Objective To investigate the regulatory effects of endogenous sulfur dioxide (SO2) on collagen accumulation in pulmonary arterial fibroblasts of rats and its mechanisms.Methods Primary rat pulmonary artery fibroblasts were used in the experiment and were divided into 3 groups:the control group,the L-aspartate-beta-hydroxamate(HDX) group and the HDX ± SO2 group.SO2 content of pulmonary artery fibroblasts supernatant was detected by adopting high performance liquid chromatography (HPLC).Collagen type Ⅰ and collagen type Ⅲ in pulmonary artery fibroblasts were determined by using immunofluorescence.Phosphorylation of Smad2/3,protein expression of matrix metalloproteinase (MMP)-13 and tissue inhibitors of MMP (TIMP)-1 were detected by using Western blot.One-way ANOVA was used for multiple group comparisons followed by Bonferroni test for each group.P ＜ 0.05 was considered as significant difference.Results Compared with control group,endogenous SO2 content in HDX group was significantly decreased [(14.30-± 0.48) μmol/L vs.(20.14 ± 0.49) μμmol/L,P ＜ 0.01],the level of Smad2/3 increased (1.03 ±0.31 vs.0.48 ± 0.20,P ＜ 0.01),protein expressions of MMP-13 and TIMP-1 in pulmonary artery fibroblasts were decreased (MMP-13:0.28 ± 0.06 vs.0.75 ± 0.11,P ＜ 0.01;TIMP-1:0.40 ± 0.05 vs.0.66 ± 0.20,P ＜ 0.01),and the ratio of MMP-13/TIMP-1 was decreased (0.71 ± 0.12 vs.1.23 ± 0.45,P ＜0.01).However,contents of collagen Ⅰ and collagen Ⅲ were significantly increased.Compared with HDX group,the level of Smad2/3 phosphorylation in HDX ± SO2 group decreased (0.57 ± 0.16 vs.1.03 ± 0.31,P ＜ 0.01),protein expression of MMP-13 and TIMP-1 upregulated (MMP-13:0.63 ± 0.06 vs.0.28 ± 0.06,P ＜ 0.01;0.59 ± 0.11 vs.0.40 ± 0.05,P =0.015),the ratio of M MP-13/TIMP-1 (1.10 ± 0.22 vs.0.71 ± 0.12,P =0.033) increased,but contents of collagen type Ⅰ and type Ⅲ were reduced obviously.Conclusions SO2 promotes the degradation of collagen and collagen accumulation in pulmonary artery fibroblasts of rats probably by inhibiting Smad2/3 signal pathway,increasing protein expression of MMP-13 and TIMP-1,and upregulating the ratio of MMP-13/TIMP-1.

Objective To investigate the production of hydrogen sulfide (H2S) in rats with monocrotaline (MCT)-induced pulmonary hypertension(PAH).Methods Fourteen male Wistar rats were randomly divided into 2 groups (7 cases for each group):control group and MCT group.Rats in the MCT group were intraperitoneally injected with MCT (60 mg/kg) on day 1 while rats in the control group received only the same volume of 9 g/L saline.And then the conventional breeding was given for 21 days.Mean pulmonary artery pressure (mPAP) was evaluated via right cardiac catheterization procedure.The ratio of right ventricular mass (RV) and left ventricular plus septal mass (LV + SP) [RV/(LV + SP)] was calculated.The morphological change of pulmonary artery was observed by applying hematoxylin-eosin (HE) staining on the lung tissue paraffin section by measuring relative media thickness (RMT) and relative media area (RMA) of pulmonary aetery.H2S contents in serum and lung tissue rat were detected by using free radicals detector.The expression of cystathionine-γ-lyase (CSE),a key enzyme catalyzing endogenous H2S generaion,in lung tissue was detected by using Western blot method.CSE mRNA level in lung was detected by adopting the real-time polymerase chain reaction (RT-PCR) method.Results Compared with control group,mPAP of rats in MCT group was increased significantly [(49.31 ±3.67) mmHg vs (14.31 ±2.07) mmHg(1 mm Hg =0.133 kPa),P ＜ 0.01] and the ratio of RV/(LV + SP) was increased (0.43 ± 0.03 vs 0.21 ± 0.03,P ＜ 0.01),while RMT and RMA of pulmonary artery were enlarged [(43.46 ± 1.94) μm vs (13.16 ± 1.48) μm,P ＜ 0.01;(3 321.10 ± 318.20) μm2 vs (963.40 ± 127.26) μm2,P ＜0.01].The evaluation of microstructure of pulmonary artery showed that the vessel wall of pulmonary artery in control rats was thin and no inflammatory cell was observed around artery.However,the vessel wall of pulmonary artery in rats of MCT group was thickened and there was evident infiltration of inflammatory cells in perivascular area.Compared with control group,H2S contents in serum and lung tissue of rats in MCT group were markedly decreased [(9.28 ± 0.94) μmol/L vs (14.20 ± 1.21) μmol/L,P ＜ 0.01;(0.43 ± 0.08) μmol/g protein vs (0.87 ±0.17) μmol/g protein,P ＜0.01],while CSE protein and mRNA expression in lung tissue of rats in MCT group were downregulated (0.14 ± 0.02 vs 0.28 ± 0.09,P ＜ 0.01;0.84 ± 0.06 vs 1.12 ± 0.04,P ＜ 0.01).Condusion Endogenous H2S pathway is significantly downregulated in rats with monocrotaline-induced pulmonary hypertension.

Objective To investigate the effects of endogenous sulfur dioxide (SO2) on the oxidative stress induced by cobalt chloride (CoCl2) in the rat pulmonary artery smooth muscle cells (PASMCs).Methods Rat PASMCs were treated with 200 μ mol/L CoCl2 to mimic the hypoxia insult.Endogenous SO2 generating enzyme aspartate aminotransferase 1 (AAT1) expression was upregulated or downregulated (AAT1 sh) by transfection with lentivirus.Rat PASMCs were randomly divided into 8 groups:vehicle group,vehicle + CoCl2 group,AAT1 group,AAT1 + CoCl2 group,scramble group,scramble + SO2 group,AAT1 sh group and AAT1 sh + SO2 group.SO2 donor Na2 SO3/NaHSO3 at concentration of 100 μ mol/L were added in scramble + SO2 group and AAT1sh + SO2 group.The expressions of AAT1,superoxide dismutase 1 (SOD1) and SOD2 in PASMCs were detected by Western blot method.In situ SO2 content in PASMCs was detected by fluorescent probe.The superoxide anions in PASMCs were labeled by dihydroethidium (DHE) probe under fluorescent microscope.Results Compared with the vehicle group,the levels of SO2 and the expressions of AAT1 (0.221 ± 0.002 vs.0.446 ± 0.004),SOD1 (0.076 ± 0.028 vs.0.171 ± 0.019) and SOD2 (0.080 ± 0.031 vs.0.196 ± 0.018) significantly decreased (all P ＜ 0.01),and superoxide anion increased in rat PASMCs of vehicle + CoCl2 group.Meanwhile,compared with vehicle + CoCl2 group,the levels of SO2 and the expressions of AAT1 (0.839 ± 0.056 vs.0.221 ± 0.002),SOD1 (0.177 ± 0.020 vs.0.076 ± 0.028) and SOD2 (0.195 ±0.018 vs.0.080-± 0.031) markedly increased (all P ＜ 0.01),and superoxide anion decreased in rat PASMCs of AAT1 + CoCl2 group.On the contrary,compared with the scramble group,the levels of SO2 and the expressions of AAT1 (0.062 ±0.017 vs.0.354 ±0.034),SOD1 (0.054 ±0.029 vs.0.157 ±0.023) and SOD2(0.180 ±0.100 vs.0.586 ± 0.176)significantly decreased (all P ＜ 0.01),and superoxide anion increased in rat PASMCs of AAT1sh group.Furthermore,compared with the AAT1 sh group,the levels of SO2 and the expressions of SOD1 (0.155 ± 0.022vs.0.054 ± 0.029) and SOD2 (0.578 ± 0.200 vs.0.180 ± 0.100) significantly increased (all P ＜ 0.01),and superoxide anion decreased in rats PASMCs of AAT1sh + SO2 group.Conclusion Endogenous SO2/AAT1 inhibits CoCl2-induced oxidative stress in rat PASMCs.

Objective To explore the effect of endogenous sulfur dioxide (SO2)on the apoptosis induced by cobalt chloride (CoCl2)in the human pulmonary arterial endothelial cells (HPAECs).Methods CoCl2was used in the primary HPAECs to mimize hypoxia-induced cell apoptosis.The aspartate aminotransferase 1(AAT1),and the key enzyme generating endogenous SO2 were over -expressed by transfecting HPAECs with lentivirus containing AAT1 cDNA.HPAECs were divided into 4 groups:vehicle group,vehicle + CoCl2 group,AAT1 group and AAT1 + CoCl2 group.The expressions of AAT1,B-cell lymphoma-2 (bcl-2),bcl-associated X protein (bax),Caspase-3 and activated Caspase-3 (cleaved Caspase-3)in the HPAECs were measured by Western blot.The AAT activity was assessed with colorimetry method.The SO2 content in the HPAECs was in situ observed by SO2-specific fluorescent probe.The HPAECs apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)assay.Results There were significant differences in the endogenous SO2 content,the expre-ssions of AAT1 and bcl-2,and the ratio of cleaved Caspase-3/Caspase-3 among 4 groups of HPAECs were (F＝147.364,23.738,6.521,64.884,all P＜0.05).However,there was no difference in the expression of bax among 4 groups of HPAECs (F＝1.620,P＞0.05).Compared with vehicle group,AAT activity [(0.96 ± 0.24)Carmen's unit/μg vs.(2.21 ± 0. 60)Carmen's unit/μg],endogenous SO2 content (40.71 ± 7.72 vs.105.60 ± 16.20)and bcl-2 expression (0.59 ± 0.19 vs.1.02 ± 0.20)in the HPAECs of vehicle +CoCl2 group were significantly de-creased,while the cell apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (1.56 ± 0.25 vs.0.95 ± 0.13)were significantly increased (all P＜0.05).However,there were no differences in the expression of AAT1 (0. 50 ± 0.12 vs.0.53 ± 0.11)in the HPAECs between vehicle group and vehicle+CoCl2 group (P＞0.05). The SO2 content (351.50 ± 42.43 vs.105.60 ± 16.20)and AAT1 expression (1.22 ± 0.33 vs.0.53 ± 0.11)in the HPAECs of AAT1 group were higher than those of vehicle group (all P ＜0. 05 ). Compared with AAT1 group, endogenous SO2content (333.50 ± 46.22 vs.351.50 ± 42.43)and the expression of AAT1 (1.26 ± 0.36 vs.1.22 ± 0.33)and bcl-2 (1.14 ± 0.38 vs.1.03 ± 0.27)in the HPAECs of AAT1 +CoCl2group did not change (all P＞0. 05).Moreover,no difference was observed in the HPAECs apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (0.51 ± 0.17 vs.0.50 ± 0.11)between the two AAT1 -overexpressed groups (all P ＞0. 05).Conclusion Endo-genous SO2inhibited the hypoxic HPAECs apoptosis stimulated by the treatment of CoCl2.

Gastrointestinal motility disorder is an important cause of digestive system diseases. Patients often suffer from nausea， vomiting， gastric retention， gastroparesis， constipation， and many other symptoms， and their quality of life is seriously reduced. Prokinetic agents are routinely used in clinical practice， but their long-term use is prone to problems such as reduced efficacy and increased adverse reactions. Since the incidence of gastrointestinal diseases has continued to rise globally in recent years， there is an urgent need for clinical development of safe and effective treatment strategies. Aurantii Fructus， a traditional Chinese medicine， has the effect of smoothing Qi and eliminating distention， and it has been used to treat gastrointestinal diseases for thousands of years. In modern clinical practice， it is mainly used for the treatment and auxiliary treatment of various gastrointestinal diseases such as functional dyspepsia， functional constipation， and irritable bowel syndrome. The efficacy is remarkable， and no adverse reactions have been reported at conventional doses. Therefore， it can greatly improve the symptoms of patients with gastrointestinal diseases and improve their quality of life. Modern research has revealed that there are many active components in Aurantii Fructus， among which flavonoids have the highest content and the most types. Flavonoids are the main active components in Aurantii Fructus to regulate gastrointestinal motility. Aurantii Fructus and its active components can affect gastrointestinal hormones， neural pathways， Cajal mesenchymal cells， and other multiple mechanisms. They can adjust gastrointestinal motility and correct gastrointestinal motility disorders， showing potential application value in the treatment of gastrointestinal motility disorders. However， a comprehensive analysis of Aurantii Fructus in this aspect is still lacking. This study summarized the pharmacological activities of active components of Aurantii Fructus extract and its flavonoids， volatile oils， alkaloids， and coumarin on the regulation of gastrointestinal motility and explored the latest research progress on its mechanism. Finally， the adverse reactions of Aurantii Fructus were summarized. It aims to provide a scientific basis for the research and clinical application of Aurantii Fructus and its active components in the regulation of gastrointestinal motility.