Xianling Gubao Capsule Prevents Cadmium-Induced Kidney Injury

Xianling Gubao Capsule (XGC), a kind of capsule preparation of Chinese herbal officially approved for sale by the National Medical Products Administration (NMPA), has the effect of tonifying kidney and strengthening bones. Although the impact of XGC in treating bone diseases has been widely studied, the effect of XGC in kidney injury is unknown yet. The kidney injury model is established by intraperitoneal injection with cadmium chloride (CdCl2). Before model establishment, each XGC group was pregavaged with XGC for 10 d. After 10 d, CdCl2 was injected intraperitoneally into the model group and each XGC group, each XGC group continued to be gavaged with XGC for 4 weeks, and the control group was gavaged with equal doses of distilled water once daily. The level of serum urea nitrogen (BUN) and serum creatinine (Cr) is evaluated by kit. The effect of XGC on protecting kidney injury in mice with kidney injury is analyzed by histopathology (HE stain), immunohistochemistry (IHC), and real-time fluorescence quantitative PCR (RT-qPCR). The results show that CdCl2 significantly increases the level BUN and Cr in serum and results in remarkable pathological changes in the nephron, including tubule edema, congestion, and necrosis. While oral administration of XGC can significantly decrease BUN and Cr in serum and prevent and protect the kidney from the above injuries. In addition, the protein expression of p-mTOR was remarkably reduced, and the ratio of LC3II/LC3I protein and mRNA was significantly increased in mice with oral administration of XGC. Our findings suggest that XGC can prevent and protect kidney injury by improving the state of renal tubular hyperemia and necrosis and reduce the level of BUN and Cr in cadmium poisoning mice.

Feasibility study of fluorescence quantitative PCR for the detection of microecological dynamics in fermented maize stover feeds

During the fermentation of corn stalk bio-feed, the quantity of bacteria in corn stalk bio-feed was counted by fluorescence quantitative PCR and plate colony counting respectively. The comparative analysis of these studies was used to explore the feasibility of fluorescence quantification methods and changes in microbiota during fermentation. The results showed that the standard deviation of fluorescence quantitative method was smaller than that of plate method, but the trend was similar. The biomass of Bacillus subtilis, Lactobacillus plantarum and Saccharomyces cerevisiae reached their maximum on the third, fifth and fifth day respectively, and then decreased gradually and maintained at a certain level. The experiment showed that the fluorescence quantitative PCR method can accurately quantify the number of bacteria in corn stalk bio-feed, and it is a better method to quantitatively detect the dynamic changes of different kinds of bacteria in corn stalk bio-feed.

Identification of Lactic Acid Bacteria in the corn stalk Fermentation by Fluorescence Quantitative PCR

Lactic acid bacteria play an important role in the fermentation of biological feed. In this experiment, Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus casei and Lactobacillus rhamnosus were used to ferment corn stalk by single and mixed bacteria. The changes of Lactobacillus flora during fermentation were analyzed by fluorescence quantitative PCR. The results showed that the maximum number of lactic acid bacteria appeared on the 7th day of mixed fermentation, and the total bacteria content could be significantly increased by adding five kinds of lactic acid bacteria at the same time, and the number of each bacteria in mixed fermentation was close to that measured by single fermentation, which indicated that these five kinds of lactic acid bacteria could cooperate during fermentation and help to improve the quality of stalk fermented feed.

Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV). Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR. Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19×102 copies/μL, 3.89×101 copies/μL and 3.74×101 copies/μL, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.

The Downregulation of Placental Lumican Promotes the Progression of Preeclampsia

Multiple pieces of evidence illustrate that impaired trophoblast function results in preeclampsia (PE), and migration/invasion of human trophoblast cells is stringently regulated by extracellular matrix (ECM) components. Many studies have indicated abnormal expressions of placental ECM components are associated with preeclampsia. However, the change and influence of lumican, a vital member of extracellular matrix (ECM) molecules, on trophoblast cells during preeclampsia remain unclear. This study examines the possibility that the roles of lumican in trophoblast cells contribute to PE. To address this issue, the expression of lumican in human placental tissues was observed using immunohistochemistry, fluorescence quantitative PCR, and Western blot technology. After the HTR-8/SVneo cell line was transfected with pcDNA3.1-human lumican, pGPU6-human lumican shRNA, and their negative controls, the impact of lumican on the HTR-8/SVneo cell line was investigated. Lumican was expressed in human placental tissues. Compared with the control group, its expression was significantly lower in PE placentas. Lumican downregulation inhibited cell proliferation significantly and reduced Bcl-2 expression, but increased P53 expression. These results indicate that the downregulation of placental lumican may drive PE development via promoting the downregulation of Bcl-2 expression and upregulation of P53.

Method for Solving Non-specific Amplification Interference of Fluorescence Quantitative PCR in Gene Detection

Objective: To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection. Method: A three-step method was used for amplification, and the quantitative fluorescence signal collection process was set in the extension stage. Results: Three-step amplification has the advantages of wide application range; improved accuracy; and reduced primer design requirements. Conclusion: The interference of non-specific amplification signals was effectively avoided, the melting curve plotting process was omitted, the reaction time was shortened, and the detection accuracy was improved.

Selection and Validation of the Optimal Panel of Reference Genes for RT-qPCR Analysis in the Developing Rat Cartilage

Real-time fluorescence quantitative PCR (RT-qPCR) is widely used to detect gene expression levels, and selection of reference genes is crucial to the accuracy of RT-qPCR results. Minimum Information for Publication of RT-qPCR Experiments (MIQE) proposes that using the panel of reference genes for RT-qPCR is conducive to obtaining accurate experimental results. However, the selection of the panel of reference genes for RT-qPCR in rat developing cartilage has not been well documented. In this study, we selected eight reference genes commonly used in rat cartilage from literature (GAPDH, ACTB, 18S, GUSB, HPRT1, RPL4, RPL5, and SDHA) as candidates. Then, we screened out the optimal panel of reference genes in female and male rat cartilage of fetus (GD20), juvenile (PW6), and puberty (PW12) in physiology with stability analysis software of genes expression. Finally, we verified the reliability of the selected panel of reference genes with the rat model of intrauterine growth retardation (IUGR) induced by prenatal dexamethasone exposure (PDE). The results showed that the optimal panel of reference genes in cartilage at GD20, PW6, and PW12 in physiology was RPL4 + RPL5, which was consistent with the IUGR model, and there was no significant gender difference. Further, the results of standardizing the target genes showed that RPL4 + RPL5 performed smaller intragroup differences than other panels of reference genes or single reference genes. In conclusion, we found that the optimal panel of reference genes in female and male rat developing cartilage was RPL4 + RPL5, and there was no noticeable difference before and after birth.