggesting that it is essential for protocatechuate catabolism. Its function is unknown as recombinant NRRL3_00837 did not affect the in vitro conversion of protocatechuic acid to ß-ketoadipate.

Efficient production of protocatechuic acid using systems engineering of Escherichia coli.

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is widely used in the pharmaceuticals, health food, and cosmetics industries owing to its diverse biological activities. However, the inhibition of 3-dehydroshikimate dehydratase (AroZ) by PCA and its toxicity to cells limit the efficient production of PCA in Escherichia coli. In this study, a high-level strain of 3-dehydroshikimate, E. coli DHS01, was developed by blocking the carbon flow from the shikimate-overproducing strain E. coli SA09. Additionally, the PCA biosynthetic pathway was established in DHS01 by introducing the high-activity ApAroZ. Subsequently, the protein structure and catalytic mechanism of 3-dehydroshikimate dehydratase from Acinetobacter pittii PHEA-2 (ApAroZ) were clarified. The variant ApAroZR363A, achieved by modulating the conformational dynamics of ApAroZ, effectively relieved product inhibition. Additionally, the tolerance of the strain E. coli PCA04 to PCA was enhanced by adaptive laboratory evolution, and a biosensor-assisted high-throughput screening method was designed and implemented to expedite the identification of high-performance PCA-producing strains. Finally, in a 5 L bioreactor, the final strain PCA05 achieved the highest PCA titer of 46.65 g/L, a yield of 0.23 g/g, and a productivity of 1.46 g/L/h for PCA synthesis from glucose using normal fed-batch fermentation. The strategies described herein serve as valuable guidelines for the production of other high-value and toxic products.

Menadione and protocatechuic acid: A drug combination with antitumor effects in murine osteosarcoma cells.

Osteosarcoma (OS) is a primary malignant bone tumor that has an abnormal expression of oncogenesis and tumor suppressors and causes dysregulation of various signaling pathways. Thus, novel therapeutic strategies for OS are needed to overcome the resistance of traditional treatments. This study evaluated the cytotoxic and anticancer effects of the association between menadione (MEN) and protocatechuic acid (PCA) in murine OS cells (UMR-106). The concentrations were 3.12 µM of isolated MEN, 500 µM of isolated PCA, and their associations. We performed cell viability assays, morphology modification analysis, cell migration by the wound-healing method, apoptosis by flow cytometry, reactive oxygen species (ROS) production, gene expression of NOX by RT-qPCR, and degradation of MMP-2 and 9 by zymography. Our results showed that the association of MEN+PCA was more effective in OS cells than the compounds alone. The association decreased cell viability, delayed cell migration, and decreased the expression of NOX-2 and ROS. In addition, the MEN+PCA association induced a slight increase in the apoptotic process. In summary, the association can enhance the compound's antitumor effects and establish a higher selectivity for tumor cells, possibly caused by significant mitochondrial damage and antioxidant properties.

Biodegradation of DEP, DIBP, and BBP by a psychrotolerant Sphingobium yanoikuyae strain P4: Degradation potentiality and mechanism study.

Phthalic acid esters (PAEs) are human made chemicals widely used as plasticizers to enhance the flexibility of plastic products. Due to the lack of chemical bonding between phthalates and plastics, these materials can easily enter the environment. Deleterious effects caused by this chemo-pollutant have drawn the attention of the scientific community to remediate them from different ecosystem. In this context, many bacterial strains have been reported across different habitats and Sphingobium yanoikuyae strain P4 is among the few psychrotolerant bacterial species reported to biodegrade simple and complex phthalates. In the present study, biodegradation of three structurally different PAEs viz., diethyl phthalate (DEP), di-isobutyl phthalate (DIBP), and butyl benzyl phthalate (BBP) have been investigated by the strain P4. Quantitative analyses through High-performance liquid chromatography (HPLC) revealed that the bacterium completely degraded 1 g/L of DEP, DIBP, and BBP supplemented individually in minimal media pH 7.0 within 72, 54, and 120 h of incubation, respectively, at 28 °C and under shake culture condition (180 rpm). In addition, the strain could grow in minimal media supplemented individually with up to 3 g/L of DEP and 10.0 g/L of DIBP and BBP at 28 °C and pH 7.0. The strain also could grow in metabolites resulting from biodegradation of DEP, DIBP, and BBP, viz. n-butanol, isobutanol, butyric acid, ethanol, benzyl alcohol, benzoic acid, phthalic acid, and protocatechuic acid. Furthermore, phthalic acid and protocatechuic acid were also detected as degradation pathway metabolites of DEP and DIBP by HPLC, which gave an initial idea about the biodegradation pathway(s) of these phthalates.

Exploring the Efficacy of Hydroxybenzoic Acid Derivatives in Mitigating Jellyfish Toxin-Induced Skin Damage: Insights into Protective and Reparative Mechanisms.

The escalation of jellyfish stings has drawn attention to severe skin reactions, underscoring the necessity for novel treatments. This investigation assesses the potential of hydroxybenzoic acid derivatives, specifically protocatechuic acid (PCA) and gentisic acid (DHB), for alleviating Nemopilema nomurai Nematocyst Venom (NnNV)-induced injuries. By employing an in vivo mouse model, the study delves into the therapeutic efficacy of these compounds. Through a combination of ELISA and Western blot analyses, histological examinations, and molecular assays, the study scrutinizes the inflammatory response, assesses skin damage and repair mechanisms, and investigates the compounds' ability to counteract venom effects. Our findings indicate that PCA and DHB significantly mitigate inflammation by modulating critical cytokines and pathways, altering collagen ratios through topical application, and enhancing VEGF and bFGF levels. Furthermore, both compounds demonstrate potential in neutralizing NnNV toxicity by inhibiting metalloproteinases and phospholipase-A2, showcasing the viability of small-molecule compounds in managing toxin-induced injuries.

Elucidation of 4-Hydroxybenzoic Acid Catabolic Pathways in Pseudarthrobacter phenanthrenivorans Sphe3.

4-hydroxybenzoic acid (4-HBA) is an aromatic compound with high chemical stability, being extensively used in food, pharmaceutical and cosmetic industries and therefore widely distributed in various environments. Bioremediation constitutes the most sustainable approach for the removal of 4-hydroxybenzoate and its derivatives (parabens) from polluted environments. Pseudarthrobacter phenanthrenivorans Sphe3, a strain capable of degrading several aromatic compounds, is able to grow on 4-HBA as the sole carbon and energy source. Here, an attempt is made to clarify the catabolic pathways that are involved in the biodegradation of 4-hydroxybenzoate by Sphe3, applying a metabolomic and transcriptomic analysis of cells grown on 4-HBA. It seems that in Sphe3, 4-hydroxybenzoate is hydroxylated to form protocatechuate, which subsequently is either cleaved in ortho- and/or meta-positions or decarboxylated to form catechol. Protocatechuate and catechol are funneled into the TCA cycle following either the ß-ketoadipate or protocatechuate meta-cleavage branches. Our results also suggest the involvement of the oxidative decarboxylation of the protocatechuate peripheral pathway to form hydroxyquinol. As a conclusion, P. phenanthrenivorans Sphe3 seems to be a rather versatile strain considering the 4-hydroxybenzoate biodegradation, as it has the advantage to carry it out effectively following different catabolic pathways concurrently.

The Cancer-Protective Potential of Protocatechuic Acid: A Narrative Review.

Cancer is one of the leading causes of death worldwide, making the search for alternatives for its control a critical issue. In this context, exploring alternatives from natural sources, such as certain vegetables containing a variety of secondary metabolites with beneficial effects on the body and that play a crucial role in the fight against cancer, is essential. Among the compounds with the greatest efficacy in controlling this disease, those with antioxidant activity, particularly phenolic com-pounds, stand out. A remarkable example of this group is protocatechuic acid (PCA), which has been the subject of various revealing research on its activities in different areas. These studies sustain that protocatechuic acid has anti-inflammatory, antimutagenic, antidiabetic, antiulcer, antiviral, antifibrogenic, antiallergic, neuroprotective, antibacterial, anticancer, antiosteoporotic, anti-aging, and analgesic properties, in addition to offering protection against metabolic syndrome and con-tributing to the preservation of hepatic, renal, and reproductive functionality. Therefore, this paper aims to review the biological activities of PCA, focusing on its anticancer potential and its in-volvement in the control of various molecular pathways involved in tumor development, sup-porting its option as a promising alternative for cancer treatment.

Plant-Derived Phenolic Acids Limit the Pathogenesis of Salmonella Typhimurium and Protect Intestinal Epithelial Cells during Their Interactions.

The incidence of gastrointestinal illness attributable to Salmonella enterica serovar Typhimurium (ST) remains a concern for public health worldwide, as it can progress into systemic infections mediated by the type-three secretion system (T3SS), which allows for adherence and invasion to intestinal epithelial cells. The current study evaluates the ability of gallic acid (GA), protocatechuic acid (PA), and vanillic acid (VA) to impair the adhesion and invasion abilities of ST to a human epithelial (INT-407) cell monolayer while also assessing their cytotoxicity. GA, PA, and VA inhibited detectable ST growth at specific concentrations but showed cytotoxicity against INT-407 cells (>20% reduction in viability) after 3 h of treatments. Adjusting the pH of the solutions had a neutralizing effect on cytotoxicity, though it did reduce their antimicrobial potency. Adhesion of ST was reduced significantly when the cells were treated with 4.0 mg/mL of VA, whereas invasion was reduced in all treatments, with GA requiring the lowest concentration (0.5 mg/mL). Relative gene expression of virulence genes after treatment with GA showed downregulation in the T3SS regulator and effector hilA and sipA, respectively. These findings suggest further use of phenolic acids in reducing the activity of key virulence factors critical during ST infection.

Biological Valorization of Lignin-Derived Aromatics in Hydrolysate to Protocatechuic Acid by Engineered Pseudomonas putida KT2440.

Alongside fermentable sugars, weak acids, and furan derivatives, lignocellulosic hydrolysates contain non-negligible amounts of lignin-derived aromatic compounds. The biological funnel of lignin offers a new strategy for the "natural" production of protocatechuic acid (PCA). Herein, Pseudomonas putida KT2440 was engineered to produce PCA from lignin-derived monomers in hydrolysates by knocking out protocatechuate 3,4-dioxygenase and overexpressing vanillate-O-demethylase endogenously, while acetic acid was used for cell growth. The sugar catabolism was further blocked to prevent the loss of fermentable sugar. Using the engineered strain, a total of 253.88 mg/L of PCA was obtained with a yield of 70.85% from corncob hydrolysate 1. The highest titer of 433.72 mg/L of PCA was achieved using corncob hydrolysate 2 without any additional nutrients. This study highlights the potential ability of engineered strains to address the challenges of PCA production from lignocellulosic hydrolysate, providing novel insights into the utilization of hydrolysates.

Protocatechuic acid prevents isoproterenol-induced heart failure in mice by downregulating kynurenine-3-monooxygenase.

Protocatechuic acid (3,4-dihydroxybenzoic acid) prevents oxidative stress, inflammation and cardiac hypertrophy. This study aimed to investigate the therapeutic effects of protocatechuic acid in an isoproterenol-induced heart failure mouse model and to identify the underlying mechanisms. To establish the heart failure model, C57BL/6NTac mice were given high-dose isoproterenol (80 mg/kg body weight) for 14 days. Echocardiography revealed that protocatechuic acid reversed the isoproterenol-induced downregulation of fractional shortening and ejection fraction. Protocatechuic acid attenuated cardiac hypertrophy as evidenced by the decreased heart-weight-to-body-weight ratio and the expression of Nppb. RNA sequencing analysis identified kynurenine-3-monooxygenase (Kmo) as a potential target of protocatechuic acid. Protocatechuic acid treatment or transfection with short-interfering RNA against Kmo ameliorated transforming growth factor ß1-induced upregulation of Kmo, Col1a1, Col1a2 and Fn1 in vivo or in neonatal rat cardiac fibroblasts. Kmo knockdown attenuated the isoproterenol-induced increase in cardiomyocyte size, as well as Nppb and Col1a1 expression in H9c2 cells or primary neonatal rat cardiomyocytes. Moreover, protocatechuic acid attenuated Kmo overexpression-induced increases in Nppb mRNA levels. Protocatechuic acid or Kmo knockdown decreased isoproterenol-induced ROS generation in vivo and in vitro. Thus, protocatechuic acid prevents heart failure by downregulating Kmo. Therefore, protocatechuic acid and Kmo constitute a potential novel therapeutic agent and target, respectively, against heart failure.

Protocatechuic acid inhibits LPS-induced mastitis in mice through activating the pregnane X receptor.

Mastitis refers to the inflammation in the mammary gland caused by various reasons. Protocatechuic acid (PCA) exerts anti-inflammatory effect. However, no studies have shown the protective role of PCA on mastitis. We investigated the protective effect of PCA on LPS-induced mastitis in mice and elucidated its possible mechanism. LPS-induced mastitis model was established by injection of LPS into the mammary gland. The pathology of mammary gland, MPO activity and inflammatory cytokine production were detected to evaluate the effects of PCA on mastitis. In vivo, PCA significantly attenuated LPS-induced mammary pathological changes, MPO activity, TNF-α and IL-1ß production. In vitro, the production of inflammatory cytokines TNF-α and IL-1ß was significantly reduced by PCA. Furthermore, LPS-induced NF-κB activation was also inhibited by PCA. In addition, PCA was found to activate pregnane X receptor (PXR) transactivation and PCA dose-dependently increased the expression of PXR downstream molecule CYP3A4. In addition, the inhibitory effect of PCA on inflammatory cytokine production was also reversed when PXR was knocked down. In conclusion, the protective effects of PCA on LPS-induced mastitis in mice through regulating PXR.

Protocatechuic Acid and Syringin from Saussurea neoserrata Nakai Attenuate Prostaglandin Production in Human Keratinocytes Exposed to Airborne Particulate Matter.

Saussurea neoserrata Nakai offers a reliable and efficient source of antioxidants that can help alleviate adverse skin reactions triggered by air pollutants. Air pollutants, such as particulate matter (PM), have the ability to infiltrate the skin and contribute to the higher occurrence of cardiovascular, cerebrovascular, and respiratory ailments. Individuals with compromised skin barriers are particularly susceptible to the impact of PM since it can be absorbed more readily through the skin. This study investigated the impact of protocatechuic acid and syringin, obtained from the n-BuOH extract of S. neoserrata Nakai, on the release of PGE2 and PGD2 induced by PM10. Additionally, it examined the gene expression of the synthesis of PGE2 and PGD2 in human keratinocytes. The findings of this research highlight the potential of utilizing safe and efficient plant-derived antioxidants in dermatological and cosmetic applications to mitigate the negative skin reactions caused by exposure to air pollution.

The Skin-Whitening and Antioxidant Effects of Protocatechuic Acid (PCA) Derivatives in Melanoma and Fibroblast Cell Lines.

The skin is the most voluminous organ of the human body and is exposed to the outer environment. Such exposed skin suffers from the effects of various intrinsic and extrinsic aging factors. Skin aging is characterized by features such as wrinkling, loss of elasticity, and skin pigmentation. Skin pigmentation occurs in skin aging and is caused by hyper-melanogenesis and oxidative stress. Protocatechuic acid (PCA) is a natural secondary metabolite from a plant-based source widely used as a cosmetic ingredient. We chemically designed and synthesized PCA derivatives conjugated with alkyl esters to develop effective chemicals that have skin-whitening and antioxidant effects and enhance the pharmacological activities of PCA. We identified that melanin biosynthesis in B16 melanoma cells treated with alpha-melanocyte-stimulating hormone (α-MSH) is decreased by PCA derivatives. We also found that PCA derivatives effectively have antioxidant effects in HS68 fibroblast cells. In this study, we suggest that our PCA derivatives are potent ingredients for developing cosmetics with skin-whitening and antioxidant effects.

Protocatechuic acid reverses myocardial infarction mediated by ß-adrenergic agonist via regulation of Nrf2/HO-1 pathway, inflammatory, apoptotic, and fibrotic events.

Myocardial infarction (MI) is an instant ischemic death of cardiomyocytes that remains a major global cause of mortalities. MI is accompanied by oxidative, inflammatory, apoptotic, and fibrotic insults. Protocatechuic acid (PCA) is a polyphenolic compound with various potent biological activities. In this study, we explored the possible cardioprotective role of PCA against isoproterenol (ISO)-mediated MI. Rats were either injected with ISO (85 mg/kg, subcutaneously) or pretreated with PCA (100 or 200 mg/kg, orally). PCA supplementation markedly normalized ISO-induced disturbed cardiac function markers (creatine kinase-MB, lactate dehydrogenase, and troponin T). Notably, PCA administration exerted remarkable increases in glutathione and its derived enzymes, superoxide dismutase, and catalase, as well as decreases in malondialdehyde and nitric oxide levels in the injured cardiac tissue. The molecular findings validated the augmented cellular antioxidative capacity by PCA via increasing the gene expressions of nuclear factor erythroid 2-related factor 2 and heme oxygenase-1. The cardioprotective efficacy of PCA extended to suppress cardiac inflammation as demonstrated by the decreased levels of tumor necrosis factor-alpha, interleukin-1 beta, and nuclear factor kappa B. Additionally, PCA prevented cardiomyocyte loss and fibrosis by decreasing Bax, caspase-3, transforming growth factor-ß1 and matrix metalloproteinase-9, and enhancing B-cell lymphoma 2 and tissue inhibitors of metalloproteinase-3. The cardiac histological screening further confirmed the PCA's protective action. The obtained data recommend PCA as an alternative therapeutic agent to attenuate the molecular, biochemical, and histological alterations associated with MI development.

Copper-inducible expression system for metabolic engineering of Escherichia coli.

AIMS: The inducible expression system plays an important role in engineering Escherichia coli for chemical production. However, it still heavily relies on expensive chemical inducers, like IPTG. There is a pressing need to develop alternative expression systems with more affordable inducers. MATERIALS AND RESULTS: We herein report a copper-inducible expression system in E. coli based on the two-component Cus system and T7 RNA polymerase (RNAP). By integrating the gene encoding T7 RNAP at the CusC locus, we managed to program eGFP expression under the T7 promoter in response to different concentrations of Cu2+ (0-20 µM). Subsequently, we demonstrated that the copper-inducible expression system was suitable for the metabolic engineering of E. coli toward protocatechuic acid overproduction, and the resulting strain with combined manipulation of the central metabolism via CRISPRi produced 4.12 g L-1 PCA under the optimal copper concentration and induction time. CONCLUSIONS: We have established a copper-inducible T7 RNAP expression system in E. coli. The copper-inducible expression system could rationally control metabolic pathways in a temporal and dose-dependent manner. The gradient expression system based on copper inducer could be widely used in E. coli cell factories, and the design principle reported here would also be applicable in other prokaryotes.

Protocatechuic acid alleviates polycystic ovary syndrome symptoms in mice by PI3K signaling in granulosa cells to relieve ROS pressure and apoptosis.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a complicated gynecological endocrine disease that occurs in women of childbearing age. Protocatechuic acid is a phenol-rich compound derived from herbs and owns vital functions in numerous diseases. Howbeit, protocatechuic acid's impact on PCOS is unknown. METHODS: A combination of in vivo and in vitro models was examined in this study. C57BL/6 mice were injected subcutaneously daily with dehydroepiandrosterone to establish a PCOS mouse model, and protocatechuic acid was intraperitoneally injected into PCOS mice. Granulosa cells of PCOS ovaries were also isolated. The function of protocatechuic acid was appraised using enzyme-linked immunosorbent assay, hematoxylin-eosin staining, reactive oxygen species (ROS) and LC3 levels analysis, flow cytometry, quantitative real-time PCR, and western blot. Meanwhile, the mechanism of protocatechuic acid was assessed with a series of molecular experiments. RESULTS: Protocatechuic acid owned no apparent toxic effect on mice. Functionally, protocatechuic acid owned a function of mitigating PCOS in vivo. Meanwhile, protocatechuic acid repressed ROS, autophagy, and apoptosis of PCOS ovarian granulosa cells in vitro. Mechanistically, rescue assays elucidated that the protective function of protocatechuic acid against PCOS was interrelated to the activation of the PI3K/AKT/mTOR axis. CONCLUSION: Protocatechuic acid alleviated PCOS symptoms in mice through PI3K signaling in granulosa cells to reduce ROS levels and apoptosis.

Protocatechuic acid ameliorates lipopolysaccharide-induced kidney damage in mice via downregulation of TLR-4-mediated IKBKB/NF-κB and MAPK/Erk signaling pathways.

Acute kidney injury (AKI) is a very critical cause of death in the whole world. Lipopolysaccharide (LPS) induces kidney damage by activating various deleterious inflammatory and oxidative pathways. Protocatechuic acid, a natural phenolic compound, has shown to exert beneficial effects against oxidative and inflammatory responses. The study aimed to clarify the nephroprotective activity of protocatechuic acid in LPS-induced acute kidney damage in mice. Forty male Swiss mice were allocated in four groups as follows: normal control group; LPS (250 µg/kg, ip)-induced kidney injury group; LPS-injected mice treated with protocatechuic acid (15 mg/kg, po), and LPS-injected mice treated with protocatechuic acid (30 mg/kg, po). Significant toll-like receptor 4 (TLR-4)-mediated activation of IKBKB/NF-κB and MAPK/Erk/COX-2 inflammatory pathways has been observed in kidneys of mice treated with LPS. Oxidative stress was revealed by inhibition of total antioxidant capacity, catalase, nuclear factor erythroid 2-related factor 2 (Nrf2), and NAD(P)H quinone oxidoreductase (NQO1) enzyme along with increased nitric oxide level. In parallel, focal inflammatory effects were shown in between the tubules and glomeruli as well as in the perivascular dilated blood vessels at the cortex affecting the normal morphology of the kidney tissues of LPS-treated mice. However, treatment with protocatechuic acid reduced LPS-induced changes in the aforementioned parameters and restored normal histological features of the affected tissues. In conclusion, our study uncovered that protocatechuic acid has nephroprotective effects in mice with AKI through opposing different inflammatory and oxidative cascades.

Protocatechuic acid reduces H2O2-induced migration and oxidative stress of fibroblast-like synoviocytes in rheumatoid arthritis by activating Nrf2-Keap1 signaling pathway.

Honeycomb (Nidus vespae) is traditional Chinese medicine and can treat rheumatoid arthritis (RA), and protocatechuic acid (PCA) is a bioactive component of honeycomb. This study aimed to investigate whether PCA could reduce the H2O2-induced migration and oxidative stress of RA fibroblast-like synoviocytes (RA-FLSs). H2O2-induced RA-FLSs were used to simulate the in vitro model of RA. The viability, apoptosis, migration, invasion, and oxidative stress of RA-FLSs were detected by Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, wound healing, transwell assays, DCFDA staining, and malonaldehyde and superoxide dismutase enzyme-linked immunosorbent assay kits. The expression of migration and invasion-related proteins and Nrf2/Keap1 signaling pathway-related proteins was analyzed by western blotting. As a result, PCA suppressed the viability, migration, invasion, and oxidative and promoted apoptosis of H2O2-induced RA-FLSs by activating the Nrf2/Keap1 signaling pathway. ML-385, an Nrf2 inhibitor, could enhance the viability, migration, invasion, and oxidative and inhibited apoptosis of H2O2-induced RA-FLSs. In conclusion, PCA reduced H2O2-induced migration and oxidative stress of RA-FLSs by activating the Nrf2-Keap1 signaling pathway.

The Muscle-Conditioned Medium Containing Protocatechuic Acid Improves Insulin Resistance by Modulating Muscle Communication with Liver and Adipose Tissue.

Diabetes mellitus is a public health concern, affecting 10.5% of the population. Protocatechuic acid (PCA), a polyphenol, exerts beneficial effects on insulin resistance and diabetes. This study investigated the role of PCA in improving insulin resistance and the crosstalk between muscle with liver and adipose tissue. C2C12 myotubes received four treatments: Control, PCA, insulin resistance (IR), and IR-PCA. Conditioned media from C2C12 was used to incubate HepG2 and 3T3-L1 adipocytes. The impact of PCA was analyzed on glucose uptake and signaling pathways. PCA (80 µM) significantly enhanced glucose uptake in C2C12, HepG2, and 3T3-L1 adipocytes (p < 0.05). In C2C12, PCA significantly elevated GLUT-4, IRS-1, IRS-2, PPAR-γ, P-AMPK, and P-Akt vs. Control (p ≤ 0.05), and modulated pathways in IR-PCA. In HepG2, PPAR-γ and P-Akt increased significantly in Control (CM) vs. No CM, and PCA dose upregulated PPAR-γ, P-AMPK, and P-AKT (p < 0.05). In the 3T3-L1 adipocytes, PI3K and GLUT-4 expression was elevated in PCA (CM) vs. No CM. A significant elevation of IRS-1, GLUT-4, and P-AMPK was observed in IR-PCA vs. IR (p ≤ 0.001). Herein, PCA strengthens insulin signaling by activating key proteins of that pathway and regulating glucose uptake. Further, conditioned media modulated crosstalk between muscle with liver and adipose tissue, thus regulating glucose metabolism.

Impact of protocatechuic acid on alleviation of pulmonary damage induced by cyclophosphamide targeting peroxisome proliferator activator receptor, silent information regulator type-1, and fork head box protein in rats.

Cyclophosphamide (CP) is a chemotherapeutic agent that causes pulmonary damage by generating free radicals and pro-inflammatory cytokines. Pulmonary damage has a high mortality rate due to the severe inflammation and edema occurred in lung. PPARγ/Sirt 1 signaling has been shown to be cytoprotective effect against cellular inflammatory stress and oxidative injury. Protocatechuic acid (PCA) is a potent Sirt1 activator and exhibits antioxidant as well as anti-inflammatory properties. The current study aims to investigate the therapeutic impacts of PCA against CP-induced pulmonary damage in rats. Rats were assigned randomly into 4 experimental groups. The control group was injected with a single i.p injection of saline. CP group was injected with a single i.p injection of CP (200 mg/kg). PCA groups were administered orally with PCA (50 and 100 mg/kg; p.o.) once daily for 10 consecutive days after CP injection. PCA treatment resulted in a significant decrease in the protein levels of MDA, a marker of lipid peroxidation, NO and MPO along with a significant increase in GSH and catalase protein levels. Moreover, PCA downregulated anti-inflammatory markers as IL-17, NF-κB, IKBKB, COX-2, TNF-α, and PKC and upregulated cytoprotective defenses as PPARγ, and SIRT1. In addition, PCA administration ameliorated FoxO-1 elevation, increased Nrf2 gene expression, and reduced air alveoli emphysema, bronchiolar epithelium hyperplasia and inflammatory cell infiltration induced by CP. PCA might represent a promising adjuvant to prevent pulmonary damage in patients receiving CP due to its antioxidant and anti-inflammatory effects with cytoprotective defenses.