Expression, purification, and functional verification of recombinant human glycoprotein hormone beta 5/alpha 2 fusion protein in CHO-S cells / 中国药理学通报

Aim To express and purify recombinant hCGH-CTP fusion protein in high-density suspension culture of Chinese hamster ovary cells (CHO-S), and to verify the lipid accumulation effect of rhCGH-CTP on 3T3-L1 mature adipocytes. Methods The recombinant protein expression vector (pcDNA3. 1-rhCGH-CTP) was constructed, achieved by fusing the human glycoprotein hormone beta 5/alpha 2 cDNA with CTP Linker. The expression plasmid was transiently transfected into the suspended CHO-S to express rhCGH-CTP protein and then purified, and the protein biological activity was verified. Intervention with 3T3-L1 mature adipocyte cells for 24 h was performed to detect the changes of intracellular triglyceride (TG) level. Results Western blot results showed that rhCGH-CTP protein was successfully expressed in CHO-S cells, and the yield was up to 715. 4 mg • L~ . The secreted protein was purified by AKTA pure system with higher purity that was up to 90% as identified by SDS-PAGE. In addition, the intracellular cAMP content of mature adipocytes with high expression of TSHR gene significantly increased after intervention with different concentrations of rhCGH-CTP protein by ELISA kit, indicating that rhCGH-CTP protein had biological activity. Oil red 0 staining showed that compared with the control group, the lipid content of mature adipocytes in the intervention groups with different concentrations of rhCGH-CTP protein significantly decreased (P < 0. 05) . Conclusions The rhCGH-CTP protein has been successfully expressed and purified with biological activity, and effectively reduce TG. This research provides an important theoretical basis for further revealing the physiological role of CGH protein and its potential application in clinical practice.

Effect of a novel phosphodiesterase type 5 inhibitor, CPD1, on paraquat-induced lung fibrosis in rats / 中国药理学通报

Aim To investigate the effects of CPD1, a novel phosphodiesterase 5 inhibitor, on lung pathological phenotype and epithelial-mesenchymal transition of alveolar epithelial cells in lung fibrosis model rats caused by paraquat (PQ). Methods Lung fibrosis model was constructed by a single intraperitoneal injection of PQ (30 mg·kg

Effect of a novel phosphodiesterase type 5 inhibitor,CPD1,on unilateral renal interstitial fibrosis caused by ureteric obstruction in mice / 中国药理学通报

Aim To investigate the effects of CPD1,a novel phosphodiesterase 5 inhibitor,on renal pathological phenotype and fibrotic protein expression in renal fibrosis model mice. Methods Male C57BL/6 J mice were divided into three groups randomly(sham group,UUO group and UUO+CPD1 group). Unilateral ureteric obstruction model was constructed by surgery,and CPD1(5 mg·kg-1·d-1)was administered by intragastric administration two hours after the modeling for seven days. HE and Sirius Red staining were used to observe the distribution of tissue structural lesions and fibrosis. Immunohistochemical staining and Western blot were used to detect the expression of fibronectin(FN),α-SMA,collagen-I and kidney injury molecule-1(Kim-1). Results Compared with sham operation group,the renal tubules of mice were dilated and accompanied by a large amount of inflammatory infiltration. Moreover,the expressions of FN,α-SMA,collagen-I and Kim-1 proteins increased significantly(P<0.05)in UUO group. CPD1 treatment improved the kidney structure and decreased the expression of collagen fibers. Furthermore,CPD1 inhibited the expression of FN,α-SMA,collagen-I and Kim-1 markedly(P<0.05). Conclusions Phosphodiesterase 5 inhibitor CPD1 alleviates the progression of renal fibrosis induced by unilateral ureteral obstruction through down-regulating ECM deposition in the extracellular matrix and expression of Kim-1. The specific mechanism remains to be further studied.

Effect of a novel phosphodiesterase type 5 inhibitor, CPD1, on carbon tetrachloride-induced liver fibrosis in mice / 中国药理学通报

Aim To investigate the effects of CPD1, a novel phosphodiesterase 5 inhibitor, on liver pathological phenotype and hepatic stellate cells (HSCs) activation in hepatic fibrosis model mice caused by carbon tetrachloride ( CCl

Expression, purification and functional verification of recombinant human α-galactosidase A in suspension CHO-S / 中国药理学通报

Aim To express and purify rhα-Gal A with a 6 X His tag via using a serum-free expression system in high-density suspension culture of Chinese hamster ovary cells ( CHO-S) , and to verify the scavenging effect of rhα-Gal A on globular trisaccharide ceramide (Gb3 or GL3) . Methods The construction of recombinant protein expression vector, pcDNA4-GLA, was achieved by fusing the human α-galactosidase cDNA, gla, with 6 X His tag and artificial DNA synthesis. The expression plasmid was transfected into the suspended CHO-S to express rhα-Gal A and then purified. Following this procedure, we determined rhα-Gal A's expression, the enzymatic activity, and the glycosylation of the recombinant enzyme. Co-incubation with cultured cells was performed to examine whether rhα-Gal A could be taken up into the cells and effectively remove Gb3 substrates. Results rhα-Gal A was successfully expressed and purified after transiently transfecting pcDNA4-GLA into the suspended CHO-S, and the yield was up to (100 ±20. 6) mg • L

Effect of menthol, a transient receptor potential melastatin-8 channel activator, on pulmonary arterial hypertensive rats / 中国药理学通报

Aim To investigate the effects of menthol, a transient receptor potential melastatin-8 channel activator, on treating pulmonary arterial hypertension (PAH) in PAH model rats caused by monocrotaline (MCT). Methods Male Sprague-Dawley rats were divided into six groups randomly (control group, MCT group, MCT + menthol 1 mg • kg

Effect of FKBP38 protein on endometrial precancerous lesions and mechanism / 中国药理学通报

Aim To build the model of the gene FKBP38(FK506 binding protein 38)conditional knock out in uterus and then investigate the effect on endometrial precancerous lesions and the underlying mechanism.Methods Transgenic mice whose FKBP38 gene was flanked with loxP were constructed by embryo microinjection. The conditional knockout of FKBP38 was obtained by breeding mice harboring two loxP sites in FKBP38(FKBP38

Effect of a novel phosphodiesterase 5 inhibitor, CPD1, on renal interstitial fibrosis after unilateral renal ischemia-reperfusion injury / 生理学报

This study was designed to evaluate the protective effect of CPD1, a novel phosphodiesterase 5 inhibitor, on renal interstitial fibrosis after unilateral renal ischemia-reperfusion injury (UIRI). Male BALB/c mice were subjected to UIRI, and treated with CPD1 once daily (i.g, 5 mg/kg). Contralateral nephrectomy was performed on day 10 after UIRI, and the UIRI kidneys were harvested on day 11. Hematoxylin-eosin (HE), Masson trichrome and Sirius Red staining methods were used to observe the renal tissue structural lesions and fibrosis. Immunohistochemical staining and Western blot were used to detect the expression of proteins related to fibrosis. HE, Sirius Red and Masson trichrome staining showed that CPD1-treated UIRI mice had lower extent of tubular epithelial cell injury and deposition of extracellular matrix (ECM) in renal interstitium compared with those in the fibrotic mouse kidneys. The results from immunohistochemistry and Western blot assay indicated significantly decreased protein expressions of type I collagen, fibronectin, plasminogen activator inhibitor-1 (PAI-1) and α-smooth muscle actin (α-SMA) after CPD1 treatment. In addition, CPD1 dose-dependently inhibited the expression of ECM-related proteins induced by transforming growth factor β1 (TGF-β1) in normal rat kidney interstitial fibroblasts (NRK-49F) and human renal tubular epithelial cell line (HK-2). In summary, the novel PDE inhibitor, CPD1, displays strong protective effects against UIRI and fibrosis by suppressing TGF-β signaling pathway and regulating the balance between ECM synthesis and degradation through PAI-1.

Effect of FKBP38 expression on occurrence and development of nonalcoholic fatty liver disease in mice / 中国药理学通报

Aim To investigate the effects of FKBP38 gene on nonalcoholic fatty liver disease ( NAFLD ) model induced by methionine and choline deficiency j J diet (MCD) in mice.Methods The mutant model of hepatocellular specific deletion of FKBP38 gene was successfully established.The mice were divided into wild-type group ( WT) and homologous knockout group (L-FKBP38 ).Mice were fed with MCD for four weeks to construct NAFLD model.Liver injury was e- valuated by the contents of alanine transaminase (ALT), aspartate transaminase (AST) in the serum samples.We also performed HE staining, examined lipid accumulation by triglyceride (TG) and total cholesterol (CHO) and oil red staining, as well as macrophage infiltration by F4/80 immunohistochemical stai-ning of the liver sections.Fatty acid metabolism-relat ed genes were quantifier] by Quantitative Real-time PCR assays.Results Comparer] with WT group, the levels of ALT, AST, TG and CHO in serum signifi- eantly inereased ( P < 0.05 ) ; liver damage , lipid ac- eumulation, and maerophage infiltration were markedly more severe, and the expressions of fatty aeirl oxidation related genes PPARa, ACOX-1 , CPT-la and SIRT3 markedly rleereaserl ( P < 0.05) in the liver samples of L-FKBP38 group.Conclusions Hepatocellular speeifie deletion of FKBP38 intensifies lipid accumula- tion by inhibiting fatty aeid oxidation in the liver, thus exaeerbating nonaleoholie fatty liver disease.

Effect of a novel phosphodiesterase type 5 inhibitor, CPD1, on vasoconstriction in rats with pulmonary arterial hypertension / 中国药理学通报

Aim To investigate the effect of CPD1 , a novel phosphodiesterase 5 inhibitor, on contractile ten- sion of pulmonary artery and aorta in rats with pulmonary arterial hypertension ( PAH ) .Methods MCT- induced PAH was generated by a single intraperitoneal injection of MCT(50 mg • kg"1) in rats.Seven days after MCT injection, the rats were treated with CPD1 ( 10 mg • kg-1 • d"1) for 14 days.The tension of vascular rings was examined in MCT-induced PAH rats.Results MCT treated rats exhibited profound PAH when examined 3 weeks after injection.In contrast, gavage administration of CPD1 led to significant decrease in the right ventricle systolic pressure ( RVSP) and right ventricular mass index (RVMI), as well as MCT-induced pulmonary arterial wall thinning and enlarged lumen, indicating that CPD1 inhibited the de- velopment of PAH.Cavage administration of CPD1 also reduced phenylephrine and endothelin-1-induced pulmonary artery contraction and aorta contraction in MCT-treated rats.Conclusions Treatment with CPD1 attenuates vascular reactivity, lessens vascular smooth muscle cell proliferation and remodeling, and inhibits PAH via inhibition of non-voltage dependent Ca2∗ channels in normal and PAH rats.

Impact of autophagy gene Atg5 deficiency on sperm maturation / 中国药理学通报

Aim To investigate the role of autophagy core protein Atg5 in maintaining epididymal physiological functions and sperm maturation. Methods The Atg5 conditional knockout mouse model of principle cells in the 4 - 5 segments of the epididymal caput was constructed by Cre/LoxP system. The mice were divided into three groups according to genotype: Atg5v(, Atg5u~ and Atg5~/~ . The pregnancy rate and litter sizes were recorded by mating experiments at the age of 8 weeks-old. Sperm motility, sperm counts were evaluated with computer-aided sperm analysis ( CASA) after 14 weeks of feeding. HE staining was conducted to observe the morphology of the epididymal caput. Western blot and immunofluorescence technologies were applied to verify the expression level of Atg5 and the impediment of autophagy after Atg5 conditional knock out. Results After tissue specific knockout of Atg5 in the 4-5 epididymal principle cells, the autophagy marker proteins, LC3- I and p62, were accumulated and autophagy was successfully blocked. There was no significant difference in the morphology of epididymal tissues of the three genotypes of mice, nor any statistically significant difference in sperm motility, sperm count, litter size and pregnancy rate. Conclusions Under normal conditions without external challenge, Atg5 conditional knockout led to autophagy blocking of epididymal caput, but had no effect on mouse epididymal function and sperm maturation process.

Alteration of tetraethylammonium-sensitive potassium current in pulmonary artery smooth muscle cells of pulmonary hypertension rats / 中国药理学通报

Aim To investigate the alteration of volt-age-depending potassium channel(KV) current in pul-monary arterial smooth muscle cells(PASMCs) of pul-monary hypertension (PH) rats, and the effect of tet-raethylammonium (TEA,a blocker of KV) on potassi-um channel current in different PH models. Methods The whole-cell patch clamp techniques were applied to record the KVcurrents from PASMCs cultured with Ham's F-12 (1% FBS). Furthermore, the effects of TEA on the KVcurrents were examined in different PH models. Results The whole-cell KVcurrents were ob-viously inhibited in PASMCs of chronic hypoxia (CH)and monocrotaline (MCT)-treated rats. TEA signifi-cantly decreased the whole-cell KVcurrents in PASMCs of control and PH rats,and the inhibitory effect of TEA was dramatically reduced in PH group. Conclusions The degree of the voltage-dependent potassium chan-nels opening is significantly inhibited in PASMCs of CH and MCT-treated rats,accordingly,the TEA-sen-sitive KVcurrents obviously decrease.

Comparison of the synchronous changes of vascular tension and intracellular Casignal in third-order branches of mesenteric arteries under the different objective amplification of confocal microscope / 生理学报

This study was aimed to establish an optimized method to observe the synchronous changes of vascular tension and intracellular Casignal in the third-order branches of mesenteric arteries (sMA, diameter: 100-300 μm). The vascular tension and intracellular Casignal changes in response to potassium chloride (KCl), endothelin-1 (ET-1) and Gdwere detected using confocal wire myograph system and confocal laser scanning microscopy imaging technique, respectively. The experimental results were analyzed to explore the optimal experimental conditions. The results showed that KCl caused contraction in sMA significantly, and the intracellular Calevel of vascular smooth muscle cells (VSMCs) was also increased under 20× and 40× objective lens. Compared with those under the 40× objective lens, the Casignal change was larger and the fluorescence value was more stable under the 20× objective lens, whereas the Casignal change was not obvious under the 10× objective lens. ET-1 (1-10 nmol/L) caused concentration dependent contraction in sMA significantly, and the intracellular Casignal of VSMCs was also enhanced in a concentration dependent manner. Additionally, Gdsignificantly reduced the contraction of sMA and the intracellular Casignal of VSMCs caused by ET-1. The results suggest that the intracellular Casignal of VSMCs changes with vascular contraction or relaxation caused by the agonists or antagonists of Cachannels. We successfully recorded both changes synchronously using confocal wire myograph system and confocal laser scanning microscopy imaging technique at the same time. Based on the analysis of the experimental results, we concluded that 20× objective lens provides the best experimental condition. Compared to combination of vascular tone detection method and real-time cellular fluorescence imaging technique, the present synchronous method is convenient and helpful to reduce experimental error.

Comparison of agonists-induced contraction between main and the third-order branches of pulmonary arteries in rats / 生理学报

This study was designed to observe the differences between main pulmonary arteries and the third-order branches of pulmonary arteries in the contractile response to phenylephrine (Phen), endothelin-1 (ET-1) and potassium chloride (KCl). The vascular tension changes of main and the third-order branches of pulmonary arteries induced by KCl, ET-1 and Phen were recorded by traditional vascular tone detection methods and microvascular ring technique, respectively. The results showed that Phen could cause a significant contraction in main pulmonary arteries, but did not induce apparent contraction in the third-order branches of pulmonary arteries. Compared with main pulmonary arteries, ET-1 contracted the third-order branches of pulmonary arteries with reduced maximal response value and PDvalue. In comparison with the main pulmonary arteries, contraction caused by KCl was enhanced in the third-order branches of pulmonary arteries. The results suggest that the vascular reactivity of main and the third-order branches of pulmonary arteries is different and it is important to study the vascular function of small branches of pulmonary arteries. This study could provide an important experimental basis for the further study on vascular function of small branches of pulmonary arteries and the functional changes in pulmonary hypertension.