Targeting FoxO proteins induces lytic reactivation of KSHV for treating herpesviral primary effusion lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus consisting of both latent and lytic life cycles. Primary effusion lymphoma (PEL) is an aggressive B-cell lineage lymphoma, dominantly latently infected by KSHV. The latent infection of KSHV is persistent and poses an obstacle to killing tumor cells. Like the "shock and kill" strategy designed to eliminate latent HIV reservoir, methods that induce viral lytic reactivation in tumor latently infected by viruses represent a unique antineoplastic strategy, as it could potentially increase the specificity of cytotoxicity in cancer. Inspired by this conception, we proposed that the induction of KSHV lytic reactivation from latency could be a potential therapeutic stratagem for KSHV-associated cancers. Oxidative stress, the clinical hallmark of PEL, is one of the most prominent inducers for KSHV reactivation. Paradoxically, we found that hydrogen peroxide (H2O2) triggers robust cytotoxic effects on KSHV-negative rather than KSHV-positive B lymphoma cells in a dose-dependent manner. Mechanistically, we identified forkhead box protein O1 (FoxO1) and FoxO3 as irrevocable antioxidant defense genes and both of them are upregulated by KSHV latent infection, which is essential for the promoted ROS scavenging in KSHV-positive B lymphoma cells. Pharmacological inhibition or functional knockdown of either FoxO1 or FoxO3 is sufficient to ablate the antioxidant ability and therefore increases the intracellular ROS level that further reverses KSHV from latency to active lytic replication in PEL cells, resulting in tremendous cell death both in vitro and in vivo. Additionally, the elevated level of ROS by inhibiting FoxO proteins further sensitizes PEL cells to ROS-induced apoptosis. Our study therefore demonstrated that the lytic reactivation of KSHV by inhibiting FoxO proteins is a promising therapeutic approach for PEL, which could be further extended to other virus-associated diseases.

Effects of atorvastatin on the function of Tenon's capsule fibroblasts in human eyes.

PURPOSE: This study aimed to explore the role of atorvastatin (ATO) in the prevention and treatment of the scarring of filtration channels after glaucoma surgery. METHODS: Human Tenon's capsule fibroblasts (HTFs) were co-cultured with various concentrations of ATO. First, Cell Counting Kit-8 assay was performed to evaluate the effects of various concentrations of ATO on the viability of HTFs. Then, after the ATO stimulated the HTFs for 24 h, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to evaluate the apoptosis of HTFs. Transwell assay was also performed to evaluate the migration of HTFs. Moreover, enzyme-linked immunosorbent assay (ELISA) was performed to detect the protein expression levels of transforming growth factor-ß1 (TGF-ß1) and TGF-ß2 in the cell culture supernatant of HTFs. Western blot was carried out to detect the protein expression levels of smooth muscle actin (SMA), p38, Smad3, fibronectin, collagen I and collagen III in different groups. RESULTS: The results revealed that ATO could inhibit the proliferation and migration of HTFs. Based on the TUNEL assay, 100 µM and 150 µM ATO could induce cell apoptosis. The ELISA results indicated that ATO could down-regulate the expression level of TGF-ß2, and western blot analysis revealed that the protein expression levels of SMA, p38, Smad3, fibronectin, collagen I and collagen III in the TGF-ß2 group were all up-regulated compared with the control group, whereas the addition of ATO could reverse this up-regulation. CONCLUSIONS: ATO could inhibit the proliferation and migration of HTFs and induce their apoptosis. It was preliminary proven that ATO could inhibit the signaling pathway induced by TGF-ß. It is suggested that ATO could be a basis for the treatment of the scarring of filtration channels after glaucoma surgery.

Autophagy is involved in the neuroprotective effect of nicotiflorin.

ETHNOPHARMACOLOGICAL RELEVANCE: Nicotiflorin is a flavonoid glycoside derived from the traditional Chinese medicine FlosCarthami, dried petals of Carthamus tinctorius L., and has been confirmed to be a promising novel drug candidate for ischemic stroke. Yet, the exact role of nicotiflorin in cerebral I/R injury is uncharacterized and the possible mechanisms have not been clearly expounded. AIM OF THE STUDY: The present study was designed to determine the effect of nicotiflorin on cerebral ischemia/reperfusion (I/R) injury and its relationship with autophagy. MATERIALS AND METHODS: Middle cerebral artery occlusion (MCAO) in rats and oxygen-glucose deprivation and reintroduction (OGD/R) in SH-SY5Y cells were established in in vivo and in vitro models, respectively. The severity of MCAO was assessed by brain infarct size, neurological scores and survival rate. The severity of OGD/R was evaluated by cell viability, lactate dehydrogenase (LDH) release and cell apoptosis. The level of autophagy was evaluated both in vivo and in vitro. Autophagosomes were observed using transmission electron microscopy and autophagic flux was measured using mRFP-GFP-tandem fluorescent LC3 adenovirus. Autophagy-related proteins (LC3-II/I, SQSTM1, beclin-1, Phospho-mTOR/mTOR) were measured by immunoblot. Autophagy-related mRNA levels (Becn1, Atg7) were detected by Real-Time PCR. Inhibition of autophagy was implemented by 3-Methyladenine (3-MA) or chloroquine in vitro. RESULTS: In vivo, nicotiflorin treatment alleviated brain damage and neurological deficit while it dramatically increased 72 h survival rate in rats. In vitro, nicotiflorin treatment also ameliorated the severity of OGD/R. Moreover, nicotiflorin treatment increased ischemic penumbra autophagy (autophagosomes, BECN1, LC3-II/I ratio, SQSTM1, Phospho-mTOR/mTOR, Atg7). In vitro, nicotiflorin likewise enhanced autophagy and promoted autophagy flux. Furthermore, the blockade of autophagy by 3-MA or chloroquine disabled the efficacic of nicotiflorin in preventing cell damage upon OGD/R insult. CONCLUSION: These findings suggest that autophagy plays a significant role in the protective effect of nicotiflorin against ischemic stroke.

The homogenous polysaccharide SY01-23 purified from leaf of Morus alba L. has bioactivity on human gut Bacteroides ovatus and Bacteroides cellulosilyticus.

Function of mulberry leaf (Morus alba L.) polysaccharide has been reported on antitumor, immunostimulatory and anti-inflammatory effects. However, the bioactivity on human gut microbiota is unclear so far. Here, three homogenous polysaccharides named SY01-21, SY01-22, SY01-23 were isolated from mulberry leaf with molecular weight 57 kDa, 25 kDa and 7.2 kDa, respectively. The monosaccharide composition of SY01-21 contained rhamnose, galactose and arabinose in a molar ratio of 7.60:43.52:48.88. SY01-22 contained rhamnose, galacturonic acid, glucose, galactose, xylose and arabinose in a molar ratio of 14.61:9.06:1.35:34.65:2.99:37.34. SY01-23 contained rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose in a molar ratio of 23.00:4.12:24.60:5.74:17.28:1.12:24.13. Bioactivity test showed SY01-21 promoted the growth of Bacteroides cellulosilyticus (BC) while SY01-22 benefited the growth of Bacteroides ovatus (BO). Interestingly, SY01-23 boosted the growth of both BO and BC. However, Bacteroides thetaiotamicron (BT) only grew on 5 mg/mL SY01-21. Intriguingly, the growth of co-culture of BT with BO or BC was better than monoculture. This suggested that cross-feeding might exist between them. Besides, we found BO and BC generated acetate and propionate by utilizing SY01-23. The above results suggested that SY01-23 might modify human gut microbiota by driving colonization of Bacteroides in the gut to improve wellness.

Intestinal microbes derived butyrate is related to the immunomodulatory activities of Dendrobium officinale polysaccharide.

Although immunomodulatory activities of Dendrobium officinale polysaccharide has been investigated for many years, yet the potential contribution of its metabolite derived from intestinal microbes on immunoregulation effect has not been reported. In this study, polysaccharide DOW-5B with average molecular weight of 39.4 kDa was isolated from the stem of Dendrobium officinale Kimura et Migo. The carbohydrate content was 91.97% and no protein was detected. The monosaccharide analysis showed this polysaccharide was composed of glucuronic acid and glucose at a molar ratio (M/G) of 1.2:19.4. Animal test indicated DOW-5B increased the diversity of gut microbiota on mice. Beneficial microbes such as Ruminococcus, Eubacterium, Clostridium, Bifidobacterium, Parabacteroides and Akkermansiamuciniphila increased while harmful bacteria in Proteobacteria decreased. Surprisingly, DOW-5B promoted gut microbes to generate more butyrate and mainly produced by Parabacteroides_sp_HGS0025. Further, we found the health of large intestine as well as immunity response of mice was improved. In addition, Parabacteroides_sp_HGS0025 positively correlated with butyrate, IgM, IL-10, and TNF-α products in intestine and mice blood, respectively. The data suggested that Dendrobium officinale polysaccharide has function on immunity may be mediated by butyrate. It adds new evidence to support the basis of how herbal polysaccharides affect immunity.

Effect of cytoglobin overexpression on extracellular matrix component synthesis in human tenon fibroblasts.

BACKGROUND: Conjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery. METHODS: Cygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-ß1, and HIF1α were determined by RT-PCR and western blotting respectively. RESULTS: After Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-ß1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05). CONCLUSION: Our study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-ß1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.

Effect of cytoglobin overexpression on extracellular matrix component synthesis in human tenon fibroblasts

BACKGROUND: Conjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery. METHODS: Cygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-ß1, and HIF1α were determined by RT-PCR and western blotting respectively. RESULTS: After Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-ß1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05). CONCLUSION: Our study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-ß1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.

Collagen peptide modified carboxymethyl cellulose as both antioxidant drug and carrier for drug delivery against retinal ischaemia/reperfusion injury.

Oxidative stress can cause injury in retinal endothelial cells. Carboxymethyl cellulose modified with collagen peptide (CMCC) is of a distinct antioxidant capacity and potentially a good drug carrier. In this study, the protective effects of CMCC against H2 O2 -induced injury of primary retinal endothelial cells were investigated. In vitro, we demonstrated that CMCC significantly promoted viability of H2 O2 -treated cells, efficiently restrained cellular reactive oxygen species (ROS) production and cell apoptosis. Then, the CMCC was employed as both drug and anti-inflammatory drug carrier for treatment of retinal ischaemia/reperfusion (I/R) in rats. Animals were treated with CMCC or interleukin-10-loaded CMCC (IL-10@CMCC), respectively. In comparisons, the IL-10@CMCC treatment exhibited superior therapeutic effects, including better restoration of retinal structural thickness and less retinal apoptosis. Also, chemiluminescence demonstrated that transplantation of IL-10@CMCC markedly reduced the retinal oxidative stress level compared with CMCC alone and potently recovered the activities of typical antioxidant enzymes, SOD and CAT. Therefore, it could be concluded that CMCC provides a promising platform to enhance the drug-based therapy for I/R-related retinal injury.

Celosins inhibit atherosclerosis in ApoE-/- mice and promote autophagy flow.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen celosiae is a traditional Chinese medicine for purging hepatic pathogenic fire and removing nebula to improve eyesight, treating hepatopyretic vertigo and hypertension. It possesses a serial of potential bioactivities such as hepatoprotection, anti-tumor and anti-inflammatory, anti-diabetes. The triterpenoid saponins celosins from it were proved to have hepatoprotection, lipid lowing and anti-inflammatory. However, the anti-atherosclerosis activities were not reported to date. AIM OF THE STUDY: This study was designed to examine the therapeutic effects of celosins (CES), the active constituents extracted from Semen celosiae. MATERIALS AND METHODS: Atherosclerosis model by feeding high fat diet for 12 weeks in ApoE-/- mice and foam cell model by ox-LDL-treated peritoneal macrophages were performed. The lipid plaque was measured by histopathological analysis. The LC3 dots in the aortic root lesion examined through tissue immunofluorescence. The peritoneal macrophage phagocytosis, formation of foam cells, genes associated protein expression and autophagy flux were measured on foam cell model by oxidized low-density lipoprotein (Ox-LDL) stimulating peritoneal macrophages. The mRNA expression of CD36, SR-A1, ABCA1 and ABCG1 were determined by Real-Time PCR method. The expressions of LC3 and beclin 1 were measured using Western blot. RESULTS: CES (10, 30, 90mg/kg; p.o.) administrated for 4 weeks significantly reduced the prevalence of the relative area of plaque in mouse aorta, and showed the therapeutic effect on atherosclerosis. In the tissue section of immunofluorescence for aortic root, compared with high fat diet model group, the number of autophagy bodies in CES group increased significantly, suggesting that inhibiting atherosclerosis effect of CES may be related to its promoting autophagy. In vitro, CES significantly reduced phagocytosis of macrophages on lipid and formation rate of foam cells. CES down-regulated the mRNA expression of CD36 and SR-A1 while up-regulated mRNA expression of ABCA1 and ABCG1. Further, CES increased the autophagy specific protein LC3 and beclin 1, and it also increased the level of autophagy in the cells, and promoted the process of autophagy. CONCLUSIONS: The therapeutic effect of CES on atherosclerosis may be related to the promotion of autophagy.

Bakkenolide-IIIa Protects Against Cerebral Damage Via Inhibiting NF-κB Activation.

AIMS: This study was designed to examine the neuroprotective effects of bakkenolide-IIIa, a major novel compound extracted from the rhizome of P. trichinous. METHODS: Transient focal cerebral damage model in rats and oxygen-glucose deprivation (OGD) in cultured hippocampal neurons were performed. The amount of apoptotic neurons was determined using TUNEL assay. The expressions of Bcl-2, Bax, Akt, ERK1/2, IKKß, IκBα were measured using Western blot. The nuclear translocation and activation of NF-κB was measured using a fluorescence microscope and electrophoretic mobility shift assay (EMSA). RESULTS: Bakkenolide-IIIa (4, 8, 16 mg/kg; i.g.) was administered immediately after reperfusion could reduce the brain infarct volume, and the neurological deficit, as well as a high dose of bakkenolide-IIIa, increases the 72 h survival rate in cerebrally damaged rats. In vitro data demonstrated that bakkenolide-IIIa could increase cell viability and decrease the amount of apoptotic cells in cultured primary hippocampal neurons exposed to OGD. Bakkenolide-IIIa also dose-dependently increased the ratio of Bcl-2 to Bax. These results indicated that inhibition of apoptosis partly mediated the neuroprotection of bakkenolide-IIIa. Furthermore, bakkenolide-IIIa inhibited the phosphorylation of Akt, ERK1/2, IKKß, IκBα, and p65 in cultured hippocampal neurons exposed to OGD. Bakkenolide-IIIa not only inhibited the nuclear translocation of NF-κB in cultured neurons exposed to OGD, but also inhibited the activation of NF-κB in peri-infarct area in cerebrally damaged rats. CONCLUSION: Collectively, our findings indicated that bakkenolide-IIIa protects against cerebral damage by inhibiting AKT and ERK1/2 activation and inactivated NF-κB signaling.

17ß-estradiol mediates upregulation of stromal cell-derived factor-1 in the retina through activation of estrogen receptor in an ischemia-reperfusion injury model.

PURPOSE: There is increasing evidence that suggests stromal cell-derived factor-1 (SDF-1) can induce a protective response against ischemic injury in various organs. In this study, we examined the expression of SDF-1 in a rat model of retinal ischemia-reperfusion (IR) injury. Further, we explored the effect of estrogen 17ß-estradiol (E2), and the role of estrogen receptor (ER) in regulating SDF-1 expression. METHODS: Retinal IR injury was established in Sprague-Dawley rats by elevating the intraocular pressure to 110 mmHg for 60 mins. Relative expression levels of SDF-1 mRNA and protein in the retina at 6 h, 12 h, and 24 h after reperfusion were determined by RT-PCR and western blot respectively. To investigate the influence of estrogen and ER on SDF-1 expression, E2 was administered intraperitoneally 30 mins before induction of ischemia, and the estrogen receptor antagonist ICI 182-780 was administered 1 h before E2 injection. RESULTS: SDF-1 expression in IR-injured retina is upregulated at 6 h, 12 h, and 24 h after injury, with maximum expression at 12 h. As expected, pretreatment of retinal IR rats with E2 enhanced the upregulation in SDF-1 expression after injury, through activation of the estrogen receptor. We proved this hypothesis by demonstrating that pretreatment of retinal IR rats with ICI 182-780 led to a partial decrease in E2-induced SDF-1 expression. CONCLUSIONS: Our findings suggest that 17ß-estradiol offers protection against retinal ischemic injury by inducing an upregulation in SDF-1 expression through activation of the estrogen receptor.