Antioxidants and related compounds are anti-inflammatory and exhibit great potential in promoting human health. They are also often considered to be important elements in the process of neurodegeneration. Here we describe a antioxidant blend of Curcumin and Broccoli Seed Extract (BSE). Flies treated with the blend exhibit extended lifespan. RNA-seq analysis of samples from adult fly brains reveals a wide array of new genes with differential expression upon treatment with the blend. Interestingly, abolishing expression of some of the identified genes in dopaminergic (DA) neurons does not affect DA neuron number. Taken together, our findings reveal an antioxidant blend that promotes fly longevity and exhibits protective effect over neurodegeneration, demonstrating the importance of antioxidants in health and pathology.

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase critically involved in the regulation of cellular energy homeostasis. Many studies have suggested that AMPK activation exert significant anti-inflammatory effects. In this study, we assessed the anti-inflammatory effect of AMPK activator from natural products, Broccoli seed (BS, Brassica oleracea var. italic) extract and Sweet tea (ST, Rubus Suavissmus S. Lee) extract, in RAW264.7 cells. AMPK assay Natural product samples treated purified AMPK enzyme (CycLex Co., Ltd. Japan) or cell lysates were added to the well of CycLex AMPK kinase assay kit, then performed. Cell Culture HepG2 and RAW264.7 cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with buffer. ELISA assay of IL-6 RAW264.7 cells were incubated with LPS, with or without presence of natural products, IL-6 in the supernatant was determined with Mouse IL-6 ELISA Kit. Western Blotting Cells were treated with LPS in the presence of or absence of natural products for 6 h, then lysed with buffer and a protease inhibitor. Proteins separated and blotted onto PVDF, blocked and probed with antibodies against caspase-1, finally incubated with the goat anti-rabbit horseradish-peroxidase coupled secondary antibody. The target proteins were analyzed using the ChemiDoc XRS System and quantified by ImageJ. Natural products BS and ST were identified as AMPK activators with the established AMPK activity assay model. (Fig. 1) Macrophages were stimulated with LPS, while the secretion of IL-6 was decreased in the presence of BS or ST, the protective effect observed of BS on the LPS-induced secretion of IL-6 was 0.0625–0.125 mg/mL, ST at 0.25–0.5 mg/mL. (Fig. 2) Furthermore, BS and ST suppressed the LPS-induced expression of caspase-1 gene in dose-dependent manner. (Fig. 3–4) Caspase-1 is a cysteine protease that converts the inactive form of IL-1β to the active inflammatory cytokine. Our preliminary study showed that AMPK activators, BS and ST, reduced LPS-induced inflammation in macrophages by combined inhibition of IL-6 secretion and block of inflammatory gene caspase-1 expression in RAW264.7 macrophage cell line. This result implicates anti-inflammatory effect of BS and ST, which may be induced by activation of AMPK and then regulated via NLRP3 pathway and caspase-1 expression. This study is supported by the research funding from Shanghai Anti-Aging Research Center, Nu Skin Enterprises, Shanghai, China.

Ethanol consumption is associated with a spectrum of diseases in the liver ranging from steatosis, alcoholic hepatitis to cirrhosis and hepatocellular carcinoma. We established an alcoholic liver disease model in mice to investigate the protective effects of the phytoextract blend (PB) including schisandra, grape seed and tart cherry. A total of 100 mice were randomly divided into 5 groups. The groups were: a) control group, b) ethanol group and c) three experimental groups. Animals in the control and ethanol groups were orally gavaged with distilled water, while the other groups were given varying concentrations of PB for 30 days. The ethanol group and the treatment groups were then given ethanol for 15 days. The liver/somatic index, levels of ALT, AST, TBIL and TG in serum, and the expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were measured. The liver tissues were stained with hematoxylineosin (H&E) for evaluation of histopathological changes. The liver/somatic index was increased in the ethanol group while it was decreased nearly to the normal value in treated groups. ALT increased significantly in ethanol group. PB reduced serum ALT concentrations in a dose-dependent manner. Ethanol elevated AST in the ethanol group. Although PB low dose group showed increased AST levels, AST was decreased by medium and high doses of PB compared with the ethanol group. Ethanol slightly though not significantly downregulated TBIL level in the ethanol group, while low and medium doses of PB increased TBIL. Ethanol-induced TG upregulation was completely reversed by PB treatment. The expression of hepatic IL-6 and TNF-α mRNA were elevated after ethanol consumption, whereas these inflammatory cytokines were reduced after treatment. Ethanol induced necrosis and substantial small fat droplets in the liver section. However, livers of mice in all treated groups showed noticeable recovery with fewer small fat droplets and hepatocyte necrosis. This study showed the PB containing schisandra, grape seed and tart cherry could protect liver by reducing hepatic steatosis and hepatocyte necrosis and alleviating inflammation. The PB used in this study may provide insights into the prevention and treatment of ethanol-induced liver injury. Nu Skin Research Funding.

Previous studies revealed that the combination of anthocyanins and prebiotics had protective effects on gut health. In this study, we observed the response of oral consumption of a blend of prebiotics and anthocyanins (hereafter called Balance +) on the gastrointestinal (GI) function of human volunteers. Skin status was also evaluated as a secondary indication. A total of 42 subjects (11 males and 31 females) with complaints of mild to moderate GI discomfort were recruited and provided a daily dose of 9.6 g/day of Balance + for 10 weeks. IBS-SSS and IBS-QOL questionnaires were used to evaluate GI function and quality of life of the subjects. An internally designed “Skin status questionnaire” was used to evaluate self-perception of skin conditions. Questionnaires were collected at the baseline, week-1, week-5, and week-10 after consumption. A total score of GI discomfort derived from the validated GI questionnaires revealed an improvement of GI issues compared with baseline after 5 and 10 weeks consumption of Balance + in most, but not all, symptoms. For example, satisfaction of bowel movement was improved significantly at week 1, 5, and 10; frequency of abdominal pain occurrence was decreased at week 5 and 10; abdominal symptoms affecting daily life were significantly reduced at week 5 and 10. While abdominal pain was generally improved, the improvement was not statistically significant. Moreover no changes were found in abdominal distension. The survey also revealed that quality of life was improved significantly after consumption of Balance + and subjects were less concerned about psychological reactions relevant to abdominal discomfort. Interestingly, according to our questionnaire, general skin condition was also positively impacted after Balance + consumption. Subjects were asked to self-evaluate 7 general skin attributes including dry skin, dark skin or dark patches, sensitive skin, acne, coarse pores, fine wrinkles, and couperose skin. The self evaluation of such general skin conditions revealed improvement after 10 weeks consumption of Balance +. No severe adverse effects were found except for some mild abdominal response, e.g., abdominal pain and distension in 7.1% and 9.5% of individuals studied respectively. Balance + consumption may help regulate human GI system, help improve the self-perception of quality of life as well as skin conditions that may be relevant to GI status. Nu Skin Research Funding.

Reishi (Ganoderma lucidum) is best‐known for its anti‐aging and medicinal activities. We previously reported that Reishimax (RM, a proprietary extract of Reishi, enriched in Reishi triterpenes (6.5%) and Reishi polysaccharides (13%)) has anti‐inflammatory, anti‐oxidant, and life‐extension effects in mice (FASEB Journal, 2012, 26(1):373.2). We further analyzed gene expression and epigenetic regulation of its anti‐aging effect in murine liver using a natural aging mouse model. In this study, naturally aged ICR male mice (22m of age) were randomized and treated either with RM 175mg/kg body weight or deionized distilled water control by gavage. After 13 weeks of treatment, liver samples were analyzed for gene expression, lncRNA(long‐non‐coding RNA) and DNA methylation. We also set up a young control group with male ICR mice (2m of age). IPA (Ingenuity Pathway Analysis) disease & function analysis showed RM treatment reversed or partially reversed abnormities in old vs. young organismal injury and abnormalities, small molecule biochemistry, lipid metabolism, and functions related to cancer. Pathway prediction showed opposing regulation of LPS/IL‐1‐ mediated inhibition of RXR Function and NRF2‐mediated oxidative stress response pathways. In the RM treatment group, LncRNA‐mRNA co‐analysis indicated that LncRNAs were involved in metabolic and biosynthesis pathways and may be affecting lipid metabolism and small molecular functions. It was also involved in the networks related to neurological disease, cardiovascular system development and function, developmental disorder and cellular function and maintenance. DNA methylation‐mRNA co‐analysis indicated that in the RM treated group, DNA methylations were involved in JAK2 hormone‐like cytokine signaling, systemic lupus erythematosus signaling, as well as NAD biosynthesis/salvage pathways and may affect immune functions as well as organismal injury and abnormalities and cellular development functions. Further study is needed to confirm the epigenetic regulations and to fully understand their mechanisms.

PURPOSE:To evaluate the effect of adeno-associated virus (AAV) vector-mediated viral (v)IL-10 gene expression on lacrimal gland (LG) immunopathology and ocular surface disease in a rabbit model of induced autoimmune dacryoadenitis (ID).METHODS:Autologous peripheral blood lymphocytes, activated in a mixed-cell reaction when cocultured with purified rabbit lacrimal epithelial cells, induce a Sjögren's-like autoimmune dacryoadenitis when injected directly back into the donor animal's inferior LG. Four weeks after disease induction, AAV vector expressing the vIL-10 gene under control of a tetracycline-inducible promoter was injected into the inferior LG of the treatment group (ID/Rx), and doxycycline was fed orally to induce transgene expression. The ID group serving as control also received doxycycline. All LGs were removed 16 weeks after disease induction.RESULTS:Clinical symptoms showed overall improvement in the ID/Rx group compared with the ID group. Histopathologic examination of the ID group's LG revealed scattered large lymphocytic foci and areas of altered or distorted acini, whereas the ID/Rx group had scattered small lymphocytic foci. The number of CD18(+) cells was almost fivefold lower in the ID/Rx group than in the ID group. Although the total number of RTLA(+) cells did not differ between the groups, the CD4/CD8 ratio was 16-fold smaller in the ID/Rx group.CONCLUSIONS:Animals with experimentally induced autoimmune dacryoadenitis appeared to benefit from AAV-mediated vIL-10 gene transfer therapy. Quantitative immunohistochemical analysis suggested that the therapy might not have been simply immunosuppressive but rather supported the induction of CD8(+) regulatory cells.

PURPOSE:To test whether CD4+ T cells proliferate in mixed cell reactions with autologous lacrimal gland (LG) acinar cells and whether these cells can autoadoptively transfer disease.METHODS:Purified acinar cells were gamma irradiated and cocultured with peripheral blood lymphocytes. Activated CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS). Unfractionated activated peripheral blood lymphocytes (UF), CD4+-enriched and CD4+-depleted T cells from an autologous mixed cell reaction were injected into the donor rabbit's remaining LG. After 4 weeks, ocular examinations were performed, and the rabbits were euthanized; LGs were removed for histopathology, immunohistochemistry, and real-time reverse transcription-polymerase chain reaction studies.RESULTS:CD4 T cells increased in the autologous mixed cell reaction from 20% to 80%. Tear production decreased in the induced disease/UF (ID/UF) group and declined even more in the ID/CD4+-enriched group. Tear breakup times decreased and rose bengal staining increased in all groups. All LGs exhibited significant histopathology and increased messenger RNAs for tumor necrosis factor α. The ID/UF group exhibited the largest increases of CD4+ and rabbit T-lymphocyte antigen-positive cells. The ID/CD4+-enriched group contained fewer infiltrating CD4 cells but more eosinophils, severely altered acinar morphology, and increased fibrosis. LG of the ID/CD4+-depleted group exhibited large increases of CD18, major histocompatibility complex II, and CD4+ cells. Messenger RNAs for interleukin 2, interleukin 4, and CD4+ increased in the ID/CD4+-enriched group compared with the CD4+-depleted group.CONCLUSIONS:Autoreactive CD4+ effector cells activated ex vivo and autoadoptively transferred, caused what seems to be a distinct dacryoadenitis. The CD4+-depleted cell fraction also contained pathogenic effector cells capable of inducing disease.

Purpose: To use a rabbit model of induced autoimmune dacryoadenitis to evaluate the efficacy of topical ophthalmic cyclosporine A (CsA). Methods: Autoimmune dacryoadenitis was induced by injecting autologous peripheral blood lymphocytes, which had been activated in a mixed cell reaction with acinar cells isolated from one inferior lacrimal gland (LG), back into the donor animal's remaining inferior LG. Schirmer's test, tear breakup time, and rose Bengal staining were assessed. Animals with established disease were treated topically with either CsA or Endura twice daily for 5 months. Results: Without treatment tear production and tear stability were abnormal for 6 months, and clear signs of ocular surface defects were evident. Severe immune cell infiltration was observed in the LG. Long-term CsA treatment increased tear production only slightly, but the severity of LG histopathology decreased noticeably. CD4+ T-cell infiltration of the LG was decreased and infiltration by MHC class II-expressing cells was also decreased. For the Endura-treated group tear production did not improve, rose Bengal scores remained high, and histopathology showed infiltration comparable to the untreated group, but by the end of the study the tear breakup time did improve. Conclusions: The rabbit model of autoimmune dacryoadenitis had signs of chronic dry eye disease 6 months after induction of disease. Tear production improved slightly with CsA treatment and CD4+ T-cell infiltration decreased significantly in the LG. This suggests that some Sjögren's patients may benefit from long-term CsA treatment.

In our quest to develop a tissue-engineered tear secretory system, we have tried to demonstrate active transepithelial ion fluxes across rabbit lacrimal acinar cell monolayers on polyester membrane scaffolds to evaluate the bioelectrical properties of the cultured cells. Purified lacrimal gland acinar cells were seeded onto polyester membrane inserts and cultured to confluency. Morphological properties of the cell monolayers were evaluated by transmission electron microscopy and immunofluorescence staining for Na(+),K(+)-ATPase and the tight junction-associated protein occludin. Sections revealed cell monolayers with well-maintained epithelial cell polarity, i.e., presence of apical (AP) secretory granules, microvilli, and junctional complexes. Na(+),K(+)-ATPase was localized on both the basal-lateral and apical plasma membranes. The presence of tight cell junctions was demonstrated by a positive circumferential stain for occludin. Bioelectrical properties of the cell monolayers were studied in Ussing chambers under short-circuit conditions. Active ion fluxes were evaluated by inhibiting the short-circuit current (I(sc)) with a Na(+),K(+)-ATPase inhibitor, ouabain (100 microM; basal-lateral, BL), and under Cl(-)-free buffer conditions after carbachol stimulation (CCh; 100 microM). The directional apical secretion of Cl(-) was demonstrated through pharmacological analysis, using amiloride (1 mM; BL) and bumetanide (0.1 mM; BL), respectively. Regulated protein secretion was evaluated by measuring the beta-hexosaminidase catalytic activity in the AP culture medium in response to 100 microM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl(-)-dependent, ouabain-sensitive AP --> BL I(sc) in response to CCh, consistent with current models for Na(+)-dependent Cl(-) secretion.

Gene delivery is one of the biggest challenges in the field of gene therapy. It involves the efficient transfer of transgenes into somatic cells for therapeutic purposes. A few major drawbacks in gene delivery include inefficient gene transfer and lack of sustained transgene expression. However, the classical method of using viral vectors for gene transfer has circumvented some of these issues. Several kinds of viruses, including retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus, have been manipulated for use in gene transfer and gene therapy applications. The transfer of genetic material into lacrimal epithelial cells and tissues, both in vitro and in vivo, has been critical for the study of tear secretory mechanisms and autoimmunity of the lacrimal gland. These studies will help in the development of therapeutic interventions for autoimmune disorders such as Sjögren's syndrome and dry eye syndromes which are associated with lacrimal dysfunction. These studies are also critical for future endeavors which utilize the lacrimal gland as a reservoir for the production of therapeutic factors which can be released in tears, providing treatment for diseases of the cornea and posterior segment. This review will discuss the developments related to gene delivery and gene therapy in the lacrimal gland using several viral vector systems.

Dry eye is a general term that refers to a myriad of ophthalmic disorders resulting in the inadequate wetting of the corneal surface by the tear film. Dry eyes are typically treated by the application of artificial tears. However, patients with lacrimal insufficiencies such as Stevens-Johnson syndrome, chemical and thermal injuries, or ocular cicatricial pemphigoid have very limited options because of the short duration and action of lubricating agents. As a therapeutic strategy, we are working to develop a bioengineered tear secretory system for such patients. This article describes the growth and physiological properties of purified rabbit lacrimal gland acinar cells (pLGACs) on several matrix protein-coated polymers such as silicone, collagen I, copolymers of poly-D,L-lactide-co-glycolide (PLGA; 85:15 and 50:50), poly-L-lactic acid (PLLA), and Thermanox plastic cell culture coverslips. Monolayers of acinar cells were established on all of the polymeric substrata. An assay of beta-hexosaminidase activity in the supernatant medium showed significant increases in protein secretion, following stimulation with 100 microM carbachol on matrix protein-coated and uncoated polymers such as silicone, PLGA 85:15, and PLLA. Our study demonstrates that PLLA supported the morphological and physiological properties of purified rabbit lacrimal gland epithelial cells more successfully than the others.