Data_Sheet_1_Optimization of the extraction process and metabonomics analysis of uric acid-reducing active substances from Gymnadenia R.Br. and its protective effect on hyperuricemia zebrafish.docx

BackgroundAs Gymnadenia R.Br. (Gym) has an obvious uric acid-lowering effect, but its specific bioactive substances and mechanism are still unclear. The key metabolites and pathways used by Gym to reduce uric acid (UA) were identify.MethodsAn optimized extraction process for urate-lowering active substances from Gym was firstly been carried out based on the xanthine oxidase (XOD) inhibition model in vitro; then, the Ultra-high-performance liquid chromatography and Q-Exactive mass spectrometry (UHPLC-QE-MS) based on non-targeted metabolomics analysis of Traditional Chinese Medicine were performed for comparison of Gym with ethanol concentration of 95% (low extraction rate but high XOD inhibition rate) and 75% (high extraction rate but low XOD inhibition rate), respectively; finally, the protective effect of ethanolic extract of Gym on zebrafish with Hyperuricemia (referred to as HUA zebrafish) was explored.ResultsWe found that the inhibition rate of Gym extract with 95% ethanol concentration on XOD was 84.02%, and the extraction rate was 4.32%. Interestingly, when the other conditions were the same, the XOD inhibition rate of the Gym extract with 75% ethanol concentration was 76.84%, and the extraction rate was 14.68%. A total of 539 metabolites were identified, among them, 162 different metabolites were screened, of which 123 were up-regulated and 39 were down-regulated. Besides significantly reducing the contents of UA, BUN, CRE, ROS, MDA, and XOD activity in HUA zebrafish by Gym and acutely reduce the activity of SOD.ConclusionAlong with the flavonoids, polyphenols, alkaloids, terpenoids, and phenylpropanoids, the ethanolic extract of Gym may be related to reduce the UA level of Gym.</p

Rats with Chronic, Stable Pulmonary Hypertension Tolerate Low Dose Sevoflurane Inhalation as Well as Normal Rats Do

<div><p>Background</p><p>The effects of low concentration of sevoflurane on right ventricular (RV) function and intracellular calcium in the setting of pulmonary arterial hypertension (PAH) have not been investigated clearly. We aim to study these effects and associated signaling pathways in rats with PAH.</p><p>Methods</p><p>Hemodynamics were assessed with or without sevoflurane inhalation in established PAH rats. We analysis the classic RV function parameters and RV-PA coupling efficiency using steady-state PV loop recordings. The protein levels of SERCA2, PLB and p-PLB expression was analyzed by western blot to assess their relevance in PAH.</p><p>Results</p><p>Rats with PAH presented with RV hypertrophy and increased pulmonary arterial pressure. The values of Ea, R/L ratio, ESP, SW, PRSW, +dP/dt_max and the slope of the dP/dtmax-EDV relationship increased significantly in PAH rats (<i>P</i><0.05). Sevoflurane induced a concentration-dependent decrease of systemic and pulmonary blood pressure, HR, RV contractility, and increased the R/L ratio in both groups. Sevoflurane reduced the expression of SERCA2 and increased the expression of PLB in both groups. Interestingly, sevoflurane only reduced the p-PLB/PLB ratio in PAH rats, not in normal rats.</p><p>Conclusions</p><p>Rats with chronic, stable pulmonary hypertension tolerate low concentrations of sevoflurane inhalation as well as normal rats do. It may be related to the modulation of the SERCA2-PLB signaling pathway.</p></div

Cardiac morphometric data <i>(n = 12)</i>.

<p>Cardiac morphometric data <i>(n = 12)</i>.</p

HE staining of lung (A) and right ventricle (C) from a normal rat and lung (B) and right ventricle (D) from a PAH rat.

<p>The black arrows show the pulmonary arterioles. PAH rats exhibited markedly thickened pulmonary arterioles, narrowed lumen, and hypertrophic endothelial cells, with a dense inflammatory infiltration around the arterioles. The RV myocardial cells of PAH rats were hypertrophic and exhibited a disorganized arrangement with different sizes of nuclei.</p

The effects of sevoflurane on the expression of SERCA2 (A,D), PLB (B, E), SERCA2/PLB ratio (F) p-PLB(C) and p-PLB/PLB ratio(G) in the right ventricle.

<p>The results are expressed as the mean±SD, n = 3. ^a<i>P<</i>0.05 vs. NS; ^b<i>P<</i>0.05 vs. MCT.</p

The parameters of right ventricular function.

<p>The parameters of right ventricular function.</p

Values are expressed as means±SD.

<p><i>P</i> values from repeated measures ANOVA; *, group NS compared with group NS-sevo, group MCT compared with MCT-sevo, <i>P<</i>0.05,**, group NS compared with group NS-sevo, group MCT compared with MCT-sevo, <i>P</i><0.01, NS, group NS compared with group NS-sevo, group MCT compared with MCT-sevo, no significant. #, group NS compared with group MCT at T0, <i>P</i><0.05, &, group NS-sevo compared with MCT-sevo at T0, <i>P</i><0.05. HR, heat rate; MAP, mean systemic arterial pressure; mPAP, mean pulmonary arterial pressure; R/L ratio, ratio of systolic pulmonary arterial pressure over systolic blood pressure.</p

Right ventricle (RV) pressure-volume (PV) loops.

<p>Representative recordings of RV PV loops in a normal rat (A) and a PAH rat (B) during baseline condition and n a normal rat (C) and a PAH rat (D) 1.5% sevoflurane inhalation. Solid lines (black arrows) represent end-systolic pressure volume-relationship (ESPVR). RV ESP, right ventricle end-systolic pressure.</p