The role of endoplasmic reticulum stress in pulmonary hypertension in rat induced by chronic hypoxia and hypercapnia / 中国应用生理学杂志

OBJECTIVE@#To observe the pulmonary vascular remodeling in rats with pulmonary hypertension induced by hypoxia and hypercapnia, and to explore the role of endoplasmic reticulum stress in pulmonary hypertension.@*METHODS@#Forty SD rats were random-ly divided into four groups:normoxic control group (N), hypoxia hypercapnia group (HH), ERS inhibitor 4-phenylbutyric acid group (4-PBA), endoplasmic reticulum stress (ERS) pathway agonist tunicamycin group (TM), ten rats in each group.The mean pulmona-ry artery pressure (mPAP), mean carotid artery pressure (mCAP) and right ventricular hypertrophy index of rats in each group were measured.Pulmonary artery smooth muscle cells were identified by immunofluorescence α-smooth muscle actin (α-SMA).Morphologi-cal changes of lung tissue and pulmonary artery were observed by electron microscope.The apoptotic index of pulmonary artery smooth muscle cells in each group was detected by TUNEL.Reverse transcription polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), c-Jun N-terminal kinase (JNK) and cysteinyl aspartate specific proteinase-12 (caspase-12) mRNA and protein in each group.@*RESULTS@#①Compared with the N group, the mPAP, the ratio of right ventricle weight to left ventricle plus ventricular septum weight[RV/(LV+S)]and the ratio of pulmonary artery wall area to total tube area (WA/TA) were increased (<0.01), and the ratio of pulmonary artery luminal area to total tube area (LA/TA) were decreased (<0.01), pulmonary artery smooth muscle cell apoptosis index were decreased (<0.05 or <0.01) in HH group, 4-PBA group and TM group.ERS related protein and mRNA expressions were increased, the differences were statistically significant.②Compared with the HH group, the mPAP, [RV/(LV+S)]and WA/TA of 4-PBA group were decreased ( <0.01), LA/TA and pulmonary artery smooth muscle cell apoptosis index were increased (<0.01, <0.05).The expressions of ERS related protein and mRNA were all decreased (<0.05 or <0.01).③Compared with the HH group, the mPAP, [RV/(LV+S)]and WA/TA of TM group were increased (<0.05 or <0.01), pulmonary artery middle layer thickened, LA/TA and pulmonary artery smooth muscle cell apoptotic index were decreased (<0.01).ERS related protein and mRNA expressions were increased with statistical significance except GRP78 protein.@*CONCLUSIONS@#Pulmonary vascular remodeling in rats with pulmonary hypertension induced by hypoxia and hypercapnia may be related to the excessive proliferation of pulmonary artery smooth muscle cells and too little apopto-sis;ERS related factors (JNK, caspase-12 and CHOP) are involved in the regulation of pulmonary hypertension induced by hypoxia hypercapnia.

Excessive endoplasmic reticulum stress mediates brain damage in hypoxia hypercapnia induced pulmonary hypertension rats / 生理学报

The purpose of the present study was to investigate the effect of excessive endoplasmic reticulum stress (ERS) on the brain damage in hypoxia hypercapnia induced pulmonary hypertension (HHPH) rats. Forty healthy SPF male SD rats were randomly divided into four groups (n = 10 for each): control group, hypoxia hypercapnia group, ERS pathway agonist tunicamycin (TM) group and ERS pathway inhibitor 4-phenylbutyric acid (4-PBA) group. The rats of control group lived in normal environment, while the rats of other three groups were raised for four weeks in the tank with 8.5%-11% Oand 5%-6% CO. TM (0.08 mg/kg, twice a week) and 4-PBA (80 mg/kg, daily) were respectively intraperitoneally injected into the rats of TM and 4-PBA groups, and the hypoxia hypercapnia group was given the same volume of normal saline. The mean pulmonary artery pressure and heart perfusion of the rats were determined and recorded after four-week raising. Then the brain tissue of the rats were quickly taken out for the brain water content measuring and morphological changes observing. The Caspase-3 activity and the apoptotic index of the brain cells were also determined. The protein and mRNA expressions of p-JNK, Caspase-12, CHOP and GRP78 in brain tissues were detected by Western blot and RT-PCR. The results showed that compared with the control group, the mean pulmonary artery pressure, brain water content and brain cells apoptotic index, Caspase-3 activity, the protein and mRNA levels of p-JNK, Caspase-12, CHOP and GRP78 were increased (P < 0.05), and the brain tissues of the rats were obviously damaged in the rats raised in the hypoxia hypercapnia environment; compared with hypoxia hypercapnia group, the mean pulmonary artery pressure, brain water content, brain apoptotic index and Caspase-3 activity, p-JNK, Caspase-12, CHOP, GRP78 protein and mRNA expressions in TM group were increased (P < 0.05), and the brain tissues of the rats were obviously damaged, while all above changes were relieved in 4-PBA group (P < 0.05). These results suggest that excessive ERS may participate in the brain injury induced by HHPH in rats and inhibition of excessive ERS can relieve the brain injury in the rats with HHPH.

Evaluation of Quality Coherence of Trillium tschonoskii Maxim. from Different Producing Areas Based on HPLC Fingerprint and PCA / 中国中医药信息杂志

Objective To evaluate the quality coherence of Trillium tschonoskii Maxim.from different producing areas by HPLC fingerprint and PCA; To provide a method for quality control. Methods Samples were separated by Hibar C18 (4.6 mm × 250 mm, 5 μm) with acetonitrile-water as gradient mobile phase at the flow rate of 1.0 mL/min. The wavelength was 203 nm and the temperature was 30 ℃. Chromatographic Fingerprint Similarity Evaluation System and PCA were used to analyze the data. Results The results of method validation of HPLC fingerprint met technical standards.15 common peaks was verified and the similarities of 14 batches of Trillium tschonoskii Maxim. from different producing areas were among 0.389–0.979. 3 principal components with the characteristic root cumulative contribution rate reaching 87.674% were screened out by PCA results. The composite score of S2 was the highest (4.926), and the quality was the best. Conclusion The application of HPLC combined with PCA can objectively and effectively evaluate the quality difference of Trillium tschonoskii Maxim.from different producing areas.

Evaluation of Quality Coherence of Trillium tschonoskii Maxim. from Different Producing Areas Based on HPLC Fingerprint and PCA / 中国中医药信息杂志

Objective To evaluate the quality coherence of Trillium tschonoskii Maxim.from different producing areas by HPLC fingerprint and PCA; To provide a method for quality control. Methods Samples were separated by Hibar C18 (4.6 mm × 250 mm, 5 μm) with acetonitrile-water as gradient mobile phase at the flow rate of 1.0 mL/min. The wavelength was 203 nm and the temperature was 30 ℃. Chromatographic Fingerprint Similarity Evaluation System and PCA were used to analyze the data. Results The results of method validation of HPLC fingerprint met technical standards.15 common peaks was verified and the similarities of 14 batches of Trillium tschonoskii Maxim. from different producing areas were among 0.389–0.979. 3 principal components with the characteristic root cumulative contribution rate reaching 87.674% were screened out by PCA results. The composite score of S2 was the highest (4.926), and the quality was the best. Conclusion The application of HPLC combined with PCA can objectively and effectively evaluate the quality difference of Trillium tschonoskii Maxim.from different producing areas.

Role of TRPC6 in pulmonary artery smooth muscle cells proliferation and apoptosis under hypoxia and hypercapnia / 生理学报

The present study was to investigate the role of TRPC6 in pulmonary artery smooth muscle cells (PASMCs) proliferation and apoptosis under hypoxia and hypercapnia. PASMCs were isolated from chloral hydrate-anesthetized male Sprague-Dawley (SD) rats. Cellular purity was assessed by immunofluorescence staining for smooth muscle α-actin under fluorescence microscopy. Passage 4-6 PASMCs were starved for 24 h in serum-free DMEM and divided into 5 groups randomly: normoxia, hypoxia and hypercapnia, DMSO, TRPC6 inhibitor SKF-96365 and TRPC6 activator OAG groups. The normoxic group was incubated under normoxia (5% CO, 21% O, 37 °C) for 24 h, and the others were incubated with corresponding drugs under hypoxic and hypercapnic (6% CO, 5% O, 37 °C) atmosphere for 24 h. TRPC6 mRNA was detected by reverse transcription-PCR. TRPC6 protein was detected by Western blotting. The proliferation of PASMCs was performed by CCK-8 kit. Apoptosis of the PASMCs was detected using TUNEL assay. The [Ca]in the PASMCs was measured using Fura 2-AM fluorescence. The results showed that the expressions of TRPC6 mRNA and protein, and [Ca]were upregulated under hypoxic and hypercapnic conditions. Hypoxia and hypercapnia promoted cellular proliferation and inhibited apoptosis in the PASMCs. OAG enhanced the above-mentioned effects of hypoxia and hypercapnia, whereas SKF-96365 reversed these effects. These results suggest that TRPC6 may play a role in PASMCs proliferation and apoptosis under hypoxia and hypercapnia by regulating [Ca].

Effect of ligustrazine on expression of Fas/FasL in pulmonary injury induced by ischemia/reperfusion in rabbits / 中国应用生理学杂志

<p><b>AIM</b>To investigate the effect of ligustrazine (LGT) on expression of Fas/FasL mRNA during pulmonary ischemia/reperfusion injury (PI/RI) in the rabbits.</p><p><b>METHODS</b>Single lung ischemia/reperfusion animal model was used in this study. The rabbits were randomly divided into three groups (n = 30, in each): sham operated group (Sham), I/R group (I/R) and I/R + LGT group (I/R + LGT). Changes of several parameters which included apoptotic index (AI), wet to dry ratio of lung tissue weight (W/D) and index of quantitative assessment of histologic lung injury (IQA) were measured at 1h, 3h, 5h after reperfusion in lung tissue. Meanwhile the location and expression of Fas/FasL mRNA were observed. Lung tissue was prepared for light microscopic and electron microscopic ob servation at 1 h, 3 h, 5 h after reperfusion.</p><p><b>RESULTS</b>As compared with group I/R, Fas/FasL mRNA slightly expressed in intima and extima of small pulmonary artery, alveoli, and bronchiole epithelia in group LGT. The values of AI, W/D and IQA showed significantly lower in group I/R + LGT than that in group I/R at 1 h, 3 h, 5 h after reperfusion in lung tissue (P < 0.01 and P < 0.05). Meanwhile, abnormal changes of the lung tissue in morphologically were lessen markedly in group I/R + LGT.</p><p><b>CONCLUSION</b>Ligustrazine has notable protective effects on PI/RI in rabbits by inhibiting Fas/FasL mRNA express in lung tissue and decreasing apoptosis.</p>

Effect of L-arginine on expression of PKC mRNA in pulmonary injury induced by ischemia-reperfusion in rabbits / 中华急诊医学杂志

Objective To investigate the effect of L-arginine on expression of protein kinase C(PKC)mRNA during pulmonary ischemia and reperfusion injury(PIRI)in the rabbits.Methods Single lung ischemia and reperfusion animal model was used in vivo.The rabbits were randomly divided into three groups(n=9,in each),sham operated group (Sham),PIR group(I-R)and PIR+L-arginine group(L-Arg).Changes of several rariables including malondialdehyde (MDA),superoxide dismutase(SOD),malandialde hyde(MDA),nitril oxide(NO),wet to dry ratio of lung tissue weight(W/D)and index of quantitative assessment(IQA)of histolngic lung injury were recorded at 60 minutes after reperfusion in lung tissue.Meanwhile the location and expression of PKC mRNA were observed.Lung tissue was prepared for light microscopic and electron microscopic observation at 60 minutes after reperfusion.Results In comparison with group I-R,PKC mRNA strongly expressed in intima and extima of small pulmonary artery as well as thin-waU vessels (mostly small pulmonary veins).The average optical density values of PKC-?,?and?mRNA in small pulmonary veins in L-Arg group had significance(all P＜0.01);SOD increased while MDA,W/D and IQA decreased at 60 minutes after reperfusion in lung tissue(P＜0.01 and P＜0.05).A morphologically abnormal changes of the lung tissue,were lessen markedly in L-Arg group.Conclusion L-arginine possess notably protective effects on PIRI in rabbits by activating PKC-?,?and?mRNA expression in lung tissue,raising NO level,dropping oxygen free radical level and decreasing lipid peroxidation.

Site-saturation Mutagenesis Technology and Its Application in Protein Engineering / 中国生物工程杂志

Site-saturation mutagenesis is a newly-developed technology in protein engineering.By manipulating the encoding genes,it can rapidly obtain the mutants of desired proteins whose target residues are substituted by 19 other common amino acids.Site-saturation mutagenesis could serve not only as a powerful tool in protein engineering,but also as an important method in exploring the structure-function relationship of proteins.Several techniques were summarized to achieve site-saturation mutagenesis and introduce their application status in the protein engineering.The problem and promising future of its application were also discussed.

DNA damage, Bcl-2, Bax expression and ultrastructure change in spermatogenic cell of mice exposed to cadmium / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study DNA damage, Bcl-2 and Bax expression, and ultrastructure change in spermatogenic cell of mice by cadmium exposure.</p><p><b>METHODS</b>Twenty-four male mice were divided into 4 groups: 3 groups treated with cadmium chloride of 1, 5, 10 micromol x kg(-1) x d(-1) i.p. respectively for 5 days, and one normal saline control group. The DNA damage of spermatogenic cell by single-cell gel electrophoresis technology was detected. The expression positive rate of Bcl-2, Bax protein in spermatogenic cell by the immunohistochemical method was assayed, and the ultrastructural change of spermatogenic cell by the transmission electron microscope was observed.</p><p><b>RESULTS</b>DNA damage rates of of spermatogenic cell in 1, 5, 10 micromol/kg cadmium chloride groups were higher than that of normal group (P < 0.001). Bcl-2 protein expression positive rates were lower than that of normal group (P < 0.001). Bax protein positive expression rate in 5 micromol/kg group was higher than those in normal group, and 1, 10 micromol/kg groups. The ultrastructure of karyotis, karyotheca, mitochondria, endoplasmic reticulum in three treated groups had different degree of damage and the degree of ultrastructural change was increasing with rising concentration of cadmium.</p><p><b>CONCLUSION</b>Cadmium exposure will cause the DNA break, Bcl-2 and Bax protein abnormal expression and ultrastructural change in spermatogenic cell.</p>