Recombinant bivalent subunit vaccine combining truncated VP4 from P[7] and P[23] induces protective immunity against prevalent porcine rotaviruses.

Porcine rotaviruses (PoRVs) cause severe economic losses in the swine industry. P[7] and P[23] are the predominant genotypes circulating on farms, but no vaccine is yet available. Here, we developed a bivalent subunit PoRV vaccine using truncated versions (VP4*) of the VP4 proteins from P[7] and P[23]. The vaccination of mice with the bivalent subunit vaccine elicited more robust neutralizing antibodies (NAbs) and cellular immune responses than its components, even at high doses. The bivalent subunit vaccine and inactivated bivalent vaccine prepared from strains PoRVs G9P[7] and G9P[23] were used to examine their protective efficacy in sows and suckling piglets after passive immunization. The immunized sows showed significantly elevated NAbs in the serum and colostrum, and the suckling piglets acquired high levels of sIgA antibodies from the colostrum. Challenging subunit-vaccinated or inactivated-vaccinated piglets with homologous virulent strains did not induce diarrhea, except in one or two piglets, which had mild diarrhea. Immunization with the bivalent subunit vaccine and inactivated vaccine also alleviated the microscopic lesions in the intestinal tissues caused by the challenge with the corresponding homologous virulent strain. However, all the piglets in the challenged group displayed mild to watery diarrhea and high levels of viral shedding, whereas the feces and intestines of the piglets in the bivalent subunit vaccine and inactivated vaccine groups had lower viral loads. In summary, our data show for the first time that a bivalent subunit vaccine combining VP4*P[7] and VP4*P[23] effectively protects piglets against the diarrhea caused by homologous virulent strains.IMPORTANCEPoRVs are the main causes of diarrhea in piglets worldwide. The multisegmented genome of PoRVs allows the reassortment of VP4 and VP7 genes from different RV species and strains. The P[7] and P[23] are the predominant genotypes circulating in pig farms, but no vaccine is available at present in China. Subunit vaccines, as nonreplicating vaccines, are an option to cope with variable genotypes. Here, we have developed a bivalent subunit candidate vaccine based on a truncated VP4 protein, which induced robust humoral and cellular immune responses and protected piglets against challenge with homologous PoRV. It also appears to be safe. These data show that the truncated VP4-protein-based subunit vaccine is a promising candidate for the prevention of PoRV diarrhea.

Isolation and characterization of a G9P[23] porcine rotavirus strain AHFY2022 in China.

Rotavirus group A (RVA) is a main pathogen causing diarrheal diseases in humans and animals. Various genotypes are prevalent in the Chinese pig herd. The genetic diversity of RVA lead to distinctly characteristics. In the present study, a porcine RVA strain, named AHFY2022, was successfully isolated from the small intestine tissue of piglets with severe diarrhea. The AHFY2022 strain was identified by cytopathic effects (CPE) observation, indirect immunofluorescence assay (IFA), electron microscopy (EM), high-throughput sequencing, and pathogenesis to piglets. The genomic investigation using NGS data revealed that AHFY2022 exhibited the genotypes G9-P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1, using the online platform the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (https://www.bv-brc.org/). Moreover, experimental inoculation in 5-day-old and 27-day-old piglets demonstrated that AHFY2022 caused severe diarrhea, fecal shedding, small intestinal villi damage, and colonization in all challenged piglets. Taken together, our results detailed the virological features of the porcine rotavirus G9P[23] from China, including the whole-genome sequences, genotypes, growth kinetics in MA104 cells and the pathogenicity in suckling piglets.

Classic congenital adrenal hyperplasia with unilateral functional adrenal cortical adenoma: case report.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders related to adrenal steroid biosynthesis, and mainly caused by mutations in the CYP21A2 gene encoding 21-hydroxylase. Adrenal tumors are common in CAH, but functional adrenal tumors are rare. Here, we report a 17-year-old female with virilized external genitalia and primary amenorrhea, accompanied by a right adrenal tumor. Her 17-OHP level was normal, cortisol and androgen levels were significantly elevated, and the tumor pathology showed adrenal cortical adenoma. Gene testing for CYP21A2 showed c.518T > A in exon 4 and c.29313C > G in intron 2. The possibility of untreated classic CAH with 21-OH deficiency causing functional adrenal cortical adenoma should be considered. When clinical diagnosis highly considers CAH and cannot rule out the influence of functional adrenal tumors' secretion function on 17-OHP, gene mutation analysis should be performed.

Normalizing Tumor Blood Vessels to Improve Chemotherapy and Inhibit Breast Cancer Metastasis by Multifunctional Nanoparticles.

The abnormal tumor blood vessels with high leakage can promote tumor cells to infiltrate into the systemic circulation and increase the risk of tumor metastasis. In addition, chemotherapy may destroy tumor blood vessels and further aggravate metastasis. Normalizing tumor blood vessels can reduce vascular leakage and increase vascular integrity. The simultaneous administration of vascular normalization drugs and chemotherapy drugs may resist the blood vessels' destruction of chemotherapy. Here, multifunctional nanoparticles (CCM@LMSN/DOX&St), which combined chemotherapy with tumor blood vessel normalization, were prepared for the treatment of breast cancer. The results showed that CCM@LMSN/DOX&St-loaded sunitinib (St) promoted the expression of junction proteins Claudin-4 and VE-cadherin of endothelial cells, reversed the destruction of DOX to the endothelial cell layer, protected the integrity of the endothelial cell layer, and inhibited the migration of 4T1 tumor cells across the endothelial cell layer. In vivo experiments showed that CCM@LMSN/DOX&St effectively inhibited tumor growth in situ; what is exciting was that it also inhibited distal metastasis of breast cancer. CCM@LMSN/DOX&St encapsulated with St can normalize tumor blood vessels, reverse the damage of DOX to tumor blood vessels, increase the integrity of blood vessels, and prevent tumor cell invasion into blood vessels, which can inhibit breast cancer spontaneous metastasis and reduce chemotherapy-induced metastasis. This drug delivery platform effectively inhibited the progression of tumors and provided a promising solution for effective tumor treatment.

GSH-Responsive Polymeric Micelles for Remodeling the Tumor Microenvironment to Improve Chemotherapy and Inhibit Metastasis in Breast Cancer.

The tumor microenvironment (TME) of breast cancer is hypoxic, which can promote tumor progression, including invasion and metastasis, and limit the efficacy of anti-tumor treatment. Nitric oxide (NO) can dilate blood vessels, effectively alleviate hypoxia, and regulate the TME, which has the potential to improve the anti-tumor therapeutic efficacy. Here, chitosan (CO) and octadecylamine (ODA) were linked by the disulfide bond, and the LinTT1 peptide was linked onto CO-SS-ODA for targeting tumor cells and endothelial cells in tumors. The NO donor S-nitroso-N-acetylpenicillamine (SNAP) was connected to CO. Doxorubicin (DOX) was encapsulated, and GSH hierarchically responsive polymer micelles (TSCO-SS-ODA/DOX) were constructed for the treatment of breast cancer. The micelles had differently responsive drug release in different GSH concentrations. In endothelial cells, the micelles rapidly responded to release NO. In tumor cells, the disulfide bond rapidly broke and released DOX to effectively kill tumor cells. The disulfide bond was not sensitive to GSH concentration in endothelial cells, which had less release of DOX. The killing effect of the micelles to endothelial cells was much lower than that to tumor cells. The cell selective drug release of the drug delivery systems enabled safe and effective treatment of drugs. TSCO-SS-ODA/DOX, which had the excellent ability to target tumors, can alleviate tumor hypoxia, decrease the infiltration of M2 macrophages in tumors, increase the infiltration of M1 macrophages in tumors, and remodel the TME. Notably, TSCO-SS-ODA/DOX can significantly inhibit the growth of the primary tumor and effectively inhibit tumor metastasis. The drug delivery system provided a potential solution for effectively treating breast cancer.

Aucubin alleviates methotrexate-induced enteritis in rats by inducing autophagy.

One of the toxic side effects of methotrexate (MTX) is enteritis. Aucubin, an iridoid glycoside derived from traditional medicinal herbs, has been proven to have anti-inflammation, anti-apoptosis and anti-oxidation properties. This work explored the effect and mechanism of aucubin in treating MTX-induced enteritis in a rat model. Two doses of aucubin (5 and 10 mg/kg) were adopted for the assessment of its pharmacological activity. We observed that in rats with MTX-induced enteritis, the body weight and small intestinal weight decreased. The intestine barrier was injured, as reflected by pathological examinations and an increase in D-lactate and diamine oxidase concentration in serum. Intestinal inflammation was shown by the observation of macrophages in the intestine and the concentrations of inflammatory cytokines tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in serum. The NLR family pyrin domain containing 3 (NLRP3) inflammasome was shown to be activated by the enhancement of NLRP3, cleaved-caspase 1, IL-18 and IL-1ß. Moreover, autophagy was reflected by transmission electron microscopy as slightly induced, along with changes in autophagy-related markers microtubule-associated protein 1 light chain 3 (LC3) and Beclin1. Remarkably, aucubin treatment attenuated the MTX-induced disease activity index increase, intestinal damage, inflammatory response and NLRP3 inflammasome activation, but provoked autophagy. Rapamycin, an autophagy activator, showed similar therapeutic effects to aucubin on MTX-induced enteritis. However, 3-methyladenine, an autophagy inhibitor, reversed the protective effects of aucubin. These findings prompted the hypothesis that aucubin alleviates MTX-induced enteritis by aggravating autophagy. This study might provide evidence for further investigation on the therapeutic role of aucubin in MTX-resulted enteritis.

Corynoline ameliorates dextran sulfate sodium-induced colitis in mice by modulating Nrf2/NF-κB pathway.

OBJECTIVE: Corynoline is an active substance extracted from Corydalis bungeana Turcz and exerts a therapeutic effect in multiple diseases by alleviating inflammatory response. The present study sought to elucidate the role of corynoline in ulcerative colitis (UC). METHODS: The experimental colitis models were induced in BALB/c mice via receiving a drinking water supplemented with 3.5% (I) dextran sulfate sodium (DSS) ad libitum for 7 days. RESULTS: Corynoline administration inhibited body weight loss, colon shortening, disease activity index and colonic pathomorphological changes in DSS-treated mice. Besides, corynoline down-regulated the levels of pro-inflammatory interleukin (IL)-1ß, IL-6 and tumor necrosis factor Alpha (TNF-α), as well as decreased myeloperoxidase (MPO) activity in the colon of DSS-treated mice. In addition, severe oxidative stress in the colonic tissues of DSS-treated was mitigated by corynoline treatment. However, these beneficial effects were reversed by a specific nuclear factor E2-related factor 2 (Nrf2) inhibitor ML385 intervention. Further evidence confirmed that corynoline promoted Nrf2 nuclear migration and heme oxygenase-1 gene expression in the colonic tissues of UC mice. Besides, corynoline treatment restrained colonic nuclear factor-kappa B (NF-κB) activation as proved by the decrease in phosphorylation and nuclear translocation of NF-κB. CONCLUSIONS: Corynoline ameliorates DSS-induced mouse colitis, which may provide a promising therapeutic strategy for UC treatment.

Formation of volatiles in response to tea green leafhopper (Empoasca onukii Matsuda) herbivory in tea plants: a multi-omics study.

KEY MESSAGE: Combined transcriptome and metabolome analysis of fresh leaf infestation by tea green leafhoppers (Empoasca (Matsumurasca) onukii Matsuda) suggests roles for alternative pre-mRNA splicing and mRNAs in the regulation of aroma formation in tea plants. Oriental Beauty is a high-grade, oolong tea with a pronounced honey-like aroma and rich ripe fruit flavor that develops primarily as a result of the infestation of the fresh leaves by tea green leafhoppers (Empoasca (Matsumurasca) onukii Matsuda). Here, we used PacBio Iso-Seq and RNA-seq analyses to determine the full-length transcripts and gene expression profiles of fresh tea leaves in response to E. (M.) onukii herbivory. We investigated the relationship between RNA-seq, tea metabolites, and aroma response mechanisms in leaves infested by leafhoppers. We found 3644 differentially expressed genes, of which 2552 were up- and 1092 were down-regulated. A total of 49,913 alternative splicing events were predicted, including 324 differential AS events. Moreover, 3105 differentially expressed transcripts were also identified, of which 2295 were up- and 810 were down-regulated. The characterization of expression patterns of the key gene transcript isoforms involved in the aroma formation pathways identified 130 differentially expressed metabolites, 97 of which were up- and 33 were down-regulated. Two key aroma compounds (phenylacetaldehyde and 4-hydroxybenzaldehyde) were highly correlated with genes of the aroma formation pathways. Our results revealed that pre-mRNA AS plays a crucial role in the metabolic regulation surrounding aroma formation under leafhopper herbivory in tea plants.

Quantitative Structure-Activity Relationship Model to Predict Antioxidant Effects of the Peptide Fraction Extracted from a Co-Culture System of Chlorella pyrenoidosa and Yarrowia lipolytica.

In this study, the antioxidant components in co-culture of Chlorella pyrenoidosa and Yarrowia lipolytica (3:1 ratio) were confirmed as trypsin-hydrolyzed peptides (EHPs). The EHPs were composed of 836 different peptides with molecular weights ranging from 639 to 3531 Da and were mainly composed of hydrophobic amino acids (48.1%). These peptides showed remarkable protective effects against oxidative stress in HepG2, which may be attributed to their structures. Furthermore, the mRNA and protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) were significantly lower in the peptide-treated group than in the control group, suggesting that the antioxidant enzyme-coding genes were not activated. The EC50 value of three peptides in the EHPs were in the order of AGYSPIGFVR (0.04 ± 0.002 mg/mL) > VLDELTLAR (0.09 ± 0.001 mg/mL) > LFDPVYLFDQG (0.41 ± 0.03 mg/mL); these results agreed with the prediction of the model (R2 > 0.9, Q2 > 0.5). Thus, EHPs show potential as potent new antioxidant agents.

Organic zinc absorption by the intestine of broilers in vivo.

In Expt 1, a Zn-unsupplemented basal diet (control) and the basal diet supplemented with one of four different Zn sources, including ZnSO4, Zn-amino acid chelate with a weak chelation strength (Zn-AA W), Zn-protein chelate with a moderate chelation strength (Zn-Pro M) and Zn-protein chelate with a strong chelation strength (Zn-Pro S) were fed to broiler chickens from days 14 to 28. On day 28, Zn content in plasma from the hepatic portal vein increased (P0·05) and Zn-AA W(P<0·04) were higher than those for ZnSO4. These findings indicate that organic Zn absorption (especially Zn-Pro S) in intact living broilers was more effective than that of inorganic Zn; organic Zn absorption in the ligated duodenal segment was a saturable carrier-mediated process similar to that of ZnSO4. Moreover, except for MT, there might be other Zn transporters involved in Zn absorption that are affected by different Zn sources.

Determination of Dietary Iron Requirements by Full Expression of Iron-Containing Enzymes in Various Tissues of Broilers.

BACKGROUND: The current dietary iron requirement (80 mg/kg) of broilers is mainly based on growth, hemoglobin concentration, or hematocrit data obtained in a few early studies; however, expressions of iron-containing enzymes might be more sensitive novel criteria to evaluate dietary iron requirements. OBJECTIVE: The objective of this study was to determine dietary iron requirements of broilers for the full expression of succinate dehydrogenase (SDH), catalase, and cytochrome c oxidase (COX) in various tissues. METHODS: A total of 336 1-d-old Arbor Acres male chicks were randomly assigned to 1 of 7 treatments with 6 replicates and fed a basal corn and soybean-meal diet (control, containing 67 mg Fe/kg) and the basal diet supplemented with 20, 40, 60, 80, 100, or 120 mg Fe/kg from FeSO4 ⋅ 7H2O for 21 d. Regression analysis was performed to estimate the optimal dietary iron concentration with the use of broken-line or quadratic models. RESULTS: SDH activity in the liver and heart, COX and catalase activity in the liver, Sdh mRNA levels in the liver, and Cox mRNA levels in the liver and heart of broilers were affected (P < 0.027) by supplemental iron concentration, and increased quadratically (P < 0.004) as dietary iron concentration increased. Dietary iron requirements estimated on the basis of fitted broken-line or quadratic-curve models (P < 0.005) of the above indexes were 97-136 mg/kg. CONCLUSIONS: SDH activity in the liver and heart, COX and catalase activity in the liver, Sdh mRNA levels in the liver, and Cox mRNA levels in the liver and heart are, to our knowledge, new and sensitive criteria to evaluate the dietary iron requirements of broilers, and the dietary iron requirements would be 97-136 mg/kg to support the full expression of the above iron-containing enzymes in various tissues of broiler chicks from 1 to 21 d of age, which are higher than the current NRC iron requirement.

Manganese elevates manganese superoxide dismutase protein level through protein kinase C and protein tyrosine kinase.

Three experiments were conducted to investigate the effects of inorganic and organic Mn sources on MnSOD mRNA, protein and enzymatic activity and the possible signal pathways. The primary broiler myocardial cells were treated with MnCl2 (I) or one of organic chelates of Mn and amino acids with weak, moderate (M) or strong (S) chelation strength for 12 and 48 h. Cells were preincubated with superoxide radical anions scavenger N-acetylcysteine (NAC) or specific inhibitors for MAPKs and protein tyrosine kinase (PTK) or protein kinase C (PKC) for 30 min before treatments of I and M. The MnSOD mRNA, protein and enzymatic activity, phosphorylated MAPKs or protein kinases activations were examined. The results showed that additions of Mn increased (P < 0.05) MnSOD mRNA levels and M was more effective than I. Additions of Mn elevated (P < 0.05) MnSOD protein levels and enzymatic activities, and no differences were found among I and M. Addition of NAC did not decrease (P > 0.05) Mn-induced MnSOD mRNA and protein levels. None of the three MAPKs was phosphorylated (P > 0.05) by Mn. Additions of Mn decreased (P < 0.05) the PTK activities and increased (P < 0.05) the membrane PKC contents. Inhibitors for PTK or PKC decreased (P < 0.05) Mn-induced MnSOD protein levels. The results suggested that Mn-induced MnSOD mRNA and protein expressions be not related with NAC, and MAPK pathways might not involve in Mn-induced MnSOD mRNA expression. PKC and PTK mediated the Mn-induced MnSOD protein expression.

An attenuated Listeria monocytogenes vector primes more potent simian immunodeficiency virus-specific mucosal immunity than DNA vaccines in mice.

A human immunodeficiency virus type 1 (HIV-1) vaccine that induces potent immune responses in the gastrointestinal mucosa would be highly desirable. Here we show that attenuated recombinant Listeria monocytogenes, administered orally utilizing its natural route of infection, induces potent mucosal as well as systemic immune responses in mice. Moreover, these responses can be boosted efficiently with replication-incompetent adenoviral vectors. L. monocytogenes elicited more potent simian immunodeficiency virus (SIV) Gag-specific CD8(+) T lymphocyte responses in mucosal compartments than DNA vaccines.

The synergy of recombinant NSP4 and VP4 from porcine rotavirus elicited a strong mucosal response.

Porcine rotavirus (PoRV) is one of the main pathogens causing diarrhea in piglets, and multiple genotypes coexist. However, an effective vaccine is currently lacking. Here, the potential adjuvant of nonstructural protein 4 (NSP4) and highly immunogenic structural protein VP4 prompted us to construct recombinant NSP486-175aa (NSP4*) and VP426-476aa (VP4*) proteins, combine them as immunogens to evaluate their efficacy. Results indicated that NSP4* enhanced systemic and local mucosal responses induced by VP4*. The VP4*-IgG, VP4*-IgA in feces and IgA-secreting cells in intestines induced by the co-immunization were significantly higher than those induced by VP4* alone. Co-immunization of NSP4* and VP4* also induced strong cellular immunity with significantly increased IFN-λ than the single VP4*. Summarily, the NSP4* as a synergistical antigen exerted limited effects on the PoRV NAbs elevation, but conferred strong VP4*-specific mucosal and cellular efficacy, which lays the foundation for the development of a more effective porcine rotavirus subunit vaccine.

Combining Multifunctional Delivery System with Blood-Brain Barrier Reversible Opening Strategy for the Enhanced Treatment of Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative illness characterized by intracellular tau-phosphorylation, ß-amyloid (Aß) plaques accumulation, neuroinflammation, and impaired behavioral ability. Owing to the lack of effective brain delivery approaches and the presence of the blood-brain barrier (BBB), current AD therapeutic endeavors are severely limited. Herein, a multifunctional delivery system (RVG-DDQ/PDP@siBACE1) is elaborately combined with a protein kinase B (AKT) agonist (SC79) for facilitating RVG-DDQ/PDP@siBACE1 to target and penetrate BBB, enter brain parenchyma, and further accumulate in AD brain lesion. Moreover, compared with the unitary dose of RVG-DDQ/PDP@siBACE1, this collaborative therapy strategy exhibits a distinctive synergistic function including scavenging reactive oxygen species (ROS), decreasing of Aß production, alleviating neuroinflammation by promoting the polarized microglia into the anti-inflammatory M2-like phenotype and significantly enhancing the cognitive functions of AD mice. More strikingly, according to these results, an innovative signaling pathway "lncRNA MALAT1/miR-181c/BCL2L11" is found that can mediate the neuronal apoptosis of AD. Taken together, combining the brain targeted delivery system with noninvasive BBB opening can provide a promising strategy and platform for targeting treatment of AD and other neurodegenerative diseases.

The protective effects of Axitinib on blood-brain barrier dysfunction and ischemia-reperfusion injury in acute ischemic stroke.

BACKGROUND AND PURPOSE: The pathophysiological features of acute ischemic stroke (AIS) often involve dysfunction of the blood-brain barrier (BBB), characterized by the degradation of tight junction proteins (Tjs) leading to increased permeability. This dysfunction can exacerbate cerebral injury and contribute to severe complications. The permeability of the BBB fluctuates during different stages of AIS and is influenced by various factors. Developing effective therapies to restore BBB function remains a significant challenge in AIS treatment. High levels of vascular endothelial growth factor (VEGF) in the early stages of AIS have been shown to worsen BBB breakdown and stroke progression. Our study aimed to investigate the protective effects of the VEGF receptor inhibitor Axitinib on BBB dysfunction and cerebral ischemia/reperfusion-induced injury. METHODS: BEnd3 cell exposed to oxygen-glucose deprivation (OGD) model was constructed to estimate pharmacological activity of Axitinib (400 ng/ml) on anti-apoptosis and pathological barrier function recovery. In vivo, rats were subjected to a 1 h transient middle cerebral artery occlusion and 23 h reperfusion (tMCAO/R) to investigate the permeability of BBB and cerebral tissue damage. Axitinib was administered through the tail vein at the beginning of reperfusion. BBB integrity was assessed by Evans blue leakage and the expression levels of Tjs claudin-5 and occludin. RESULTS: Our research revealed that co-incubation with Axitinib enhanced the cell viability of OGD-insulted bEnd3 cells, decreased LDH leakage rate, and suppressed the expression of apoptosis-related proteins cytochrome C and Bax. Axitinib also mitigated the damage to Tjs and facilitated the restoration of transepithelial electrical resistance in OGD-insulted bEnd.3 cells. In vivo, Axitinib administration reduced intracerebral Evans blue leakage and up-regulated the expression of Tjs in the penumbra brain tissue in tMCAO/R rats. Notably, 10 mg/kg Axitinib exerted a significant anti-ischemic effect by decreasing cerebral infarct volume and brain edema volume, improving neurological function, and reducing pro-inflammatory cytokines IL-6 and TNF-α in the brain. CONCLUSIONS: Our study highlights Axitinib as a potent protectant of blood-brain barrier function, capable of promoting pathological blood-brain barrier recovery through VEGF inhibition and increased expression of tight junction proteins in AIS. This suggests that VEGF antagonism within the first 24 h post-stroke could be a novel therapeutic approach to enhance blood-brain barrier function and mitigate ischemia-reperfusion injury.

Astragaloside IV alleviates renal fibrosis by inhibiting renal tubular epithelial cell pyroptosis induced by urotensin II through regulating the cAMP/PKA signaling pathway.

OBJECTIVE: To explore the molecular mechanism of Astragaloside IV (AS-IV) in alleviating renal fibrosis by inhibiting Urotensin II-induced pyroptosis and epithelial-mesenchymal transition of renal tubular epithelial cells. METHODS: Forty SD rats were randomly divided into control group without operation: gavage with 5ml/kg/d water for injection and UUO model group: gavage with 5ml/kg/d water for injection; UUO+ AS-IV group (gavage with AS-IV 20mg/kg/d; and UUO+ losartan potassium group (gavage with losartan potassium 10.3mg/kg/d, with 10 rats in each group. After 2 weeks, Kidney pathology, serum Urotensin II, and cAMP concentration were detected, and the expressions of NLRP3, GSDMD-N, Caspase-1, and IL-1ß were detected by immunohistochemistry. Rat renal tubular epithelial cells were cultured in vitro, and different concentrations of Urotensin II were used to intervene for 24h and 48h. Cell proliferation activity was detected using the CCK8 assay. Suitable concentrations of Urotensin II and intervention time were selected, and Urotensin II receptor antagonist (SB-611812), inhibitor of PKA(H-89), and AS-IV (15ug/ml) were simultaneously administered. After 24 hours, cells and cell supernatants from each group were collected. The cAMP concentration was detected using the ELISA kit, and the expression of PKA, α-SMA, FN, IL-1ß, NLRP3, GSDMD-N, and Caspase-1 was detected using cell immunofluorescence, Western blotting, and RT-PCR. RESULTS: Renal tissue of UUO rats showed renal interstitial infiltration, tubule dilation and atrophy, renal interstitial collagen fiber hyperplasia, and serum Urotensin II and cAMP concentrations were significantly higher than those in the sham operation group (p <0.05). AS-IV and losartan potassium intervention could alleviate renal pathological changes, and decrease serum Urotensin II, cAMP concentration levels, and the expressions of NLRP3, GSDMD-N, Caspase-1, and IL-1ß in renal tissues (p <0.05). Urotensin II at a concentration of 10-8 mol/L could lead to the decrease of cell proliferation, (p<0.05). Compared with the normal group, the cAMP level and the PKA expression were significantly increased (p<0.05). After intervention with AS-IV and Urotensin II receptor antagonist, the cAMP level and the expression of PKA were remarkably decreased (p<0.05). Compared with the normal group, the expression of IL-1ß, NLRP3, GSDMD-N, and Caspase-1 in the Urotensin II group was increased (p<0.05), which decreased in the AS-IV and H-89 groups. CONCLUSION: AS-IV can alleviate renal fibrosis by inhibiting Urotensin II-induced pyroptosis of renal tubular epithelial cells by regulating the cAMP/PKA signaling pathway.

Astragaloside IV Alleviates Podocyte Injury in Diabetic Nephropathy through Regulating IRE-1α/NF-κ B/NLRP3 Pathway.

OBJECTIVE: To investigate the effects of astragaloside IV (AS-IV) on podocyte injury of diabetic nephropathy (DN) and reveal its potential mechanism. METHODS: In in vitro experiment, podocytes were divided into 4 groups, normal, high glucose (HG), inositol-requiring enzyme 1 (IRE-1) α activator (HG+thapsigargin 1 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups. Additionally, podocytes were divided into 4 groups, including normal, HG, AS-IV (HG+AS-IV 20 µmol/L), and IRE-1α inhibitor (HG+STF-083010, 20 µmol/L) groups, respectively. After 24 h treatment, the morphology of podocytes and endoplasmic reticulum (ER) was observed by electron microscopy. The expressions of glucose-regulated protein 78 (GRP78) and IRE-1α were detected by cellular immunofluorescence. In in vivo experiment, DN rat model was established via a consecutive 3-day intraperitoneal streptozotocin (STZ) injections. A total of 40 rats were assigned into the normal, DN, AS-IV [AS-IV 40 mg/(kg·d)], and IRE-1α inhibitor [STF-083010, 10 mg/(kg·d)] groups (n=10), respectively. The general condition, 24-h urine volume, random blood glucose, urinary protein excretion rate (UAER), urea nitrogen (BUN), and serum creatinine (SCr) levels of rats were measured after 8 weeks of intervention. Pathological changes in the renal tissue were observed by hematoxylin and eosin (HE) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expressions of GRP78, IRE-1α, nuclear factor kappa Bp65 (NF-κBp65), interleukin (IL)-1ß, NLR family pyrin domain containing 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), and nephrin at the mRNA and protein levels in vivo and in vitro, respectively. RESULTS: Cytoplasmic vacuolation and ER swelling were observed in the HG and IRE-1α activator groups. Podocyte morphology and ER expansion were improved in AS-IV and IRE-1α inhibitor groups compared with HG group. Cellular immunofluorescence showed that compared with the normal group, the fluorescence intensity of GRP78 and IRE-1α in the HG and IRE-1α activator groups were significantly increased whereas decreased in AS-IV and IRE-1α inhibitor groups (P<0.05). Compared with the normal group, the mRNA and protein expressions of GRP78, IRE-1α, NF-κ Bp65, IL-1ß, NLRP3, caspase-1 and GSDMD-N in the HG group was increased (P<0.05). Compared with HG group, the expression of above indices was decreased in the AS-IV and IRE-1α inhibitor groups, and the expression in the IRE-1α activator group was increased (P<0.05). The expression of nephrin was decreased in the HG group, and increased in AS-IV and IRE-1α inhibitor groups (P<0.05). The in vivo experiment results revealed that compared to the normal group, the levels of blood glucose, triglyceride, total cholesterol, BUN, blood creatinine and urinary protein in the DN group were higher (P<0.05). Compared with DN group, the above indices in AS-IV and IRE-1α inhibitor groups were decreased (P<0.05). HE staining revealed glomerular hypertrophy, mesangial widening and mesangial cell proliferation in the renal tissue of the DN group. Compared with the DN group, the above pathological changes in renal tissue of AS-IV and IRE-1α inhibitor groups were alleviated. Quantitative RT-PCR and Western blot results of GRP78, IRE-1α, NF-κ Bp65, IL-1ß, NLRP3, caspase-1 and GSDMD-N were consistent with immunofluorescence analysis. CONCLUSION: AS-IV could reduce ERS and inflammation, improve podocyte pyroptosis, thus exerting a podocyte-protective effect in DN, through regulating IRE-1α/NF-κ B/NLRP3 signaling pathway.

Kinetics of manganese transport and gene expressions of manganese transport carriers in Caco-2 cell monolayers.

Two experiments were conducted to investigate the kinetics of manganese (Mn) transport in Caco-2 cell monolayers and the gene expressions of Mn transport carriers in apical (AP) and basolateral (BL) membranes. In experiment 1, the cells were treated with the medium containing 146 µmol/L of Mn (MnSO4·H2O). Both the uptake and transport of Mn from AP-BL or from BL-AP at different time-points were assessed to determine the optimal time for kinetics of Mn transport. The transport of Mn increased linearly with higher efficiency values in AP-BL than in BL-AP direction, however, the uptake of Mn revealed an asymptotic pattern within 120 min. In experiment 2, the kinetics of Mn transport in AP-BL was determined with media containing Mn concentrations from 0 to 2,500 µmol/L at 40 and 120 min, respectively, and mRNA levels of divalent metal transporter 1 (DMT1) and ferroportin (FPN1) were determined in Caco-2 cells treated with the medium containing 0 or 800 µmol/L of Mn for 120 min. The kinetics of Mn transport showed a carrier-mediated process when Mn concentrations were lower than 1,000 µmol/L and a linear increment when Mn concentrations exceeded 1,000 µmol/L at either 40 or 120 min. Mn treatment decreased (P < 0.01) DMT1 mRNA level and increased (P < 0.01) FPN1 mRNA level. The results from the present study suggested that Mn transport in AP-BL fit both carrier-mediated saturable and non-saturable diffusion processes, and Mn transport carriers DMT1 and FPN1 mediate the apical uptake and basolateral exit of Mn in Caco-2 cells.

Adult upper-limb forearm lymphoma hygroma (cystic lymphangioma): A case report.

INTRODUCTION: Lymphoma hygroma (LH) that is the most common type of lymphangioma, but it rarely occurs in the forearm. It may show localized invasive behavior, but is benign. CASE PRESENTATION: A 42-year-old woman presented to our hospital with a growing strip-like mass in the right forearm that had been detected 3 years earlier. Ultrasound examination showed a subcutaneous strip of low back vocal cords on the right forearm. Further magnetic resonance imaging (MRI) showed irregular strip-like dilated lymphatic vessels characteristic of LH with low T1 signal intensity and high T2 signal intensity. After radical surgical resection, hematoxylin-eosin (H & E) and immunohistochemical (IHC) staining of cystic LH endothelial cells labeled with monoclonal antibody D2-40 showed a dilated lymphangioma with no evidence of malignancy. After 7 months of follow-up, no tumor recurrence was seen and the effect was satisfactory. CLINICAL DISCUSSION: A combination of previous trauma history, signs and symptoms, and imaging evaluation are necessary to provide clues to LH, but the final diagnosis is likely to be made by pathologic evaluation of the resected specimen. Although there are many treatment modalities, all also have different outcomes. The absence of complete resection resulting in a tumor remnant is the foremost cause of LH recurrence, so we believe that the preferred approach against LH remains complete surgical resection. CONCLUSIONS: LH is benign and generally asymptomatic lesions with mild bio-behavior. As there are occasional confusing presentations, similar cystic lesions should still be considered with caution for the disease. Although MRI provides superior advantages for its diagnosis, the confirmation of diagnosis still requires histological examination. Radical lesion resection is a very safe and effective option for the treatment of LH.