Fluorescence Quantitative PCR Research Articles

The regulatory effect of electroacupuncture on endometrial M1-type macrophages in rats with intrauterine adhesions.

To observe the effect of electroacupuncture(EA) on endometrial fibrosis and M1-type macrophages in rats with intrauterine adhesions(IUA), so as to explore the possible mechanism of EA in the treatment of IUA. Fifteen female SD rats were randomly divided into blank group, model group and EA group, with 5 rats in each group. The IUA rat model was established by double damage method using mechanical scraping combined with lipopolysaccharide infection. Rats in the EA group were treated with acupuncture at "Guanyuan"(CV4), and EA at bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6)for 20 minutes each time, once a day, for 3 consecutive cycles of estrus. Five rats in each group were sampled during the estrous period, and the endometrial morphology, endometrial thickness and the number of blood vessels and glands were observed after HE staining. The fibrotic area of the uterus was observed after Masson staining. The positive expressions of Runt-related transcription factor(RUNX1), transforming growth factor-β1(TGF-β1), connective tissue growth factor(CTGF), α-smooth muscle actin(α-SMA), collagen type I(Col-Ⅰ), cluster of differentiation 86(CD86), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) in endometrial tissue were detected by immunohistochemistry. Western blot was used to detect relative protein expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF receptor 2 (TNFR2), and real-time fluorescence quantitative PCR was used to detect mRNA expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF-α in the endometrium. During the estrous phase, the endometrial layer in the model group was damaged, with reduced folds, disordered arrangement of epithelial cells, loose fibrous connective tissue, significant narrowing and adhesions in the uterine cavity, interstitial congestion, edema, and a significant infiltration of inflammatory cells with sparse glands. While uterine tissue structure of the EA group was basically intact, resembling a normal uterus, with more newly formed glands and a small amount of inflammatory cell infiltration. In comparison with the blank group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly decreased(P<0.001) in the model group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β, and TNF-α, the protein relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expression levels of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly increased (P<0.001, P<0.01). Compared to the model group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly increased(P<0.01, P<0.05) in the EA group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β and TNF-α in the endometrial tissue, the protein expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly decreased (P<0.001, P<0.01, P<0.05). EA can improve endometrial fibrosis in IUA rats, which may be related to its function in decreasing the level of endometrial M1-type macrophages and the secretion of related inflammatory factors.

The influence of knocking down the expression of low-density lipoprotein receptor associated proteins on the vascular abnormalities in hepatocellular carcinoma and its mechanisms

Objectives: To investigate the effect of the expression of low-density lipoprotein receptor associated protein (LDLR) on the vascular abnormalities in hepatocellular carcinoma (HCC) and its mechanisms. Methods: Based on the information of Oncomine Cancer GeneChip database, we analyzed the correlation between the expression level of LDLR and the expression level of carcinoembryonic antigen (CEA) and CD31 in hepatocellular carcinoma tissues. Lentiviral transfection of short hairpin RNA target genes was used to construct LDLR-knockdown MHCC-97H and HLE hepatocellular carcinoma cells. The differential genes and their expression level changes in LDLR-knockdown hepatocellular carcinoma cells were detected by transcriptome sequencing, real-time fluorescence quantitative polymerase chain reaction, and protein immunoblotting. The gene-related signaling pathways that involve LDLR were clarified by enrichment analysis. The effect of LDLR on CEA was assessed by the detection of CEA content in conditioned medium of hepatocellular carcinoma cells. Angiogenesis assay was used to detect the effect of LDLR on the angiogenic capacity of human umbilical vein endothelial cells, as well as the role of CEA in the regulation of angiogenesis by LDLR. Immunohistochemical staining was used to detect the expression levels of LDLR in 176 hepatocellular carcinoma tissues, and CEA and CD31 in 146 hepatocellular carcinoma tissues, and analyze the correlations between the expression levels of LDLR, CEA, and CD31 in the tissues, serum CEA, and alanine transaminase (ALT). Results: Oncomine database analysis showed that the expressions of LDLR and CEA in the tissues of hepatocellular carcinoma patients with portal vein metastasis were negatively correlated (r=-0.64, P=0.001), whereas the expressions of CEA and CD31 in these tissues were positively correlated ( r=0.46, P=0.010). The transcriptome sequencing results showed that there were a total of 1 032 differentially expressed genes in the LDLR-knockdown group and the control group of MHCC-97H cells, of which 517 genes were up-regulated and 515 genes were down-regulated. The transcript expression level of CEACAM5 was significantly up-regulated in the cells of the LDLR-knockdown group. The Gene Ontology (GO) function enrichment analysis showed that the differential genes were most obviously enriched in the angiogenesis function. The Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis showed that the relevant pathways involved mainly included the cellular adhesion patch, the extracellular matrix receptor interactions, and the interactions with the extracellular matrix receptors. The CEA content in the conditioned medium of the LDLR-knockdown group was 43.75±8.43, which was higher than that of the control group (1.15±0.14, P＜0.001). The results of angiogenesis experiments showed that at 5 h, the number of main junctions, the number of main segments, and the total area of the lattice formed by HUVEC cells cultured with the conditioned medium of MHCC-97H cells in the LDLR-knockdown group were 295.3±26.4, 552.5±63.8, and 2 239 781.0±13 8211.9 square pixels, which were higher than those of the control group (113.3±23.5, 194.8±36.5, and 660 621.0±280 328.3 square pixels, respectively, all P＜0.01).The number of vascular major junctions, the number of major segments, and the total area of the lattice formed by HUVEC cells cultured in conditioned medium with HLE cells in the LDLR-knockdown group were 245.3±42.4, 257.5±20.4, and 2 535 754.5±249 094.2 square pixels, respectively, which were all higher than those of the control group (113.3±23.5, 114.3±12.2, and 1 565 456.5±219 259.7 square pixels, respectively, all P＜0.01). In the conditioned medium for the control group of MHCC-97H cells,the number of main junctions, the number of main segments, and the total area of the lattice formed by the addition of CEA to cultured HUVEC cells were 178.9±12.0, 286.9±12.3, and 1 966 990.0±126 249.5 spixels, which were higher than those in the control group (119.7±22.1, 202.7±33.7, and 1 421 191.0±189 837.8 square pixels, respectively). The expression of LDLR in hepatocellular carcinoma tissues was not correlated with the expression of CEA, but was negatively correlated with the expression of CD31 (r=-0.167, P=0.044), the level of serum CEA (r=-0.061, P=0.032), and the level of serum ALT(r=-0.147,P=0.05). The expression of CEA in hepatocellular carcinoma tissues was positively correlated with the expression of CD31 (r=0.192, P=0.020). The level of serum CEA was positively correlated with the level of serum ALT (r=0.164, P=0.029). Conclusion: Knocking down LDLR can promote vascular abnormalities in HCC by releasing CEA.

Study of metal organic framework with siRNA for overcoming matrix barrierin breast cancer

Objective: This study aimed to develop a new delivery strategy that utilized metal organic framework (MOF) loaded with small-interfering RNA (siRNA) targeting ITGAV to overcome tumor matrix barrier, and thus enhance drug penetration and immune accessibility in breast cancer. Methods: MOF@siITGAV particles were constructed and characterized. The uptake of MOF@siITGAV in breast cancer cell line 4T1 was observed by the cellular uptake assay. The toxicity of MOF@siITGAV was detected by cell counting kit 8 (CCK-8). The blank control group, naked siITGAV group and MOF@siITGAV group were set. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blot were used to detect the expressions of ITGAV. The level of transforming growth factor β1 (TGF-β1) in the cell culture medium was detected by enzyme-linked immunosorbent assay (ELISA). The penetration of MOF@siITGAV in 4T1 cells was tested by constructing 3D spheroids. Mouse models of triple negative breast cancer were established. The effect of MOF@siITGAV on the growth of transplanted tumors and main organs was verified. Imminohistochemical (IHC) was used to test the expression of collagen and CD8. Results: MOF@siITGAV particles were constructed with sizes of (198.0±3.3) nm and zeta potential of -(20.2±0.4) mV. MOF@siITGAV could be engulfed by 4T1 cells and triggered to release siRNA. Compared to the blank control group, the expression of ITGAV in the MOF@siITGAV group [(46.5±11.3)%] and the naked siITGAV group [(109.9±19.0)%] was lower. TGF-β1 in the cell culture medium of the blank control group, naked siITGAV group, and MOF@siITGAV group was (474.5±34.4) pg/ml, (437.2±16.5) pg/ml, and (388.4±14.4) pg/ml, respectively. MOF@siITGAV could better penetrate into 4T1 spheroids and exhibit no obvious toxicity. The cell viability was (99.7±3.5)%, (98.2±5.2)%, (97.3±6.6)%, (92.1±8.1)%, and (92.4±4.1)%, respectively, after MOF@siITGAV treatment with the concentration of 0, 10, 20, 40, 80, and 160 μg/ml, respectively, for 24 h. The tumor growth in the MOF@siITGAV group was suppressed significantly. After 15-day treatment, the tumor volume of the MOF@siITGAV group was (135.3±41.9) mm3, smaller than that of the blank control group [(691.1±193.0) mm3] (P=0.025). The expression of collagen and the number of CD8 positive cells of the MOF@siITGAV group were lower than those of the other two groups. No significant abnormalities were observed in the main organs of mice. Conclusions: Targeting the integrinαv on the surface of cancer cells could destroy extracellular matrix, improve drug delivery, and increase immune infiltration.

Clinical and functional characterization of a novel KCNJ11 (c.101G > A, p.R34H) mutation associated with maturity-onset diabetes mellitus of the young type 13.

This study aimed to describe the clinical features, diagnostic and therapeutic course of a patient with MODY13 caused by KCNJ11 (c.101G > A, p.R34H) and how it contributes to the pathogenesis of MODY13, and to explore new therapeutic targets. Whole-exome sequencing was used to screen prediagnosed individuals and family members with clinically suspected KCNJ11 mutations. Real-time fluorescence quantitative PCR, western blotting, thallium flux of potassium channels, glucose-stimulated insulin secretion (GSIS), and immunofluorescence assays were used to analyze the regulation of insulin secretion by the KCNJ11 mutant in MIN6 cells. Daily blood glucose levels were continuously monitored for 14 days in the proband using the ambulatory blood glucose meter (SIBIONICS). Mutation screening of the entire exon of the gene identified a heterozygous KCNJ11 (c.101G > A, p.R34H) mutation in the proband and his mother. Cell-based GSIS assays after transfection of MIN6 using wild-type and mutant plasmids revealed that this mutation impaired insulin secretory function. Furthermore, we found that this impaired secretory function is associated with reduced functional activity of the mutant KCNJ11 protein and reduced expression of the insulin secretion-associated exocytosis proteins STXBP1 and SNAP25. For the first time, we revealed the pathogenic mechanism of KCNJ11 (c.101G > A, p.R34H) associated with MODY13. This mutant can cause alterations in KATP channel activity, reduce sensitivity to glucose stimulation, and impair pancreatic β-cell secretory function by downregulating insulin secretion-associated exocytosis proteins. Therefore, oral sulfonylurea drugs can lower blood glucose levels through pro-insulinotropic effects and are more favorable for patients with this mutation.

Study on the gut symbiotic microbiota in long- and short-winged brown planthopper, Nilaparvata lugens (Stål).

The brown planthopper (BPH), Nilaparvata lugens (Stål), is one of the most important rice pests in Asia rice regions. BPH has monophagy, migration, rapid reproduction and strong environmental adaptability, and its control is a major problem in pest management. Adult BPH exhibit wing dimorphism, and the symbiotic microbiota enriched in the gut can provide energy for wing flight muscles as a source of nutrition. In order to study the diversity of symbiotic microbiota in different winged BPHs, this paper takes female BPH as the research object. It was found that the number of symbiotic microbiota of different winged BPHs would change at different development stages. Then, based on the 16S rRNA and ITS sequences, a metagenomic library was constructed, combined with fluorescent quantitative PCR and high-throughput sequencing, the dominant symbiotic microbiota flora in the gut of different winged BPHs was found, and the community structure and composition of symbiotic microbiota in different winged BPHs were further determined. Together, our results preliminarily revealed that symbiotic microbiota in the gut of BPHs have certain effects on wing morphology, and understanding the mechanisms underlying wing morph differentiation will clarify how nutritional factors or environmental cues alter or regulate physiological and metabolic pathways. These findings also establish a theoretical basis for subsequent explorations into BPH-symbiont interplay.

Transcriptome analysis reveals key genes and pathways associated with heat stress in Pleurotus pulmonarius.

Pleurotus pulmonarius is a medium temperature edible mushroom, and its yield and quality are severely affected by high temperature. However, the molecular mechanism of Pleurotus pulmonarius response to heat stress remains unknown. In this study, transcriptome sequencing and analysis of Pleurotus pulmonarius mycelia under heat stress were performed, related differentially expressed genes (DEGs) were verified by fluorescence quantitative PCR (qPCR) and the reduced glutathione content was detected. 5906 DEGs, including 1086 upregulated and 4820 downregulated, were identified by RNA-Seq. GO analysis revealed that DEGs were mainly enriched in the pathways of Aminoacyl-tRNA biosynthesis, pyrimidine metabolism, arginine and proline metabolism, fructose and mannose metabolism, and glutathione metabolism. qPCR analysis showed that the expression of ggt decreased after heat stress treatment, while gst2 and gst3 increased. The glutathione content in mycelia after heat stress was significantly higher than that in the control group. These results suggest that glutathione metabolism may play an important role in the response to heat stress. Our study will provide a molecular-level perspective on fungal response to heat stress and a basis for research on fungal environmental response and molecular breeding.

Acupuncture modulates the AMPK/PGC-1 signaling pathway to facilitate mitochondrial biogenesis and neural recovery in ischemic stroke rats

AimsThe main objective of this study was to investigate the role and mechanism of acupuncture on anti-nerve injury in the acute phase by regulating mitochondrial energy metabolism via monophosphate-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) axis in rat ischemic stroke.Main methodsMiddle cerebral artery occlusion (MCAO) was established by middle cerebral artery occlusion/reperfusion. One-week of acupuncture was performed during the acute phase of ischemic stroke. The neurological function and brain tissue integrity were evaluated. Mitochondrial function (intracellular ATP level and the activity of mitochondrial respiratory chain complex I) and the level of NADH oxidase (NOX) were detected by enzymatic chemistry. Next, the potential molecular mechanisms were explored by western blotting, fluorescence quantitative PCR and immunohistochemistry method.Key findings(1) Acupuncture treatment for MCAO/R rats showed a significant improvement in the infarcted tissue accompanied by functional recovery in Zea-Longa score and balance beam score outcomes, motor function performances. (2) Acupuncture increased the levels of ATP and mitochondrial respiratory chain complex I, decreased the NOX levels in cerebral ischemia established by suture-occluded method. (3) Acupuncture reduced the necrosis dissolution of neuronal cells and meningeal edema, while promoting angiogenesis. (4) Quantitative immunohistochemical staining results showed acupuncture can increase the expression of AMPK, p-AMPK and the mitochondrial transcription factor PGC-1α, NRF2, TFAM and uncoupling protein 2 (UCP2). Meanwhile, acupuncture treatment up-regulated the expression of the corresponding protein. (5) Subsequently, acupuncture enhanced AMPK phosphorylation as well as the expression of PGC-1α, NRF2, TFAM and UCP2, implicated in mitochondrial synthesis and cellular apoptosis. (6) Finally, injections of AMPK antagonists and activators confirmed AMPK as a therapeutic target for the anti-nerve damage effects of acupuncture.SignificanceAcupuncture intervention relieved ischemic stroke progression in MCAO rats by promoting energy metabolism and mitochondrial biogenesis in the brain and alleviating neuronal apoptosis, which was mediated by eliciting AMPK/PGC-1α axis, among them AMPK is a therapeutic target.

A comprehensive evaluation of serum circCSPP1 as a novel diagnostic and prognostic biomarker for gastric cancer

PurposeGastric cancer (GC) has high incidence and mortality due to its low early screening efficiency. Circular RNAs (CircRNAs) are a new class of non-coding RNAs which is closely related to GC. Nevertheless, the clinical application value of circRNAs in GC are largely unknown. Therefore, we studied the role of a novel circRNA named circCSPP1 in patients with GC. MethodsCircRNA sequencing was performed to screen out the target molecule. Real-time fluorescent quantitative PCR (RT-qPCR) was utilized to detect the expression level of circCSPP1 in GC tissues, cells, and serum. Gel and Sanger sequencing were utilized to verify the ring structure of circCSPP1. RNase R enzyme digestion experiment and actinomycin D experiment were verifed the advantage of circCSPP1 as a diagnostic biomarker in patients with GC when that compared with linear RNA. The correlation between the expression level of serum circCSPP1 and clinicopathological data of GC patients was further analyzed. Receiver operating characteristic curve (ROC) and the area under ROC curve (AUC) were utilied to evaluate the diagnostic performance. ResultsCircCSPP1 has a circular structure which with resistance to RNA exonuclease digestion and long half-life compared with linear RNA. In our study, circCSPP1 was first found up-regulated in patients with GC. Serum circCSPP1 level was decreased significantly after surgical resection whereas increased after recurrence. High expression of circCSPP1 was associated with poor survival rates. The expression level of circCSPP1 was significantly correlated to tumor size, T stage, lymph node metastasis, and TNM stage. The AUC of serum circCSPP1 was 0.834, with high sensitivity and specificity in discriminating patients with GC from healthy donors. More importantly, the combined diagnosis of circCSPP1, CEA, and CA19–9 achieved the superior AUC of 0.882, with the highest specificity. ConclusionSerum circCSPP1 may prove to be a potential non-invasive auxiliary diagnostic biomarker for patients with GC.

The effect of miR-23b-3p on regulating GH by targeting POU1F1 in Yanbian yellow cattle

This study aimed to evaluate the effect of miR-23b-3p on growth hormone (GH) in pituitary cells of Yanbian yellow cattle. The mRNA and protein levels of GH and miR-23b-3p target genes were measured by real time fluorescence quantitative PCR (qPCR) and Western blot, respectively. The target relationship of miR-23b-3p was validated by double luciferase reporter gene system. The results showed that GH mRNA and protein levels in pituitary cells of Yanbian yellow cattle were significantly lower in the miR-23b-3p-mi group than in the NC group (P<0.01), while GH mRNA and protein levels were higher in the miR-23b-3p-in group than in the iNC group (P<0.05). The result of bioinformatics analysis and double luciferase reporter gene system validation proved that miR-23b-3p targeted 3′UTR of pituitary specific transcription factor 1 (POU1F1). POU1F1 mRNA and protein levels were lower miR-23b-3p-mi group than in the NC group (P<0.01), while POU1F1 mRNA and protein levels were higher in the miR-23b-3p-in group than in the iNC group (P<0.01). These results demonstrated that miR-23b-3p could regulate GH expression in pituitary cells by regulating POU1F1 gene.

Antioxidative effect of wheat-grain moxibustion on cyclophosphamide-induced liver injury in mice based on Nrf2-Keap1 signaling pathway

To observe the protective effect of wheat-grain moxibustion on cyclophosphamide (CTX)-induced liver injury in mice, and explore its mechanism based on the nuclear factor E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signaling pathway. Twenty-four male CD-1 (ICR) mice were randomly divided into a blank group, a model group, and a moxibustion group, with 8 mice in each group. The mice in the model group and the moxibustion group were intraperitoneally injected with CTX (80 mg/kg) to induce liver injury. The mice in the moxibustion group were treated with wheat-grain moxibustion at "Guanyuan" (CV 4) and bilateral "Zusanli" (ST 36) and "Sanyinjiao" (SP 6), with each acupoint being treated by 3 cones, approximately 30 seconds per cone, once daily for 7 days. After intervention, the general condition of the mice was observed; the liver mass was measured and the liver index was calculated; HE staining was used to observe the morphology of the liver, and the liver tissue pathological score was assessed; ELISA was used to detect the serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH) and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) in the liver; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of Nrf2, Keap1, and quinione acceptor oxidoreductase 1 (NQO1) in the liver. Compared with the blank group, the mice in the model group showed sluggishness, unsteady gait, and decreased body weight; liver index was increased (P<0.01); liver cells were loosely arranged, with a small number of cell swollen and exhibiting balloon-like changes; liver tissue pathological score was increased (P<0.05); the serum levels of AST, ALT, GLDH, and level of MDA in the liver were increased (P<0.05), and the levels of SOD and GSH-Px in the liver were decreased (P<0.05); protein and mRNA expression of Nrf2 and NQO1 in the liver was decreased (P<0.01), protein and mRNA expression of Keap1 in the liver was increased (P<0.01). Compared with the model group, the mice in the moxibustion group showed improvement in general condition; liver index was decreased (P<0.01); liver cell structure was relatively intact and clear, and liver tissue pathological score was decreased (P<0.05); the serum levels of AST, ALT, GLDH, and level of MDA in the liver were decreased (P<0.05), and the levels of SOD and GSH-Px in the liver were increased (P<0.05, P<0.01); protein and mRNA expression of Nrf2 and NQO1 in the liver was increased (P<0.05), protein and mRNA expression of Keap1 in the liver was decreased (P<0.05). The wheat-grain moxibustion may alleviate CTX-induced liver injury by activating the Nrf2-Keap1 signaling pathway and enhancing the expression of antioxidative enzyme system in the body.

Genetic analysis for a female carrying idic(Y)(p11.32) with Disorders of sex development

To explore the genetic basis for a patient with Disorders of sex development (DSD). A female patient who had presented at the Linyi People's Hospital due to primary amenorrhea on April 6, 2022 was selected as the study subject. Conventional chromosomal karyotyping, fluorescence in situ hybridization (FISH), chromosomal microarray analysis (CMA), fluorescence quantitative PCR and Sanger sequencing were carried out for the patient. The patient, a 14-year-old female, had featured short statue, multiple nevi, and primary amenorrhea. She was found to have a karyotype of 46,X,idic(Y)(p11.3)[59]/45,X[39]/47,X,idic(Y)(p11.3)×2[2]. The result of FISH assay was 46,X,der(Y).ish idic(Y)(p11.3)(SRY+)[59]/45,X[39]/47,X,der(Y)×2.ish idic(Y)(p11.3)(SRY+)[2]. That of CMA was arr[GRCh37](X)×1,(Y)×0-1,arr[GRCh37]Yp11.32(118552_472090)×1. The patient had no deletion in the AZF region of Y chromosome, and was negative for variant of SRY gene. Combining the above results, her molecular karyotype was determined as mos 46,X,idic(Y)(p11.32)[59]/45,X[39]/47,X,idic(Y)(p11.32)×2[2].ish 46,X,idic(Y)(p11.32)(DXZ1+,DYZ1++,DYZ3++,SRY+)[59]/45,X(DXZ1+,DYZ1-,DYZ3-,SRY-)[39]/47,X,der(Y)×2.ish idic(Y)(p11.32)(DXZ1+,DYZ1++,DYZ3++,SRY+)[2].arr[GRCh37](X)×1, (Y)×0-1，arr[GRCh37]Yp11.32(118552_472090)×1. The patient was diagnosed with mosaicism DSD with idic(Y)(p11.32). The abnormal mosaicism karyotype probably underlay the DSD in this patient.

N1F(Improved-Nephropathy 1 Formula) Ameliorates Renal Interstitial Fibrosis via Inhibiting Extracellular Matrix Deposition and Regulating the FGF23/P38MAPK/Wnt Pathway.

Renal fibrosis is the primary pathway in the progression of chronic kidney disease (CKD) towards end-stage renal failure. The currently used drugs currently are ineffective, and their mechanisms of action remain unclear. This study aims to investigate the nephroprotective effect of Improved-Nephropathy 1 Formula (N1F) in a rat model of unilateral ureteral obstruction (UUO) and explore the potential mechanisms of N1F-containing serum in treating TGF-ß1-induced human renal tubular epithelial cells (HK-2). SD rats received 2-week continuous N1F gavage starting on day 2 after UUO. HK-2 cells were pretreated with a P38MAPK inhibitor for 1 h in vitro, followed by induction of the cells with TGF-ß1 and treatment with N1F 48 h later. The chemical composition of N1F was analyzed using high-performance liquid chromatography-Q-Orbitrap high-resolution liquid mass spectrometry. Renal function was assessed by measuring serum creatinine (Scr), blood urea nitrogen (BUN) and urine protein (Upro) levels. Hematoxylin and eosin (HE) and Masson's trichrome (Masson) staining were used to evaluate the extent of renal tissue damage and fibrosis. Western blotting, immunohistochemistry, and immunofluorescence were used to detect the protein levels of relevant indices. The RNA levels of the relevant indices were detected using real-time fluorescence quantitative PCR (RT-qPCR). We identified 361 chemical components in the water extract of N1F. These chemical components of N1F significantly reduced the area associated with interstitial fibrosis in the kidneys of UUO rats and the levels of serum creatinine, urea nitrogen, and urinary protein. Additionally, N1F decreased the protein levels of FGF23, Wnt1, ß-catenin and p-P38MAPK/P38MAPK, along with the expression of renalfibrosis-associated proteins, α-SMA, FN, Collagen III, and Vimentin in the renal tissues of the UUO rats, while enhancing klotho and DKK1 protein levels. In vitro experiments revealed that inhibition of P38MAPK signaling significantly suppressed the expression of proteins related to the Wnt signaling pathway, with a concomitant decrease in the expression of FGF23 and an increase in the expression of Klotho. Notably, the P38MAPK inhibitor (SB203580) had similar effects to N1F in altering the above-mentioned indices in vitro. N1F may exhibit potential therapeutic efficacy against renal fibrosis by inhibiting the FGF23/P38MAPK/Wnt signaling pathway, consequently inhibiting extracellular matrix deposition due to renal injury.

Study of proanthocyanidin promotes osteogenic differentiation of human periodontal ligament stem cells through the transcription factor EB-induced autophagy-lysosome pathway

Objective: To investigate the mechanism of proanthocyanidin (PA) in regulating the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs), and to explore the effects of PA on the expression and nuclear translocation of transcription factor EB (TFEB) and on the autophagy-lysosome pathway. Methods: PDLSCs were divided into control group and PA group, which were subjected to RNA sequencing analysis (RNA Seq) to detect differentially expressed genes. The osteogenic differentiation ability and autophagy level were observed by real-time fluorescence quantitative PCR (RT-qPCR) analysis, alkaline phosphatase (ALP) staining and transmission electron microscope (TEM), respectively. Scratch assay and Transwell assay were used to detect the migration ability of PDLSCs. Lysotracker and immunofluorescence staining were used to detect the biogenesis of lysosomes. The total protein expression of transcription factor EB (TFEB) as well as that in cytoplasm and nucleus were detected by Western blotting. Confocal laser scanning microscope (CLSM) was used to observe the nuclear translocation of TFEB. The PDLSCs were treated with small interfering RNA (siRNA) technology to knock down the expression levels of TFEB gene with or without PA treatment. Western blotting was used to analyze the expressions of autophagy-related proteins Beclin1 and microtubule-associated protein 1 light chain 3 (LC3B), as well as osteogenic-related proteins runt-related transcription factor 2 (RUNX2), ALP, and osteocalcin in PDLSCs. Results: Compared with the control group, the osteogenic-related and autophagy-related genes showed differential expression in PDLSCs after PA treatment (P<0.05). The mRNA expression levels of osteogenic-related genes RUNX2 (2.32±0.15) and collagen type Ⅰ alpha 1 (COL1α1) (1.80±0.18), as well as the autophagy related genes LC3B (1.87±0.08) and Beclin1 (1.63±0.08) were significantly increased in the PA group, compared with the control group (1.01±0.16, 1.00±0.10, 1.00±0.07, 1.00±0.06, respectively, all P<0.01). Compared with the control group, the PA group had higher ALP activity, and more autophagosomes and autophagolysosomes observed by TEM. PA promoted the migration of PDLSCs (P<0.05) and the increased number of lysosomes and the expression of lysosomal associated membrane protein 1 (LAMP1). In the PA group, the relative expression level of total TFEB protein (1.49±0.07) and the nuclear/cytoplasmic expression of TFEB protein (1.52±0.12) were significantly higher than the control group (1.00±0.11, 1.00±0.13, respectively) (t=6.43, P<0.01; t=5.07, P<0.01). The relative nuclear/cytoplasmic fluorescence intensity of TFEB in the PA group (0.79±0.09) was increased compared with the control group (0.11±0.08) (t=8.32, P<0.01). Knocking down TFEB significantly reduced the expression of TFEB (1.00±0.15 vs 0.64±0.04), LAMP1 (1.00±0.10 vs 0.69±0.09), Beclin1 (1.00±0.05 vs 0.60±0.05), and LC3B Ⅱ/Ⅰ (1.00±0.06 vs 0.73±0.07) in PDLSCs (P<0.05, P<0.05, P<0.01, P<0.01). When TFEB gene was knocked down, the expression levels of Beclin1 (1.05±0.11), LC3B Ⅱ/Ⅰ (1.02±0.09), RUNX2 (1.04±0.10), ALP (1.04±0.16), and osteocalcin (1.03±0.15) proteins were significantly decreased in the PA group compared with the pre-knockdown period (1.28±0.03, 1.44±0.11, 1.38±0.11, 1.62±0.11, 1.65±0.17, respectively) (P<0.05, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). Conclusions: PA promotes the osteogenic differentiation of PDLSCs through inducing the expression and nuclear translocation of TFEB and activating the autophagy-lysosome pathway.

Effect of iron saturation of bovine lactoferrin on the inhibition of hepatitis B virus in vitro.

Hepatitis B virus (HBV) infection poses a major public health problem worldwide. Bovine lactoferrin (bLf) is a natural product that can inhibit HBV, but the effect of iron saturation on its resistance to HBV is unknown. The purpose of this study is to investigate the impact of iron saturation of bLf against HBV. HepG2 cells were cultured in DMEM high glucose containing 10% inactivated fetal calf serum, at 37°C, in 5% CO2. MTT method was used to detect the cytotoxicity of bLf to HepG2 cells. Apo-bLf and holo-bLf were prepared from bLf. Iron saturation of these proteins was determined by atomic absorption spectrophotometry. Non-cytotoxic concentrations of candidate proteins were used in anti-HBV tests. Fluorescent quantitative polymerase chain reaction was used to detect HBV-DNA. The TC50 and TC0of bLf were 54.570mg/ml and 1.997 mg/ml, respectively. The iron saturation of bLf, apo-bLf and holo-bLf were 10.29%, 8.42% and 85.32%, respectively. In this study, four non-cytotoxic concentrations of candidate proteins (1.5, 1.0, 0.5, and 0.1 mg/ml, respectively) were used to inhibit HBV in HepG2 cells. The results showed that 1.5 mg/ml bLf and 0.1 mg/ml holo-bLf effectively impaired the HBV-DNA amplification in HBV-infected HepG2 cells (P < 0.05). However, apo-bLf, and Fe3+ did not show the anti-HBV effects. A total of 1.5 mg/ml bLf and 0.1 mg/ml holo-bLf could inhibit HBV-DNA in HepG2 cells. Complete bLf structure, appropriate concentration and iron saturation of bLf are necessary conditions for anti-HBV effects.

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1

BackgroundInfectious bovine rhinotracheitis (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain’s gC, TK, gG, gD, and gE genes.ResultsThe study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 101 DNA copies/μL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28.ConclusionsThe established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

Predicting the potential mechanism of radix chimonanthi pracecocis in treating osteoarthritis by network pharmacology analysis combined with experimental validation

Ethnopharmacological relevanceRadix Chimonanthi Pracecocis (RCP), also known as Tiekuaizi, widely used by the Miao community in Guizhou, exhibits diverse biological activities and holds promise for the treatment of osteoarthritis (OA). However, there is a lack of contemporary pharmacological research in this area. Aims of the studyThis study aims to explore the potential of targets and mechanisms of RCP in the treatment of OA. Materials and methodsThe chemical components of RCP were identified using UPLC-MS/MS, and active components were determined based on the Lipinski rule. RCP and OA-related targets were retrieved from public databases such as TCMSP and GeneCards. Network pharmacology approaches were employed to identify key genes. The limma package (version 3.40.2) in R 4.3.2 was used to screen for differentially expressed genes (DEGs) between OA and healthy individuals in GSE82107. DEGs were analyzed using an independent sample t-test and receiver operating characteristic analysis in GraphPad Prism 9.5.1. Additionally, molecular docking (SYBYL2.1.1) was used to analyze the binding interactions between the active components and target proteins. Finally, we established a papain-induced osteoarthritis (OA) rat model and treated it with RCP aqueous extract by gavage. We validated relevant indicators using real-time fluorescence quantitative polymerase chain reaction, Western blot, immunohistochemistry, and enzyme-linked immunosorbent assays. ResultsSeven active components and 53 targets were identified. The results of GO and KEGG enrichment analyses confirmed the significant role of RCP in the regulation of pyroptosis. Hypoxia-inducible factor-1α (HIF-1α) was identified as a key gene involved in the main biological functions. Molecular docking analysis revealed that Praecoxin, Isofraxidin, Esculin, and Naringenin can bind to the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) (T-Score >5). Additionally, Praecoxin can bind to HIF-1α (T-Score >5). In vivo experiments demonstrated that RCP significantly affects the NLRP3 inflammasome, which is regulated by the HIF-1α pathway. RCP inhibited pyroptosis and reduced synovial inflammation. ConclusionsThis study confirmed the efficacy of RCP aqueous extract in the treatment of OA and identified seven active components (esculin, dihydrokaempferol, naringenin, praecoxin, carnosol, hydroxyvalerenic acid, isofraxidin) that may play an anti-pyroptosis role in the treatment of OA by downregulating the expression of HIF-1α and NLRP3 inflammasome.

Analyzing the expression and clinical significance of CENPE in gastric cancer

BackgroundGastric cancer (GC) is a prevalent type of malignant gastrointestinal tumor. Many studies have shown that CENPE acts as an oncogene in some cancers. However, its expression level and clinical value in GC are not clear.MethodsObtaining clinical data information on gastric adenocarcinoma from TCGA and GEO databases. The gene expression profiling interaction analysis (GEPIA) was used to evaluate the relationship between prognosis and CENPE expression in gastric cancer patients. Utilizing the UALCAN platform, the correlation between CENPE expression and clinical parameters was examined. Functions and signaling pathways of CENPE were analyzed using the Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA). The association between immunological infiltrating cells and CENPE expression was examined using TIMER2.0. Validation was performed by real-time quantitative PCR (qPT-PCR) and immunohistochemical analysis.ResultsAccording to the analysis of the GEPIA database, the expression of CENPE is increased in gastric cancer tissues compared to normal tissues. It was also found to have an important relationship with the prognosis of the patient (p<0.05). The prognosis was worse and overall survival was lower in individuals with increased expression of CENPE. In line with the findings of the GEPIA, real-time fluorescence quantitative PCR (qPT-PCR) confirmed that CENPE was overexpressed in gastric cancer cells. Furthermore, It was discovered that H. pylori infection status and tumor grade were related to CENPE expression. Enrichment analysis revealed that CENPE expression was linked to multiple biological functions and tumor-associated pathways. CENPE expression also correlated with immune-infiltrating cells in the gastric cancer microenvironment and was positively connected to NK cells and mast cells. According to immunohistochemical examination, paracancerous tissues had minimal expression of CENPE, but gastric cancer showed significant expression of the protein.ConclusionsAccording to our findings, CENPE is substantially expressed in GC and may perhaps contribute to its growth. CENPE might be a target for gastric cancer therapy and a predictor of a bad prognosis.

High-throughput fluorescence quantification method based on inner filter effect and fluorescence imaging analysis

The application of the inner filter effect (IFE) in fluorescent substance determination is gaining popularity. In this paper, a theory of the fluorescence distribution along with the excitation light path is derived from our previous research about the spatial micro-element method. According to the relationship between the summation of fluorescence intensities along the vertical direction at a certain position on the excitation light path and the position, a high-concentration and wide-range fluorescent substance quantification method based on the IFE and fluorescence imaging analysis is proposed. Correspondingly, a high-throughput fluorescent substance quantification detection system is constructed. In order to validate the method, solutions of rhodamine B in different concentrations are used for principle validation, concentration prediction, and experimental investigation on the influence of integration time and lens distortion. The high-throughput system enables the simultaneous measurement of six samples, realizing the high-concentration and wide-range quantification of rhodamine B (100–600 mg/L) with high precision (R2 = 0.9992, MRE = 2.34 %). By setting the filter wheel, the system can measure the concentration of fluorescent substances with different emission wavelengths. The improvement of experimental device is expected to reduce the single sample capacity to tens of microliters and increase the overall sample quantity to tens or even hundreds. The proposed method and system are beneficial to fluorescence measurement in fields such as biomedicine and dye research and to the improvement of high-throughput fluorescence quantitative PCR instruments.

Pathogen identification and epidemic factor analysis of yellow catfish, Pelteobagrus fulvidraco, red lower jaw disease

Since 2020, another type of epidemic disease, ‘red lower jaw disease’ (RLJD), has broken out in yellow catfish Pelteobagrus fulvidraco every spring in China, causing large-scale deaths and significant economic losses. This new disease has the characteristics of strong infectivity, high mortality and a single host. To prevent and control RLJD, epidemiological investigation, transmission electron microscopy (TEM), virome sequencing, Koch's rule verification and epidemic factor investigation were studied in this article. The clinical symptoms of diseased yellow catfish were pale gill filaments, hyperaemia and swelling of the spleen and kidney. TEM results revealed that a large number of 35–40 nm spherical virus particles without envelope structure were found in the cytoplasm of spleen cells. The liver, spleen and kidney of the diseased fish had serious lesions. After virome sequencing, the calicivirus reads in the unique virus reads accounted for 99.5% of the unique virus reads in the diseased fish, which provided a relevant reference value for identifying the pathogen of RLJD. The full-length sequence of yellow catfish calicivirus (YCCV) obtained at 7405 bp, including the RNA-helicase domain, RdRp-1 domain and d1iqqa domain. At the same time, both PCR and TaqMan fluorescence quantitative PCR detection method of YCCV were established. Through the artificial infection test, YCCV was verified as the pathogen for RLJD. It was also found that YCCV mainly harmed the fish within 1 year, whereas the fish above 2 years had good resistance. The water temperature of the RLJD outbreak was mainly concentrated at 18 °C–24 °C. In the current study, after YCCV was verified as the pathogen of RLJD, epidemic factors including fish age and water temperature were studied, to the best of our knowledge, for the first time to provide a relevant theoretical basis for the pathogenic mechanism and prevention and control measures of yellow catfish RLJD.

PGRN is involved in macrophage M2 polarization regulation through TNFR2 in periodontitis

Background and objectiveProgranulin (PGRN), a multifunctional growth factor, plays indispensable roles in the regulation of cancer, inflammation, metabolic diseases, and neurodegenerative diseases. Nevertheless, its immune regulatory role in periodontitis is insufficiently understood. This study attempts to explore the regulatory effects of PGRN on macrophage polarization in periodontitis microenvironment.MethodsImmunohistochemical (IHC) and multiplex immunohistochemical (mIHC) stainings were performed to evaluate the expression of macrophage-related markers and PGRN in gingival samples from periodontally healthy subjects and periodontitis subjects. RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were polarized towards M1 or M2 macrophages by the addition of LPS or IL-4, respectively, and were treated with or without PGRN. Real-time fluorescence quantitative PCR (qRT-PCR), immunofluorescence staining (IF), enzyme-linked immunosorbent assay (ELISA), and flow cytometry were used to determine the expressions of M1 and M2 macrophage-related markers. Co-immunoprecipitation was performed to detect the interaction between PGRN and tumor necrosis factor receptor 2 (TNFR2). Neutralizing antibody was used to block TNFR2 to confirm the role of TNFR2 in PGRN-mediated macrophage polarization.ResultsThe IHC and mIHC staining of human gingival slices showed a significant accumulation of macrophages in the microenvironment of periodontitis, with increased expressions of both M1 and M2 macrophage markers. Meanwhile, PGRN was widely expressed in the gingival tissue of periodontitis and co-expressed mainly with M2 macrophages. In vitro experiments showed that in RAW264.7 cells and BMDMs, M1 markers (CD86, TNF-α, iNOS, and IL-6) substantially decreased and M2 markers (CD206, IL-10, and Arg-1) significantly increased when PGRN was applied to LPS-stimulated macrophages relatively to LPS stimulation alone. Besides, PGRN synergistically promoted IL-4-induced M2 markers expression, such as CD206, IL-10, and Arg1. In addition, the co-immunoprecipitation result showed the direct interaction of PGRN with TNFR2. mIHC staining further revealed the co-localization of PGRN and TNFR2 on M2 macrophages (CD206+). Blocking TNFR2 inhibited the regulation role of PGRN on macrophage M2 polarization.ConclusionsIn summary, PGRN promotes macrophage M2 polarization through binding to TNFR2 in both pro- and anti-inflammatory periodontal microenvironments.