Influence of Herba Houttuyniae added to fodder on the morphological structure of the intestinal tract, the digestive enzymes, the intestinal flora, and immune function of koi carp (Cyprinus carpio) infected with Aeromonas veronii.

This study investigated whether adding Herba Houttuyniae to feed can improve intestinal function and prevent diseases for koi carp (Cyprinus carpio) infected with Aeromonas veronii. There was a total of 168 koi carp with an average body length of (9.43 ± 0.99) cm and an average body weight of (26.00 ± 11.40) g. The K group was the control group fed with basal feed, while the C group was fed with feed with a H. houttuyniae content of six per thousand. After 14 days of feeding, the fish were fasted for a day and then intraperitoneally injected with A. veronii for artificial infection, injection dose is 0.2 mL, and the concentration is 1 × 107 CFU/mL. Samples were collected from the two groups on days 0, 1, 2, and 4. The fold height, intestinal villus width, and muscle layer thickness in the gut of the koi carp were measured. In addition, on day 4, the activities of trypsin, α-amylase, and lipase in the gut were determined, and the intestinal flora of the carp in both groups was tested. The results showed that on the second and fourth days of sampling, the fold height and muscle layer thickness in the C group were significantly higher than those in the K group (P < 0.05). The villus width in the C group was slightly higher than that in the K group, but the difference was not significant (P > 0.05). Microscopic observation revealed that the intestinal structure of the carp in the C4 (day 4 in C group) group was more intact than that in the K4 (day 4 in K group) group. Moreover, the activities of trypsin, α-amylase, and lipase in the foregut and midgut in the C4 group were higher than those in the K4 group (P < 0.05). The activities of trypsin and α-amylase in the hindgut in the C4 group were higher than those in the K4 group (P < 0.05). Furthermore, beneficial bacteria, especially those in the genus Cetobacterium, were more abundant in the intestinal tract of the carp in the C4 group compared to the K group. In addition, comparisons and tests of IL-4 and IL-10 in the intestines of the fish in both groups demonstrated that the H. houttuyniae added to feed enhanced the immune function of the fish intestines after bacterial attack. In conclusion, for koi carp infected with A.veronii, adding H. houttuyniae to their feed not only improves the activity of digestive enzymes and the morphological structure of the intestine but also optimizes the beneficial intestinal microbiota, thereby protecting the intestinal tract.

The pathogenic role of succinate-SUCNR1: a critical function that induces renal fibrosis via M2 macrophage.

BACKGROUND: Renal fibrosis significantly contributes to the progressive loss of kidney function in chronic kidney disease (CKD), with alternatively activated M2 macrophages playing a crucial role in this progression. The serum succinate level is consistently elevated in individuals with diabetes and obesity, both of which are critical factors contributing to CKD. However, it remains unclear whether elevated succinate levels can mediate M2 polarization of macrophages and contribute to renal interstitial fibrosis. METHODS: Male C57/BL6 mice were administered water supplemented with 4% succinate for 12 weeks to assess its impact on renal interstitial fibrosis. Additionally, the significance of macrophages was confirmed in vivo by using clodronate liposomes to deplete them. Furthermore, we employed RAW 264.7 and NRK-49F cells to investigate the underlying molecular mechanisms. RESULTS: Succinate caused renal interstitial macrophage infiltration, activation of profibrotic M2 phenotype, upregulation of profibrotic factors, and interstitial fibrosis. Treatment of clodronate liposomes markedly depleted macrophages and prevented the succinate-induced increase in profibrotic factors and fibrosis. Mechanically, succinate promoted CTGF transcription via triggering SUCNR1-p-Akt/p-GSK3ß/ß-catenin signaling, which was inhibited by SUCNR1 siRNA. The knockdown of succinate receptor (SUCNR1) or pretreatment of anti-CTGF(connective tissue growth factor) antibody suppressed the stimulating effects of succinate on RAW 264.7 and NRK-49F cells. CONCLUSIONS: The causative effects of succinate on renal interstitial fibrosis were mediated by the activation of profibrotic M2 macrophages. Succinate-SUCNR1 played a role in activating p-Akt/p-GSK3ß/ß-catenin, CTGF expression, and facilitating crosstalk between macrophages and fibroblasts. Our findings suggest a promising strategy to prevent the progression of metabolic CKD by promoting the excretion of succinate in urine and/or using selective antagonists for SUCNR1.

The critical role of BTRC in hepatic steatosis as an ATGL E3 ligase.

Non-alcoholic fatty liver disease (NAFLD), characterized by hepatic steatosis, is one of the commonest causes of liver dysfunction. Adipose triglyceride lipase (ATGL) is closely related to lipid turnover and hepatic steatosis as the speed-limited triacylglycerol lipase in liver lipolysis. However, the expression and regulation of ATGL in NAFLD remain unclear. Herein, our results showed that ATGL protein levels were decreased in the liver tissues of high-fat diet (HFD)-fed mice, naturally obese mice, and cholangioma/hepatic carcinoma patients with hepatic steatosis, as well as in the oleic acid-induced hepatic steatosis cell model, while ATGL mRNA levels were not changed. ATGL protein was mainly degraded through the proteasome pathway in hepatocytes. Beta-transducin repeat containing (BTRC) was upregulated and negatively correlated with the decreased ATGL level in these hepatic steatosis models. Consequently, BTRC was identified as the E3 ligase for ATGL through predominant ubiquitination at the lysine 135 residue. Moreover, adenovirus-mediated knockdown of BTRC ameliorated steatosis in HFD-fed mouse livers and oleic acid-treated liver cells via upregulating the ATGL level. Taken together, BTRC plays a crucial role in hepatic steatosis as a new ATGL E3 ligase and may serve as a potential therapeutic target for treating NAFLD.

Kallistatin Deficiency Induces the Oxidative Stress-Related Epithelial-Mesenchymal Transition of Retinal Pigment Epithelial Cells: A Novel Protagonist in Age-Related Macular Degeneration.

Purpose: Retinal pigment epithelium (RPE) dysfunction induced by oxidative stress-related epithelial-mesenchymal transition (EMT) of RPE is the primary underlying mechanism of age-related macular degeneration (AMD). Kallistatin (KAL) is a secreted protein with an antioxidative stress effect. However, the relationship between KAL and EMT in RPE has not been determined. Therefore we aimed to explore the impact and mechanism of KAL in oxidative stress-induced EMT of RPE. Methods: Sodium iodate (SI) was injected intraperitoneally to construct the AMD rat model and investigate the changes in RPE morphology and KAL expression. KAL knockout rats and KAL transgenic mice were used to explain the effects of KAL on EMT and oxidative stress. In addition, Snail overexpressed adenovirus and si-RNA transfected ARPE19 cells to verify the involvement of Snail in mediating KAL-suppressed EMT of RPE. Results: AMD rats induced by SI expressed less KAL in the retina, and KAL knockout rats showed RPE dysfunction spontaneously where EMT and reactive oxygen species (ROS) production increased in RPE. In contrast, KAL overexpression attenuated EMT and ROS levels in RPE, even in TGF-ß treatment. Mechanistically, Snail reversed the beneficial effect of KAL on EMT and ROS reduction. Moreover, KAL ameliorated SI-induced AMD-like pathological changes. Conclusions: Our findings demonstrated that KAL inhibits oxidative stress-induced EMT by downregulating the transcription factor Snail. Herein, KAL knockout rats may be an appropriate animal model for observing spontaneous RPE dysfunction for AMD-like retinopathy, and KAL may represent a novel therapeutic target for treating dry AMD.

N-cadherin cleavage: A critical function that induces diabetic retinopathy fibrosis via regulation of ß-catenin translocation.

Retinal fibrosis is a severe pathological change in the late stage of diabetic retinopathy and is also the leading cause of blindness. We have previously revealed that N-cadherin was significantly increased in type 1 and type 2 diabetic mice retinas and the fibrovascular membranes from proliferative diabetic retinopathy (PDR) patients. However, whether N-cadherin directly induces retinal fibrosis in DR and the related mechanism is unknown. Here, we investigated the pathogenic role of N-cadherin in mediating retinal fibrosis and further explored the relevant therapeutic targets. We found that the level of N-cadherin was significantly increased in PDR patients and STZ-induced diabetic mice and positively correlated with the fibrotic molecules Connective Tissue Growth Factor (CTGF) and fibronectin (FN). Moreover, intravitreal injection of N-cadherin adenovirus significantly increased the expression of FN and CTGF in normal mice retinas. Mechanistically, overexpression of N-cadherin promotes N-cadherin cleavage, and N-cadherin cleavage can further induce translocation of non-p-ß-catenin in the nucleus and upregulation of fibrotic molecules. Furthermore, we found a novel N-cadherin cleavage inhibitor, pigment epithelial-derived factor (PEDF), which ameliorated the N-cadherin cleavage and subsequent retinal fibrosis in diabetic mice. Thus, our findings provide novel evidence that elevated N-cadherin level not only acts as a classic EMT maker but also plays a causative role in diabetic retinal fibrosis, and targeting N-cadherin cleavage may provide a strategy to inhibit retinal fibrosis in DR patients.

A role for Snail-MnSOD axis in regulating epithelial-to-mesenchymal transition markers expression in RPE cells.

Age-related macular degeneration (AMD) is a common cause of vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells, accompanied by oxidative damage, plays a crucial role in AMD. It is well known that manganese superoxide dismutase (MnSOD) encoded by SOD2 is a critical molecule in fighting against oxidative stress, and Snail encoded by SNAI1 is the essential transcription factor for EMT. However, the effect of MnSOD on EMT and the underlying mechanism in RPE cells remains unknown. In this study, we found that MnSOD knockdown triggered the EMT by upregulating Snail, while MnSOD overexpression reversed EMT even with TGFß treatment in RPE cells, and the anti-oxidative stress activity of MnSOD mediated this observation. In addition, Snail depletion increased both expression and activity of MnSOD while Snail overexpression decreased MnSOD expression and activity, and Dual-luciferase reporter and ChIP assays showed that Snail directly bound to E-box (CACCTG) in the SOD2 promoter. Moreover, MnSOD over-expression and Snail interference co-treatment strengthened the anti-oxidation and EMT reversing. Therefore, our findings demonstrate that MnSOD prevents EMT of RPE cells in AMD through inhibiting oxidative injury to RPE. Moreover, a critical EMT transcription factor, Snail, functions as a new negative transcriptional factor of SOD2. Herein, the Snail-MnSOD axis forms a mutual loop in the development of AMD, which may be a novel systemic treatment target for preventing AMD.

Elevated pigment epithelium-derived factor induces diabetic erectile dysfunction via interruption of the Akt/Hsp90ß/eNOS complex.

AIMS/HYPOTHESIS: Diabetes mellitus erectile dysfunction (DMED) is a common complication of diabetes. The level of pigment epithelium-derived factor (PEDF) is significantly upregulated in the serum of individuals with obesity and diabetes. However, whether elevated PEDF levels contribute to DMED remains unknown. This study aimed to investigate the pathogenic role of PEDF and its related mechanism in DMED. METHODS: We enrolled 65 men, of whom 20 were nondiabetic control participants, 21 participants with diabetes but without erectile dysfunction, and 24 with DMED. The International Index of Erectile Function (IIEF-5) questionnaire was administered to evaluate erectile function. Plasma PEDF in diabetic participants and streptozotocin (STZ)-induced diabetic animals was detected by ELISA. Erectile function was evaluated by measuring the intracavernous pressure (ICP) and the ICP/mean arterial pressure (MAP) ratio in STZ-induced diabetic rats treated with PEDF-neutralising antibody (PEDF-Ab), db/db mice treated with PEDF-Ab, and Pedf knockout mice with STZ-induced diabetes. The overexpression of PEDF was implemented by intraperitoneal injection of recombinant PEDF and intracavernous injection of PEDF-expressing adenovirus. A mechanistic study was performed by immunofluorescence staining, bimolecular fluorescence complementation (BiFC), immunoprecipitation and western blotting. RESULTS: We found that the plasma level of PEDF was significantly higher in participants with DMED compared with diabetic counterparts without erectile dysfunction and nondiabetic controls. Interestingly, PEDF levels were negatively correlated with plasma nitrite/nitrate levels and erectile function in DMED patients and STZ-induced diabetic rats. Furthermore, overexpression of PEDF significantly suppressed ICP and endothelial nitric oxide synthase (eNOS) phosphorylation in control rats. In contrast, the PEDF-Ab and Pedf knockout ameliorated ICP and eNOS phosphorylation in diabetic rats and mice. Mechanistically, PEDF promoted the membrane translocation of Hsp90ß and directly bound to the amino acid residues 341-724 of Hsp90ß on the endothelial cell surface, subsequently blocking intracellular Hsp90ß/Akt/eNOS complex formation and downregulating eNOS phosphorylation. CONCLUSIONS/INTERPRETATION: These results indicate that elevated PEDF levels contribute to impaired erectile function by suppressing Hsp90ß-mediated eNOS phosphorylation and that PEDF may represent a novel therapeutic target for diabetic erectile dysfunction. Graphical abstract.

The lncRNA SOX2OT rs9839776 C>T Polymorphism Indicates Recurrent Miscarriage Susceptibility in a Southern Chinese Population.

Genetic susceptibility may be involved in the onset of recurrent miscarriage. Previous studies have shown that some genetic polymorphisms that regulate cell migration are associated with susceptibility to recurrent miscarriage. The SOX2 overlapping transcript (SOX2OT) may regulate the migration and invasion of multiple tumor cells and is related to susceptibility to various diseases. However, whether lncRNA SOX2OT polymorphisms are related to recurrent miscarriage susceptibility is unclear. Therefore, we investigated the relationship between the lncRNA SOX2OT rs9839776 C>T polymorphism and recurrent miscarriage susceptibility. We recruited 570 subjects with recurrent miscarriage and 578 healthy control subjects from a population in southern China and used the TaqMan method for genotyping. We found a significant association between the rs9839776 CT genotype in the SOX2OT gene and an increased risk for recurrent miscarriage (CT vs CC: adjusted OR = 1.357, 95%CI = 1.065 - 1.728, P = 0.0134). However, we did not observe any significant associations between the recurrent miscarriage risk and the number of miscarriages in different age groups. In conclusion, our study indicated that the rs9839776 CT genotype may contribute to an increased risk of recurrent miscarriage in the southern Chinese population and that rs9839776 may act as a prognostic biomarker in recurrent miscarriage patients. However, an experiment-based study with a larger sample size should be performed to confirm these results.

The lncRNA MALAT1 rs619586 G Variant Confers Decreased Susceptibility to Recurrent Miscarriage.

Cardiovascula disease and recurrent miscarriage have shared risk factors, and some cardiovascular disease-related candidate genes have been confirmed to be associated with recurrent miscarriage. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA (lncRNA) that is considered to be associated with susceptibility to cardiovascular disease. However, whether lncRNA MALAT1 polymorphisms are related to recurrent miscarriage susceptibility is unclear. We genotyped three lncRNA MALAT1 polymorphisms (rs591291, rs619586, and rs3200401) in 284 patients and 392 controls using TaqMan methods. Logistic regression was used to evaluate the odds ratios (ORs) and 95% confidence intervals (CIs) adjusted for age. Our results showed that the rs619586 G variant had protective effects against recurrent miscarriage (AG vs. AA: adjusted OR = 0.670, 95% CI = 0.457-0.982, p = 0.040; GG vs. AA: adjusted OR = 0.278, 95% CI = 0.079-0.975, p = 0.046; GG/AG vs. AA adjusted OR = 0.621, 95% CI = 0.429-0.900, p = 0.012). In a combined analyses of protective genotypes, with regard to the three single nucleotide polymorphisms (SNPs), we found that individuals with two or three protective genotypes exhibited a significantly lower risk of recurrent miscarriage than those with no or only one protective genotype (adjusted OR = 0.369, 95% CI = 0.199-0.684, p = 0.002). Moreover, the decrease in recurrent miscarriage risk with two or three protective genotypes was most pronounced in women less than 35 years of age (OR = 0.290, 95% CI = 0.142-0.589, p < 0.001) and in women with 2-3 miscarriages (adjusted OR = 0.270, 95% CI = 0.126-0.580, p < 0.001). In conclusion, our study suggests that the rs619586 G variant may have potential protective effects conferring a decreased risk of recurrent miscarriage in the southern Chinese population.

The lncRNA CCAT2 rs6983267 G allele is associated with decreased susceptibility to recurrent miscarriage.

Genetics might play various roles in susceptibility to recurrent miscarriage, and previous studies suggest that some gene polymorphisms might be associated with abortion and breast cancer onset. Colon cancer-associated transcript 2 (CCAT2) is a novel long noncoding RNA (lncRNA) transcript that might be correlated with susceptibility to multiple cancers, including breast cancer. However, whether lncRNA CCAT2 polymorphisms are related to susceptibility to recurrent miscarriage is unclear. We genotyped two lncRNA CCAT2 polymorphisms (rs6983267 and rs3843549) in 248 patients with recurrent miscarriage and 392 controls through a TaqMan real-time polymerase chain reaction assay, and the strength of each association was evaluated via 95% confidence intervals (CIs) and odds ratios (ORs). Our results showed that the rs6983267 G allele in lncRNA CCAT2 was associated with decreased susceptibility to recurrent miscarriage (TG vs. TT: adjusted OR = 0.603; 95% CI = 0.420-0.866; p = 0.0062; GG/TG vs. TT: adjusted OR = 0.620; 95% CI = 0.441-0.873; p = 0.0061). The combined analysis of the two protective polymorphisms (rs3843549 AA and rs6983267 TG/GG) revealed that individuals with two unfavorable alleles exhibited a lower risk of recurrent miscarriage than those with no or only one unfavorable allele (adjusted OR = 0.531; 95% CI = 0.382-0.739). Moreover, the decreased risk associated with the two protective alleles was most obvious in women aged less than 35 years (OR = 0.551; 95% CI = 0.378-0.8803; p = 0.0019) and in women with two to three miscarriages (adjusted OR = 0.466; 95% CI = 0.318-0.683; p < 0.0001). In conclusion, our study indicates that the rs6983267G allele might contribute to a decreased risk of recurrent miscarriage in the South Chinese population.

Immature platelets and antiplatelet therapy response to aspirin in Kawasaki disease.

INTRODUCTION: Kawasaki disease is a kind of systemic vasculitis that mainly damages moderate and small-sized blood vessels, and is a leading cause of coronary artery lesions (CAL). Antiplatelet therapy is a routine component of Kawasaki disease treatment strategies. So it is important to evaluate the antiplatelet effect of aspirin because of the individual biological variability of antiplatelet effect of aspirin. The immature platelet fraction (IPF) has attracted particular attention as it may influence the antiplatelet effect of aspirin. This study investigated the prognostic factors for evaluating the degree of vasculitis and the effect of antiplatelet therapy in children with Kawasaki disease. MATERIALS AND METHODS: Blood samples were collected from 44 patients with Kawasaki disease before aspirin treatment and 7 to 10 days after treatment. The IPF counts, percentage of the IPF, and highly fluorescent IPF were detected by a Sysmex XE-5000 instrument. The levels of 11-dehydrothromboxane B2 (11-DH-TXB2), soluble CD40 ligand (sCD40L), and soluble P-selectin (sP-selectin) were measured by ELISA. The correlation between the measured factors and the degree of coronary artery damage in Kawasaki disease was analyzed. RESULTS: We found that 11-DH-TXB2, sP-selectin, and sCD40L levels were much more elevated in the CAL group than in the non-coronary artery lesions (NCAL) group before aspirin treatment. The concentrations of 11-DH-TXB2, sCD40L, sP-selectin, and IPF were reduced after aspirin treatment in the NCAL group but not the CAL group. This is related to the degree of coronary artery damage in Kawasaki disease patients. Additionally, 11-DH-TXB2, sCD40L, sP-selectin, and IPF were positively correlated with the degree of coronary artery damage in Kawasaki disease patients. CONCLUSION: The current study suggests that the presence of high plasma concentrations of 11-DH-TXB2, sCD40L, sP-selectin, and IPF can be considered a risk factor and experimental biomarker for CAL in Kawasaki disease patients.

Kallistatin inhibits lymphangiogenesis and lymphatic metastasis of gastric cancer by downregulating VEGF-C expression and secretion.

BACKGROUND: Tumor-induced lymphangiogenesis and lymphatic metastasis are predominant during the metastasis of many types of cancers. However, the endogenous inhibitors that counterbalance the lymphangiogenesis and lymphatic metastasis of tumors have not been well evaluated. Kallistatin has been recognized as an endogenous angiogenesis inhibitor. METHODS AND RESULTS: Our recent study showed for the first time that the lymphatic vessel density (LVD) was reduced in lung and stomach sections from kallistatin-overexpressing transgenic mice. Kallistatin expresses anti-lymphangiogenic activity by inhibiting the proliferation, migration, and tube formation of human lymphatic endothelial cells (hLECs). Therefore, the present study focuses on the relationships of changes in kallistatin expression with the lymphangiogenesis and lymphatic metastasis of gastric cancer and its underlying mechanisms. Our results revealed that the expression of kallistatin in cancer tissues, metastatic lymph nodes, and plasma of gastric cancer patients was significantly downregulated and that the plasma level of kallistatin was negatively associated with the phase of lymph node metastasis. Furthermore, treatment with kallistatin recombinant protein decreased LVD and lymph node metastases in the implanted gastric xenograft tumors of nude mice. Mechanically, kallistatin suppressed the lymphangiogenesis and lymphatic metastasis by downregulating VEGF-C expression and secretion through the LRP6/IKK/IÒ¡B/NF-Ò¡B signaling pathway in gastric cancer cells. CONCLUSIONS: These findings demonstrated that kallistatin functions as an endogenous lymphangiogenesis inhibitor and has an important part in the lymphatic metastasis of gastric cancer.

Kallistatin exerts anti-lymphangiogenic effects by inhibiting lymphatic endothelial cell proliferation, migration and tube formation.

Kallistatin has been recognized as an endogenous angiogenic inhibitor. However, its effects on lymphatic endothelial cells and lymphangiogenesis remain poorly understood. Lymphangiogenesis is involved in tumor metastasis via the lymphatic vasculature in various types of tumors. The aim of this study was to investigate the effects of kallistatin on lymphangiogenesis and the mechanism of action involved. Treatment with kallistatin recombinant protein or overexpression of kallistatin inhibited the proliferation, migration and tube formation of human lymphatic endothelial cells (hLECs), and induced apoptosis of hLECs. Furthermore, our results showed that the lymphatic vessel density (LVD) was reduced in lung and stomach sections from kallistatin-overexpressing transgenic mice. Treatment with kallistatin recombinant protein decreased the LVD in the implanted gastric xenograft tumors of nude mice. To the best of our knowledge, the present study is the first to demonstrate that kallistatin possesses anti-lymphangiogenic activity in vitro and in vivo. Moreover, kallistatin inhibited proliferation and migration of hLECs by reducing the phosphorylation of ERK and Akt, respectively. These findings suggested that kallistatin may be a promising agent that could be used to suppress cancer metastasis by inhibiting both angiogenesis and lymphangiogenesis.