Berberine ameliorates renal interstitial inflammation and fibrosis in mice with unilateral ureteral obstruction.

Berberine acts via multiple pathways to alleviate fibrosis in various tissues and shows renoprotective effects. However, its role and underlying mechanisms in renal fibrosis remain unclear. Herein, we aimed to investigate the protective effects and molecular mechanisms of berberine against unilateral ureteric obstruction-induced renal fibrosis. The results indicated that berberine treatment (50 mg/kg/day) markedly alleviated histopathological alterations, collagen deposition and inflammatory cell infiltration in kidney tissue and restored mouse renal function. Mechanistically, berberine intervention inhibited NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation and the levels of the inflammatory cytokine IL-1ß in the kidneys of unilateral ureteric obstruction mice. In addition, berberine relieved unilateral ureteric obstruction-induced renal injury by activating adenosine monophosphate-activated protein kinase (AMPK) signalling and promoting fatty acid ß-oxidation. In vitro models showed that berberine treatment prevented the TGF-ß1-induced profibrotic phenotype of hexokinase 2 (HK-2) cells, characterized by loss of an epithelial phenotype (alpha smooth muscle actin [α-SMA]) and acquisition of mesenchymal marker expression (E-cadherin), by restoring abnormal fatty acid ß-oxidation and upregulating the expression of the fatty acid ß-oxidation related-key enzymes or regulators (phosphorylated-AMPK, peroxisome proliferator activated receptor alpha [PPARα] and carnitine palmitoyltransferase 1A [CPT1A]). Collectively, berberine alleviated renal fibrosis by inhibiting NLRP3 inflammasome activation and protected tubular epithelial cells by reversing defective fatty acid ß-oxidation. Our findings might be exploited clinically to provide a potential novel therapeutic strategy for renal fibrosis.

Altered microRNA expression profiles of human spermatozoa in normal fertile men of different ages.

MicroRNAs (miRNAs) are mediators of the aging process. The purpose of this work was to analyze the miRNA expression profiles of spermatozoa from men of different ages with normal fertility. Twenty-seven donors were divided into three groups by age (Group A, n = 8, age: 20-30 years; Group B, n = 10, age: 31-40 years; and Group C, n = 9, age: 41-55 years) for high-throughput sequencing analysis. Samples from 65 individuals (22, 22, and 21 in Groups A, B, and C, respectively) were used for validation by quantitative real-time polymerase chain reaction (qRT-PCR). A total of 2160 miRNAs were detected: 1223 were known, 937 were newly discovered and unnamed, of which 191 were expressed in all donors. A total of 7, 5, and 17 differentially expressed microRNAs (DEMs) were found in Group A vs B, Group B vs C, and Group A vs C comparisons, respectively. Twenty-two miRNAs were statistically correlated with age. Twelve miRNAs were identified as age-associated miRNAs, including hsa-miR-127-3p, mmu-miR-5100_L+2R-1, efu-miR-9226_L-2_1ss22GA, cgr-miR-1260_L+1, hsa-miR-652-3p_R+1, pal-miR-9993a-3p_L+2R-1, hsa-miR-7977_1ss6AG, hsa-miR-106b-3p_R-1, hsa-miR-186-5p, PC-3p-59611_111, hsa-miR-93-3p_R+1, and aeca-mir-8986a-p5_1ss1GA. There were 9165 target genes of age-associated miRNAs. Gene Ontology (GO) analysis of the target genes identified revealed enrichment of protein binding, membrane, cell cycle, and so on. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of age-related miRNAs for target genes revealed 139 enriched pathways, such as signaling pathways regulating stem cell pluripotency, metabolic pathways, and the Hippo signaling pathway. This suggests that miRNAs play a key role in male fertility changes with increasing age and provides new evidence for the study of the mechanism of age-related male fertility decline.

Naringenin regulates gut microbiota and SIRT1/ PGC-1ɑ signaling pathway in rats with letrozole-induced polycystic ovary syndrome.

PURPOSE: To evaluate the effect of naringenin on improving PCOS and explore the mechanism. METHODS: Firstly, we carried out differential gene expression analysis from transcriptome sequencing data of human oocyte to screen the KEGG pathway, then the PCOS-like rat model was induced by letrozole. They were randomly divided into four groups: Normal group (N), PCOS group (P), Diane-35 group (D), and Naringenin group (Nar). The changes of estrus cycle, body weight, ovarian function, serum hormone levels, glucose metabolism, along with the expression of SIRT1, PGC-1ɑ, claudin-1 and occludin of the ovary and colon were investigated. Furthermore, the composition of the gut microbiome of fecal was tested. RESULTS: By searching the KEGG pathway in target genes, we found that at least 15 KEGG pathways are significantly enriched in the ovarian function, such as AMPK signaling pathway, insulin secretion, and ovarian steroidogenesis. Interestingly, naringenin supplementation significantly reduced body weight, ameliorated hormone levels, improved insulin resistance, and mitigated pathological changes in ovarian tissue, up-regulated the expression of PGC-1ɑ, SIRT1, occludin and claudin-1 in colon. In addition, we also found that the abundance of Prevotella and Gemella was down-regulated, while the abundance of Butyricimonas, Lachnospira, Parabacteroides, Butyricicoccus, Streptococcus, Coprococcus was up-regulated. CONCLUSION: Our data suggest that naringenin exerts a treatment PCOS effect, which may be related to the modulation of the gut microbiota and SIRT1/PGC-1ɑ signaling pathway. Our research may provide a new perspective for the treatment of PCOS and related diseases.

High glucose­induced upregulation of CD36 promotes inflammation stress via NF­κB in H9c2 cells.

Cardiac inflammation serves an important role in the progression of diabetic cardiomyopathy. CD36 (cluster of differentiation 36) mediates inflammation stress in a variety of disease states. The present study investigated CD36 expression in high glucose (HG)­induced H9c2 cells, whether CD36 upregulation promotes inflammatory stress, and its potential mechanism. HG induced CD36 expression in a time­dependent manner in cells, which was blocked following CD36 knockout or treatment with N­acetylcysteine or MitoTEMPO. CD36 translocation to the cell membrane was increased at 72 h by HG stimulation of H9c2 cells. Moreover, CD36 knockout inhibited HG­induced reactive oxygen species (ROS) generation, tumor necrosis factor­α, interleukin (IL)­6 and IL­1ß expression, and nuclear factor (NF)­κB pathway activation. Further, CD36 knockout reversed metabolic reprogramming, lipid accumulation and AMP­activated protein kinase activation caused by HG. The aforementioned data suggest that HG­induced upregulation of CD36 promotes inflammatory stress via NF­κB in H9c2 cells, mediated by metabolism reprogramming, lipid accumulation and enhanced ROS generation.

CD36 promotes NLRP3 inflammasome activation via the mtROS pathway in renal tubular epithelial cells of diabetic kidneys.

Tubulointerstitial inflammation plays a key role in the pathogenesis of diabetic nephropathy (DN). Interleukin-1ß (IL-1ß) is the key proinflammatory cytokine associated with tubulointerstitial inflammation. The NLRP3 inflammasome regulates IL-1ß activation and secretion. Reactive oxygen species (ROS) represents the main mediator of NLRP3 inflammasome activation. We previously reported that CD36, a class B scavenger receptor, mediates ROS production in DN. Here, we determined whether CD36 is involved in NLRP3 inflammasome activation and explored the underlying mechanisms. We observed that high glucose induced-NLRP3 inflammasome activation mediate IL-1ß secretion, caspase-1 activation, and apoptosis in HK-2 cells. In addition, the levels of CD36, NLRP3, and IL-1ß expression (protein and mRNA) were all significantly increased under high glucose conditions. CD36 knockdown resulted in decreased NLRP3 activation and IL-1ß secretion. CD36 knockdown or the addition of MitoTempo significantly inhibited ROS production in HK-2 cells. CD36 overexpression enhanced NLRP3 activation, which was reduced by MitoTempo. High glucose levels induced a change in the metabolism of HK-2 cells from fatty acid oxidation (FAO) to glycolysis, which promoted mitochondrial ROS (mtROS) production after 72 h. CD36 knockdown increased the level of AMP-activated protein kinase (AMPK) activity and mitochondrial FAO, which was accompanied by the inhibition of NLRP3 and IL-1ß. The in vivo experimental results indicate that an inhibition of CD36 could protect diabetic db/db mice from tubulointerstitial inflammation and tubular epithelial cell apoptosis. CD36 mediates mtROS production and NLRP3 inflammasome activation in db/db mice. CD36 inhibition upregulated the level of FAO-related enzymes and AMPK activity in db/db mice. These results suggest that NLRP3 inflammasome activation is mediated by CD36 in renal tubular epithelial cells in DN, which suppresses mitochondrial FAO and stimulates mtROS production.

Berberine Reduces Lipid Accumulation by Promoting Fatty Acid Oxidation in Renal Tubular Epithelial Cells of the Diabetic Kidney.

Abnormal lipid metabolism in renal tubular epithelial cells contributes to renal lipid accumulation and disturbed mitochondrial bioenergetics which are important in diabetic kidney disease. Berberine, the major active constituent of Rhizoma coptidis and Cortex phellodendri, is involved in regulating glucose and lipid metabolism. The present study aimed to investigate the protective effects of berberine on lipid accumulation in tubular epithelial cells of diabetic kidney disease. We treated type 2 diabetic db/db mice with berberine (300 mg/kg) for 12 weeks. Berberine treatment improved the physical and biochemical parameters of the db/db mice compared with db/m mice. In addition, berberine decreased intracellular lipid accumulation and increased the expression of fatty acid oxidation enzymes CPT1, ACOX1 and PPAR-α in tubular epithelial cells of db/db mice. The mitochondrial morphology, mitochondrial membrane potential, cytochrome c oxidase activity, mitochondrial reactive oxygen species, and mitochondrial ATP production in db/db mice kidneys were significantly improved by berberine. Berberine intervention activated the AMPK pathway and increased the level of PGC-1α. In vitro berberine suppressed high glucose-induced lipid accumulation and reversed high glucose-induced reduction of fatty acid oxidation enzymes in HK-2 cells. Importantly, in HK-2 cells, berberine treatment blocked the change in metabolism from fatty acid oxidation to glycolysis under high glucose condition. Moreover, berberine restored high glucose-induced dysfunctional mitochondria. These data suggested that berberine alleviates diabetic renal tubulointerstitial injury through improving high glucose-induced reduction of fatty acid oxidation, alleviates lipid deposition, and protect mitochondria in tubular epithelial cells.

The antioxidant peptide SS31 prevents oxidative stress, downregulates CD36 and improves renal function in diabetic nephropathy.

Background: Oxidative stress plays an independent role in the pathogenesis of diabetic nephropathy (DN). CD36, a class B scavenger receptor, mediates reactive oxygen species (ROS) production in DN. SS31 is a mitochondria-targeted antioxidant peptide that can scavenge mitochondrial ROS. The antioxidative effects of SS31 on DN and the interaction between SS31 and CD36 remain poorly understood. Herein, we examined the effects of SS31 and investigated whether SS31 treatment attenuates CD36 expression in db/db diabetic mice and high glucose (HG)-induced HK-2 cells. Methods: Eight-week-old db/m mice and db/db mice were administered with SS31 (3 mg/kg/day) for 12 weeks by intraperitoneal injection. For the in vitro studies, HG-cultured HK-2 cells were used. Biochemical parameters, body weight and histological changes in the mice were measured. The levels of oxidative stress, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Mn superoxide dismutase (MnSOD) and catalase (CAT), and the expression of CD36, nuclear factor-κB (NF-κB) p65 in mice and HK-2 cells were also analyzed. Results: The results showed that SS31 alleviated proteinuria, glomerular hypertrophy and tubular injury, and affected creatinine level in db/db mice. SS31 suppressed the levels of oxidative stress, NADPH oxidase subunits, CD36 and NF-κB p65, and activated MnSOD and CAT in db/db mice and HG-induced HK-2 cells. Conclusion: Taken together, these data demonstrated a renoprotective role of SS31 in DN by suppression of enhanced oxidative stress and CD36 expression.

Noninvasive prenatal testing in routine clinical practice for a high-risk population: Experience from a center.

This study aimed to summarize the effects of noninvasive prenatal testing (NIPT) on aneuploidy among high-risk participants in Tangshan Maternal and Children Health Hospital.NIPT or invasive prenatal diagnosis was recommended to patients with a high risk of fetal aneuploidy from February 2013 to February 2014. Patients who exhibited eligibility and applied for NIPT from January 2012 to January 2013 were included in a comparison group. The rates of patients who underwent invasive testing, declined to undergo further testing, and manifested trisomies 21, 18, and 13 were compared between two groups. Follow-up data were obtained from the participants who underwent NIPT from 2013 to 2014.A total of 7223 patients (3018 and 4205 individuals before and after NIPT) were eligible for analysis. After NIPT was introduced in 2013 to 2014, 727 patients (17.3%) underwent invasive testing, 2828 preferred NIPT (67.3%), and 650 declined to undergo further testing (15.5%). A total of 34 cases of trisomies 21, 18, and 13 (0.8%) were found. In 2012 to 2013, 565 patients (18.7%) underwent invasive testing and 2453 declined to undergo further testing (81.3%). A total of 7 cases of trisomies 21, 18, and 13 were documented (0.2%). Of these cases, 24 were found from NIPT and 10 cases were found from invasive testing. The number of participants who declined to undergo further testing significantly decreased after NIPT was introduced (81.3% vs. 15.5%, P < 0.001). The sensitivity and specificity of NIPT for trisomies 21, 18, and 13 were 100% and 99.9%, respectively. The detection rates of NIPT for trisomies 21, 18, and 13 also significantly increased (0.2% vs. 0.8%, P < 0.001). By contrast, the overall rates of invasive testing remained unchanged (18.7% vs. 17.3%, P = 0.12). The positive predictive values of NIPT for trisomies 21, 18, and 13 were 100%, 83.3%, and 50.0%, respectively. The false positive rates of NIPT were 0% and 0.04%.With NIPT implementation in clinical practice, the rate of declining a follow-up test among high-risk women was decreased and the detection rate of prenatal chromosomal aneuploidy for trisomies 21, 18, and 13 was increased without requiring numerous invasive procedures.

[Effects of α1-PDX, a furin inhibitor, on growth, invasion, and tumorigenicity of cervical cancer HeLa cells].

OBJECTIVE: To investigate the effects of the furin inhibitor α1-PDX on the growth, invasion, and tumorigenicity of cervical cancer cells and explore the mechanisms. METHODS: The changes in the growth, migration and invasion of α1-PDX-transfected HeLa cells were observed using MTT assay, Boyden migration and invasion assay. The protein levels of furin and MT1-MMP were measured using Western blotting and furin activity was detected by enzyme activity assay in the transfected cells. HeLa cells were seeded subcutaneously in nude mice and the tumor volume changes were recorded. RESULTS: Compared with the control cells, α1-PDX-treated cells showed a significant growth inhibition by 18.4% at 24 h (P<0.01) with obviously lowered migration ability and cell invasiveness (P<0.01). Treatment with α1-PDX significantly reduced furin enzyme activity and MTI-MMP protein levels in HeLa cells. In nude mice, α1-PDX-treated HeLa cells exhibited a delayed and lowered tumorigenicity with reduced size of the tumors. CONCLUSION: α1-PDX can inhibit the growth, metastasis and tumorigenicity of HeLa cells, the mechanism of which may involve a decreased furin activity and MTI-MMP expression.

[Analysis of influence of inflammatory factor in patients with ovarian endometriosis follicular fluid on the outcome of in vitro fertilization].

OBJECTIVE: To observe the effect of follicular fluid IL-6, TNF-α on the clinical outcome of in vitro fertilization and embryo transfer (IVF-ET) in patients with ovarian endometriosis. METHOD: From June 2013 to June 2014, the data of 64 (from Tangshan Maternal and Child Health Hospital IVF center) ovarian endometriosis patients was analyzed retrospectively. 58 infertility cases caused by male side were used as control group. Oocyte retrieval rate, M II oocytes rate, fertilization rate, recovery-intracytoplasmic sperm injection (R-ICSI) rate, good quality embryo rate, biochemical pregnancy rate and clinical pregnancy rate were analyzed and compared between two groups. Changes in the expression of follicular fluid IL-6, TNF-α were detected. RESULTS: Oocyte retrieval rate, M II oocytes rate, fertilization rate, good quality embryo rate, biochemical pregnancy rate and clinical pregnancy rate in ovarian endometriosis group were significantly lower than those in the control group (all P < 0.05), while R-ICSI rate increased in ovarian endometriosis group compared with control group (P < 0.05). IL-6, TNF-α expressions of follicular fluid were higher in affected side of ovarian endometriosis patients than those in the unaffected side and those in control group. CONCLUSION: Inflammation microenvironment of the follicular fluid may influence IVF-ET outcomes in ovarian endometriosis patients.

Effects of chemical combinations on the parthenogenetic activation of mouse oocytes.

The aim of this study was to identify an optimal method for the parthenogenetic activation of mouse oocytes. Ethanol (EH), strontium chloride (SrCl2) and ionomycin calcium salt were each combined with cytochalasin B to induce the parthenogenetic activation of CD-1® mouse oocytes. Among the EH combination groups, the blastocyst formation and hatching rates of the group that was activated with EH and CB for 5 min were significantly higher compared with those of the groups that were activated for 7 and 10 min (P<0.05). Among the SrCl2 combination groups, the blastocyst formation and hatching rates of the group that was activated with SrCl2 and CB for 30 min were significantly higher compared with those of the groups that were activated for 1 and 2 h (P<0.05). Among the ionomycin calcium salt combination groups, the blastocyst formation and hatching rates of the group that was activated with ionomycin and CB for 3 min were higher compared with those of the groups that were activated for 5 and 7 min (P<0.05). Compared with the other two combinations, the experimental indicators of the EH combination groups were notably superior (P<0.05). For combined activation, simultaneous activation with two substances was significantly more effective than successive activation (P<0.05). For combined activation with EH and cytochalasin B in mouse oocytes, 5 min of parthenogenetic activation had significant advantages with regard to cleavage, blastocyst formation and blastocyst hatching rates. In addition, the activation rate of combined activation was higher than that of single activators. For combined activation, the simultaneous application of two activators has a superior effect.