[Construction and application of lentivirus overexpression vector with two labeling genes fused with CopGFP and PuroR].

Objective: To construct the lentivirus overexpression vector with two label genes fused with CopGFP and PuroR and to detect the emission of green fluorescence as well as resistance to puromycin in liver cancer cells infected with lentivirus packaged with the above vector. Methods: Firstly, two fragments containing copGFP and PuroR coding sequences were amplified from pCDH-CMV-MCS-copGFP and pLKO.1 respectively; secondly, the two amplified regions were fused with each other by recombinant PCR; thirdly, the fusion DNA fragment was cut and inserted into pCDH-CMV-MCS-copGFP vector, which was linearized with the same restriction endonuclease as used to digest fusion DNA fragment: BamH Ⅰ and Sal Ⅰ. The fusion region in the constructed vector was confirmed by DNA sequencing. The checked vector was co-transfected with package assistant plasmids, namely PLP1, PLP2 and VSVG into in 293T cells and the culture supernatant was subjected to centrifuge and infect liver cancer MHCC97H cells, which were then used to detect their resistance to puromycin (infected cells were treated with 1 mg/ml puromycin for 7 days after infection) and to observe green fluorescence emission in microscope. To determine its efficiency in expressing foreign target protein, the Sp1 coding region was inserted into the MCS sites of the vector, and Sp1 mRNA and protein expression levels were compared with the vehicle vector by RT-qPCR and Western blot. Results: The lentivirus overexpression vector with two label genes fused with CopGFP and PuroR was successfully constructed, and the liver cancer cells infected with lentivirus packaged with the vector expressing two labeling genes fused with CopGFP and PuroRshowed both emission of green fluorescence and resistance to puromycin simultaneously, while cells containing with the vector inserted with Sp1 coding region improved Sp1 mRNA level with 3.3 fold and protein level with 2.2 fold higher in comparison with cells containing the vehicle vector (P＜0.01). Conclusion: The fused label genes consisting of copGFP and PuroR are correctly cloned into the lentivirus vector and confer cells with the ability to emission of green fluorescence and resistance to puromycin, besides, the vector may promote the expression of the target gene with long coding sequence.

Cinnamaldehyde prevents intergenerational effect of paternal depression in mice via regulating GR/miR-190b/BDNF pathway.

Paternal stress exposure-induced high corticosterone (CORT) levels may contribute to depression in offspring. Clinical studies disclose the association of depressive symptoms in fathers with their adolescent offspring. However, there is limited information regarding the intervention for intergenerational inheritance of depression. In this study we evaluated the intervention of cinnamaldehyde, a major constituent of Chinese herb cinnamon bark, for intergenerational inheritance of depression in CORT- and CMS-induced mouse models of depression. Depressive-like behaviors were induced in male mice by injection of CORT (20 mg·kg-1·d-1, sc) for 6 weeks or by chronic mild stress (CMS) for 6 weeks. We showed that co-administration of cinnamaldehyde (10, 20, or 40 mg·kg-1·d-1, ig) for 6 weeks in F0 males prevented the depressive-like phenotypes of F1 male offspring. In addition, co-administration of cinnamaldehyde (20 mg·kg-1·d-1, ig) for 4 weeks significantly ameliorated depressive-like behaviors of chronic variable stress (CVS)-stimulated F1 offspring born to CMS mice. Notably, cinnamaldehyde had no reproductive toxicity, while positive drug fluoxetine showed remarkable reproductive toxicity. We revealed that CMS and CORT significantly reduced testis glucocorticoid receptor (GR) expression, and increased testis and sperm miR-190b expression in F0 depressive-like models. Moreover, pre-miR-190b expression was upregulated in testis of F0 males. The amount of GR on miR-190b promoter regions was decreased in testis of CORT-stimulated F0 males. Cinnamaldehyde administration reversed CORT-induced GR reduction in testis, miR-190b upregulation in testis and sperm, pre-miR-190b upregulation in testis, and the amount of GR on miR-190b promoter regions of F0 males. In miR-190b-transfected Neuro 2a (N2a) cells, we demonstrated that miR-190b might directly bind to the 3'-UTR of brain-derived neurotrophic factor (BDNF). In the hippocampus of F1 males of CORT- or CMS-induced depressive-like models, increased miR-190b expression was accompanied by reduced BDNF and GR, which were ameliorated by cinnamaldehyde. In conclusion, cinnamaldehyde is a potential intervening agent for intergenerational inheritance of depression, probably by regulating GR/miR-190b/BDNF pathway.

Effect of different phosphate binders on fibroblast growth factor 23 levels in patients with chronic kidney disease: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Serum intact fibroblast growth factor 23 (FGF23) levels are progressively increased in relation to the severity of kidney dysfunction. High serum intact FGF23 concentration is associated with the increased cardiovascular morbidity and mortality in patients with chronic kidney disease (CKD). Clinically, phosphate binders are commonly used to reduce serum intact FGF23 levels in CKD patients by lowering serum phosphate levels. It is not clear whether all kinds of phosphate binders can reduce serum intact FGF23 levels, or which kind of phosphate binders is more effective in reducing serum intact FGF23 levels in patients with CKD. The aim of this systematic review and meta-analysis was to compare the efficiency of different kinds of commonly used phosphate binders on serum intact FGF23 levels in patients with CKD. METHODS: Systematic searches were performed through PubMed, Cochrane Central Register of Controlled Trials and Embase from 1999 to 2020. We included the studies performed only in human subjects. All randomized clinical trials (RCTs) were included. Reviews, case reports, letters, commentaries, abstracts and unpublished articles were excluded. Risk of bias was assessed by the Cochrane Collaboration's tool. Random effect was performed in meta-analysis. Meta-regression was used to investigate heterogeneity. RESULTS: Of 1,895 articles, 15 RCTs (comprising 1,098 participants) were included. Common sources of bias were selection bias. Phosphate binders could reduce serum intact FGF23 levels in patients with CKD [standard mean difference (SMD) of total change in serum intact FGF23 levels was 0.91 PG/mL (95% confidence interval: 0.38 to 1.44 PG/mL]. Meta-regression explained 89.02% of heterogeneous sources, indicating that dietary phosphate intake could weaken the effect of phosphate binders on reducing serum intact FGF23 levels, and the effect of phosphate binders on reducing serum intact FGF23 levels in dialysis patients was better than that in early-to-middle CKD patients. DISCUSSION: Phosphate binders can effectively reduce serum intact FGF23 levels in CKD patients, and iron-based phosphate binders have better effect on reducing serum intact FGF23 levels than other phosphate binders.

Atractylodis rhizoma water extract attenuates fructose-induced glomerular injury in rats through anti-oxidation to inhibit TRPC6/p-CaMK4 signaling.

BACKGROUND: Atractylodis rhizoma, an aromatic herb for resolving dampness, is used to treat Kidney-related edema in traditional Chinese medicine for thousands years. This herb possesses antioxidant effect. However, it is not yet clear how Atractylodis rhizoma prevents glomerular injury through its anti-oxidation. PURPOSE: Based the analysis of Atractylodis rhizoma water extract (ARE) components and network pharmacology, this study was to explore whether ARE prevented glomerular injury via its anti-oxidation to inhibit oxidative stress-driven transient receptor potential channel 6 (TRPC6) and its downstream molecule calcium/calmodulin-dependent protein kinase IV (CaMK4) signaling. METHODS: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyze ARE components. Network pharmacology analysis was preliminarily performed. Male Sprague-Dawley rats were given 10% fructose drinking water (100 mL/d) for 16 weeks. ARE at 720 and 1090 mg/kg was orally administered to rats for the last 8 weeks. Hydrogen peroxide (H2O2) and malondialdehyde (MDA) level, and superoxide dismutase (SOD) activity in rat kidney cortex were detected, respectively. In rat glomeruli, redox-related factors forkhead box O3 (FoxO3), SOD2 and catalase (CAT), podocyte slit diaphragm proteins podocin and nephrin, cytoskeleton proteins CD2-associated protein (CD2AP) and α-Actinin-4, as well as TRPC6, p-CaMK4 and synaptopodin protein levels were analyzed by Western Blotting. SOD2 and CAT mRNA levels were detected by qRT-PCR. RESULTS: 36 components were identified in ARE. Among them, network pharmacology analysis indicated that ARE might inhibit kidney oxidative stress. Accordingly, ARE up-regulated nuclear FoxO3 expression, and then increased SOD2 and CAT at mRNA and protein levels in glomeruli of fructose-fed rats. It reduced H2O2 and MDA levels, and increased SOD activity in renal cortex of fructose-fed rats. Subsequently, ARE down-regulated TRPC6 and p-CaMK4, and up-regulated synaptopodin in glomeruli of fructose-fed rats. Furthermore, ARE increased podocin and nephrin, as well as CD2AP and α-Actinin-4, being consistent with its reduction of urine albumin-to-creatinine ratio and improvement of glomerular structure injury in this animal model. CONCLUSIONS: These results suggest that ARE may prevent glomerular injury in fructose-fed rats possibly by reducing oxidative stress to inhibit TRPC6/p-CaMK4 signaling and up-regulate synaptopodin expression. Therefore, ARE may be a promising drug for treating high fructose-induced glomerular injury in clinic.

[Hyperfine structure study of binary sodium phosphates by Raman spectroscopy].

Quantum chemistry ab initio calculation was applied to study the hyperfine structure of binary sodium phosphates. A series of phosphate model clusters were designed to simulate the microstructure of phosphates with different components. Closed-shell Hatree-Fock method (RHF) and the basis sets of 6-31G (d, p) were employed to optimize structures and calculate Raman frequencies of these phosphate model clusters. SIT (stress index of tetrahedron), which was well used in the study of silicate microstructures, had been adjusted before it was introduced to describe the microstructure of binary sodium phosphates. It was suggested that SIT of phosphates has good relationship with the Raman shift of the corresponding structures, which means SIT could also be used to study the hyperfine structure of binary sodium phosphates. Raman spectra of both solid and molten Na5P3O10 were determined in the range of room temperature to 1 473 K. A phase transition between 873 and 1 073 K was observed, and the main peaks of Na5P3O10 shifted to a lower frequency with increasing temperature. The microstructure units of Na5P3O10 crystal are the types of Q1(2) and Q2(11) with a ratio of 2 : 1. With the increase in the temperature, various new microstructure units including Q0 , Q1(1), Q1(2), Q2(11), Q2(12) and Q2(22) appeared while Na5 P3 O10 was heated above the melting point, which led to the broadening and asymmetry of Raman spectra of molten Na5P3O10. It came to an understanding that the microstructure units of phosphates may change at different temperature, especially above the melting points. The introduction of SIT and hyperfine structure could help analyze the Raman spectra of phosphates both qualitatively and quantitatively.