Synergistic effect of Aloe vera flower and Aloe gel on cutaneous wound healing targeting MFAP4 and its associated signaling pathway: In-vitro study.

ETHNOPHARMACOLOGICAL RELEVANCE: Aloe vera (L.) Burm. f. (Liliaceae family) is a well-known traditional medicinal plant, that has been used to treat a variety of illnesses, for decades ranging from cancer to skin disorders including wounds. It has been included in the traditional and herbal healthcare systems of many cultures around the world, as well as the pharmacopeia of different countries. Several in vitro and in vivo studies have also confirmed its potential antioxidant, anti-inflammatory, and wound-healing activities, etc. in the consistency of its historical and traditional uses. However, most studies to date are based on the A. vera gel and latex including its wound-healing effects. Very few studies have been focused on its flower, and rarely with its effects on cutaneous wound healing and its molecular mechanism. AIM OF THE STUDY: To the best of our knowledge, this is the first study to report on the synergistic effect of the A. vera flower (AVF) and Aloe gel (PAG) on cutaneous wound-healing, as well as revealing its molecular mechanism targeting microfibril-associated glycoprotein 4 (MFAP4) and its associated signaling pathway. METHODS: To investigate the synergistic effect of A. vera flower and Aloe gel in cutaneous wound healing, cell viability, and cell migration, as well proliferation assay was performed. This was followed by quantitative real-time polymerase chain reaction and Western blot analyses in wounded conditions to check the effects of this mixture on protein and mRNA levels in normal human dermal fibroblast (NHDF) cells. Moreover, small interfering RNA (siRNA) -mediated knockdown of MFAP4 in NHDF cells was performed followed by migration assay and cell cycle analysis, to confirm its role in cutaneous wound healing. Additionally, HaCaT cells were included in this study to evaluate its migratory and anti-inflammatory effects. RESULTS: Based on our obtained results, the PAG and AVF mixture synergistically induced the proliferation, migration, and especially ECM formation of NHDF cells by enhancing the expression of MFAP4. Other extracellular components associated with MFAP4 signaling pathway, such as fibrillin, collagen, elastin, TGF ß, and α-SMA, also increased at both the protein and mRNA levels. Subsequently, this mixture initiated the phosphorylation of the extracellular signal-regulated kinase (ERK) and AKT signaling pathways, and the S-phase of the cell cycle was also slightly modified. Also, the mixture induced the migration of HaCaT cells along with the suppression of inflammatory cytokines. Moreover, the siRNA-mediated knockdown highlighted the crucial role of MFAP4 in cutaneous wound healing in NHDF cells. CONCLUSION: This study showed that the mixture of PAG and AVF has significant wound healing effects targeting MFAP4 and its associated signaling pathway. Additionally, MFAP4 was recognized as a new potential biomarker of wound healing, which can be confirmed by further in vivo studies.

The Role of Processed Aloe vera Gel in Intestinal Tight Junction: An In Vivo and In Vitro Study.

Leaky gut is a condition of increased paracellular permeability of the intestine due to compromised tight junction barriers. In recent years, this affliction has drawn the attention of scientists from different fields, as a myriad of studies prosecuted it to be the silent culprit of various immune diseases. Due to various controversies surrounding its culpability in the clinic, approaches to leaky gut are restricted in maintaining a healthy lifestyle, avoiding irritating factors, and practicing alternative medicine, including the consumption of supplements. In the current study, we investigate the tight junction-modulating effects of processed Aloe vera gel (PAG), comprising 5-400-kD polysaccharides as the main components. Our results show that oral treatment of 143 mg/kg PAG daily for 10 days improves the age-related leaky gut condition in old mice, by reducing their individual urinal lactulose/mannitol (L/M) ratio. In concordance with in vivo experiments, PAG treatment at dose 400 µg/mL accelerated the polarization process of Caco-2 monolayers. The underlying mechanism was attributed to enhancement in the expression of intestinal tight junction-associated scaffold protein zonula occludens (ZO)-1 at the translation level. This was induced by activation of the MAPK/ERK signaling pathway, which inhibits the translation repressor 4E-BP1. In conclusion, we propose that consuming PAG as a complementary food has the potential to benefit high-risk patients.

Establishment of particulate matter-induced lung injury model in mouse.

BACKGROUND: Particulate matter (PM) is one of the principal causes of human respiratory disabilities resulting from air pollution. Animal models have been applied to discover preventive and therapeutic drugs for lung diseases caused by PM. However, the induced severity of lung injury in animal models using PM varies from study to study due to disparities in the preparation of PM, and the route and number of PM administrations. In this study, we established an in vivo model to evaluate PM-induced lung injury in mice. RESULTS: PM dispersion was prepared using SRM2975. Reactive oxygen species were increased in MLE 12 cells exposed to this PM dispersion. In vivo studies were conducted in the PM single challenge model, PM multiple challenge model, and PM challenge with ovalbumin-induced asthma using the PM dispersion. No histopathological changes were observed in lung tissues after a single injection of PM, whereas mild to moderate lung inflammation was obtained in the lungs of mice exposed to PM three times. However, fibrotic changes were barely seen, even though transmission electron microscopy (TEM) studies revealed the presence of PM particles in the alveolar macrophages and alveolar capillaries. In the OVA-PM model, peribronchial inflammation and mucous hypersecretion were more severe in the OVA+PM group than the OVA group. Serum IgE levels tended to increase in OVA+PM group than in OVA group. CONCLUSIONS: In this study, we established a PM-induced lung injury model to examine the lung damage induced by PM. Based on our results, repeated exposures of PM are necessary to induce lung inflammation by PM alone. PM challenge, in the presence of underlying diseases such as asthma, can also be an appropriate model for studying the health effect of PM.

Anti-Inflammatory and Anti-Vascular Leakage Effects by Combination of Centella asiatica and Vitis vinifera L. Leaf Extracts.

Venous insufficiency results from several factors responsible for the progression of inflammation and oxidative damage of veins. Recently, natural extracts have been proposed for the treatment of venous insufficiency, but their efficacies have not been fully elucidated. In the present study, we evaluate the combinatorial effects on anti-inflammatory and anti-vascular leakage potential of mixed compositions containing different proportions of Centella asiatica extract (CE) and Vitis vinifera L. leaf extract (VVE) using an inflammation model of lipopolysaccharide- (LPS-) stimulated RAW264.7 cells and various vascular permeability models in mice (acetic-acid-induced peritoneal vascular model, mustard-oil-stimulated ear vascular model, and carrageenan-induced paw edema model). Pretreatment of CE and VVE in a 1 : 3 combination dose dependently inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) and mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) through downregulation of the nuclear factor-κB (NF-κB) pathway in LPS-stimulated RAW264.7 macrophages. In vascular permeability-related mouse models, pretreatment with the CE-VVE 1 : 3 combination significantly reduced the permeability of peritoneal or ear veins caused by acetic acid and mustard oil, respectively. Furthermore, pretreatment of the CE-VVE 1 : 3 combination ameliorated inflammation and edema of the hind paw caused by carrageenan injection. Thus, the combination of CE and VVE showed significant anti-inflammatory qualities and anti-vascular leakage effects. These findings indicate that an optimal combination of CE and VVE may have a more synergistic effect than that of CE or VVE alone as a putative agent against vascular incompetence.

Effects of Aloe vera Flower Extract and Its Active Constituent Isoorientin on Skin Moisturization via Regulating Involucrin Expression: In Vitro and Molecular Docking Studies.

Skin moisturization is very crucial for maintaining the flexibility, viscoelasticity, and differentiation of the epidermis and its deprivation causes several diseases from dry skin to dermatitis. Aloe vera, a miracle plant having diverse medicinal properties including skin moisturization effects. This study investigated for the first time the molecular mechanism targeting skin moisturization effects of the Aloe vera flower and its major active constituent. By treating human epidermal keratinocytes (HaCaT cells) with Aloe vera flower water extract (AFWE), we found that AFWE upregulated epidermal involucrin by activating the expression of protein kinase C, p38, and ERK 1/2. Additionally, it modulated filaggrin, increased aquaporin expression, and hyaluronan synthesis via a balanced regulation of HAS1 and HYAL1 protein. Similarly, it was able to protect UVB-induced photodamage. Western blot analysis, ELISA, and qRT- PCR were performed to evaluate various epidermal differentiation markers and moisturization-related factors on human epidermal keratinocytes (HaCaT cells). TLC and HPLC were used to detect and analyze the chemical constituents. Among them, we found that an active component of Aloe vera flower, isoorientin (IO) has a high binding affinity to all of its targeted proteins such as involucrin, PKC, P38, etc. through molecular docking assay. This study indicated that the Aloe vera flower and its active constituent, IO can be used as a prominent ingredient to enhance skin barrier function and improve its related pathologies.

Adjuvanticity of Processed Aloe vera gel for Influenza Vaccination in Mice.

The effectiveness of current influenza vaccines is considered suboptimal, and 1 way to improve the vaccines is using adjuvants. However, the current pool of adjuvants used in influenza vaccination is limited due to safety concerns. Aloe vera, or aloe, has been shown to have immunomodulatory functions and to be safe for oral intake. In this study, we explored the potential of orally administered processed Aloe vera gel (PAG) as an adjuvant for influenza vaccines in C57BL/6 mice. We first evaluated its adjuvanticity with a split-type pandemic H1N1 (pH1N1) Ag by subjecting the mice to lethal homologous influenza challenge. Oral PAG administration with the pH1N1 Ag increased survival rates in mice to levels similar to those of alum and MF59, which are currently used as adjuvants in influenza vaccine formulations. Similarly, oral PAG administration improved the survival of mice immunized with a commercial trivalent influenza vaccine against lethal homologous and heterologous virus challenge. PAG also increased hemagglutination inhibition and virus neutralization Ab titers against homologous and heterologous influenza strains following immunization with the split-type pH1N1 Ag or the commercial trivalent vaccine. Therefore, this study demonstrates that PAG may potentially be used as an adjuvant for influenza vaccines.

Combined Extract of Vitis vinifera L. and Centella asiatica Synergistically Attenuates Oxidative Damage Induced by Hydrogen Peroxide in Human Umbilical Vein Endothelial Cells.

Endothelial cell injury caused by oxidative stress is a critical factor in the initial stage of vascular diseases. Thus, identification of more effective antioxidants is a promising strategy to protect against endothelial cell injury. Recently, synergistic effects between phytochemicals have received renewed attention for their role in the treatment of various diseases. Vitis vinifera L. and Centella asiatica are well-known medicinal plants with various biological effects. However, the combination of the two has not previously been studied. Here, we investigated the effects of V. vinifera L. leaf and C. asiatica extract combination (VCEC), a standardized herbal blend comprising V. vinifera L. leaf extract (VE) and C. asiatica extract (CE), for its antioxidant activity and for the protection of endothelial cells against hydrogen peroxide (H2O2)-mediated oxidative damage in human umbilical vein endothelial cells (HUVECs). VCEC showed higher antioxidant activity than VE or CE in oxygen radical antioxidant capacity assays. In HUVECs, VCEC significantly suppressed increases in the production of intracellular reactive oxygen species, decreased levels of nitric oxide and vascular endothelial-cadherin, and increased endothelial hyperpermeability triggered by H2O2. Treatment with VE or CE alone ameliorated HUVEC injury in a pattern similar to VCEC, although their effects were significantly weaker than VCEC. Overall, VCEC exhibited a substantial synergistic effect on protecting endothelial cells against oxidative damage through its antioxidant activity. Therefore, VCEC could be developed as a potential agent for reducing the risk of vascular diseases related to oxidative stress.

Processed Aloe vera gel attenuates non-steroidal anti-inflammatory drug (NSAID)-induced small intestinal injury by enhancing mucin expression.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are well-known for exerting numerous adverse effects on the gastrointestinal tract such as bleeding, ulceration, and perforation, thereby limiting their use. Most previous studies have focused on NSAID-induced gastropathy. However, improved diagnostic techniques have recently highlighted NSAID-induced small intestinal ulcers, which have so far been underestimated. While proton pump inhibitors are prescribed to control NSAID-induced gastropathy, few preventive strategies are existent for NSAID-induced small intestinal injury, thus requiring new methods to treat these enteropathies. Numerous studies have reported the beneficial biological effects of Aloe vera, such as wound healing, anti-cancer, immune modulation, anti-oxidant, anti-microbial, and gastroprotective effects. A previous report on the effect of Aloe vera against NSAID-induced ulcers studied only gastric ulcers and elucidated the results as an anti-inflammatory effect of Aloe vera. However, ulcer prevention cannot be justified entirely to be due to the anti-inflammatory effects of Aloe vera, since NSAIDs themselves also exert an anti-inflammatory reaction. We therefore investigated the anti-ulcer effects of Aloe vera on the small intestine, especially focusing on mucin expression. Our results indicate that processed Aloe vera gel (PAG) treatment attenuates not only the severity of intestinal ulcers but also bacterial translocation, by enhancing the mucus layer in the indomethacin-induced small intestinal damage mouse model. We further confirmed that PAG positively regulates the mucin expression in the LS174T human cell line, mainly via the ERK-dependent pathway. We propose that PAG application is a potential strategy for the alleviation of NSAID-induced small intestinal ulcers.

Baicalein Inhibits Dextran Sulfate Sodium-induced Mouse Colitis.

BACKGROUND: Baicalein is a bioactive flavone that is originally extracted from the root of Scutellaria baicalensis Georgi. This plant has long served as Chinese herbal medicine in the management of multiple diseases including inflammatory bowel diseases. Although it has been revealed that baicalein inhibits experimental colitis in mice, the molecular mechanisms still remain largely unrecognized. METHODS: The experimental colitis was induced in mice by 3% dextran sulfate sodium (DSS) in drinking water. The mice were given baicalein (10 or 25 mg/kg) by gavage for 7 days before and after DSS administration. Expression of COX-2 and inducible nitric oxide synthase (iNOS) and molecules involved in NF-κB signaling, such as inhibitor of κBα (IκBα), pIκBα, p65, and phospho-p65 was examined by Western blot analysis in the tissue of the mouse colon. Activity of IκB kinase ß (IKKß) was assessed by measuring the relative amount of radioactive γ-phosphate of ATP transferred to the IκBα substrate protein. The expression and phosphorylation of STAT3 and its target gene cyclin D1 were also measured. RESULTS: Baicalein prominently mitigated the severity of DSS-induced colitis in mice. It inhibited the expression of COX-2 and iNOS. Moreover, baicalein attenuated activity and phosphorylation of IKKß and subsequent degradation of IκBα. Baicalein suppressed the phosphorylation and nuclear translocation of p65, resulting in a reduced DNA binding activity of NF-κB. Baicalein also suppressed the phosphorylation of STAT3 and expression of cyclin D1. Baicalein exhibited the synergistic effect on inhibition of COX-2 induced by DSS with curcumin, an ingredient of turmeric. CONCLUSIONS: Protective effects of baicalein on DSS-induced colitis are associated with suppression of NF-κB and STAT3 signaling pathways, which may contribute to its cancer preventive effects on colon carcinogenesis.

Dietary Aloe Improves Insulin Sensitivity via the Suppression of Obesity-induced Inflammation in Obese Mice.

BACKGROUND: Insulin resistance is an integral feature of metabolic syndromes, including obesity, hyperglycemia, and hyperlipidemia. In this study, we evaluated whether the aloe component could reduce obesity-induced inflammation and the occurrence of metabolic disorders such as blood glucose and insulin resistance. METHODS: Male C57BL/6 obese mice fed a high-fat diet for 54 days received a supplement of aloe formula (PAG, ALS, Aloe QDM, and Aloe QDM complex) or pioglitazone (PGZ) and were compared with unsupplemented controls (high-fat diet; HFD) or mice fed a regular diet (RD). RT-PCR and western blot analysis were used to quantify the expression of obesity-induced inflammation. RESULTS: Aloe QDM lowered fasting blood glucose and plasma insulin compared with HFD. Obesity-induced inflammatory cytokine (IL-1ß, -6, -12, TNF-α) and chemokine (CX3CL1, CCL5) mRNA and protein were decreased markedly, as was macrophage infiltration and hepatic triglycerides by Aloe QDM. At the same time, Aloe QDM decreased the mRNA and protein of PPARγ/LXRα and 11ß-HSD1 both in the liver and WAT. CONCLUSION: Dietary aloe formula reduces obesity-induced glucose tolerance not only by suppressing inflammatory responses but also by inducing anti-inflammatory cytokines in the WAT and liver, both of which are important peripheral tissues affecting insulin resistance. The effect of Aloe QDM complex in the WAT and liver are related to its dual action on PPARγ and 11ß-HSD1 expression and its use as a nutritional intervention against T2D and obesity-related inflammation is suggested.

Protective effect of a mixture of Aloe vera and Silybum marianum against carbon tetrachloride-induced acute hepatotoxicity and liver fibrosis.

The hepatoprotective effects of ACTIValoe N-931 complex, a mixture of Aloe vera and Silybum marianum, against acute and chronic carbon tetrachloride (CCl(4))-induced liver injuries were investigated. Acute hepatotoxicity was induced by intraperitoneal injection of CCl(4) (50 microl/kg), and ACTIValoe N-931 complex at 85, 170, and 340 mg/kg was administered orally 48, 24, and 2 h before and 6 h after injection of CCl(4). Hepatotoxicity was assessed 24 h after CCl(4) treatment. Liver fibrosis was induced by intraperitoneal injection of CCl(4) for 8 weeks (0.5 ml/kg, twice per week), and mice were treated with ACTIValoe N-931 complex at 85, 170, or 340 mg/kg once a day (p.o.). In both acute hepatotoxicity and liver fibrosis, serum aminotransferase levels and lipid peroxidation were increased and the hepatic glutathione content was decreased. These changes were prevented by ACTIValoe N-931 complex. The ACTIValoe N-931 complex attenuated the increase in tumor necrosis factor-alpha (TNF-alpha), and inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), mRNA expressions in acute hepatotoxicity. In antifibrotic experiments, tissue inhibitor of metalloprotease-1 (TIMP-1) mRNA expression was attenuated by treatment with ACTIValoe N-931 complex. The ACTIValoe N-931 complex decreased the hepatic hydroxyproline content and the transforming growth factor-beta1 levels. Our results suggest that the ACTIValoe N-931 complex has hepatoprotective effects in both acute and chronic liver injuries induced by CCl(4).

Identification of genes induced in wound-treated wild rice (Oryza minuta).

A subtracted library was constructed from wound-treated wild rice (Oryza minuta) by suppression subtractive hybridization (SSH) in combination with mirror orientation selection (MOS). To distinguish between differentially expressed transcripts and false positive clones, DNA chips containing 960 random clones were applied as a form of reverse Northern screening. Based on the signal intensities and expression ratios obtained from experiments performed in triplicate, 371 clones were selected. ESTs produced from the subtracted library showed 63.2% redundancy, and 72% of all clones could be matched to the GenBank nonredundant database. Functional categorization placed the identified enriched genes in categories of subcellular localization, metabolism, cell rescue and defense, and transcription. These EST-related resistance mechanisms could be used in investigations into the defense mechanisms of wild species, and to provide new routes to improving the germplasm of cultivated rice.

Construction of two pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites for direct cloning of PCR products.

For applications such as sequencing, transfection, and in vitro transcription, PCR products have to be subcloned into plasmids. Many strategies are used for cloning, blunt-end ligation or the incorporation of restriction endonuclease sites into PCR primers for appropriate vectors. However, the most convenient and direct method is T/A cloning. In this study, we developed two of the pGEM-7Zf(+) phagemid T-tail vectors using AhdI-restriction endonuclease sites, and these T vectors have all the features of pGEM-7Zf(+): f1 ori, T7, and SP6 RNA polymerase promoters, the alpha-peptide coding region of beta-galactosidase for X-gal blue/white color selection, the beta-lactamase gene for recombinant colony selection, and binding sites for pUC/M13 forward and reverse sequencing primers. These AhdI-containing phagemid vectors, pGEM-NJ105 and pGEM-NJ107, are useful for the easy and inexpensive preparation of T vectors and direct cloning of PCR products.