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OBJECTIVE To establish a quantitative analysis of multi-components by single marker （QAMS） method based on a variety of internal reference substances for the content determination of 6 components in Jinlian qingre granules， such as mangiferin， 2″-O-β-L-galactopyranosylorientin， orientin， veratric acid， vitexin， harpagoside. METHODS The determination was performed on Agilent Eclipse Plus C18 column with mobile phase consisted of acetonitrile-0.1% phosphoric acid solution （gradient elution） at the flow rate of 1 mL/min. The column temperature was 30 ℃， and the detection wavelength was set at 270 nm. Taking orientin， vitexin and 2″-O-β-L-galactopyranosylorientin as internal references， the relative correction factors （RCF） of the other 5 components to be determined and internal substances were determined by QAMS. The contents of 6 components in 21 batches of Jinlian qingre granules were calculated and then compared with the results of the external standard method. RESULTS The contents of mangiferin， 2″-O-β-L-galactopyranosylorientin， orientin， veratric acid， vitexin and harpagoside in 21 batches of samples were determined by QAMS in the range of 0.234-0.516， 1.804-2.270， 2.143-2.606， 0.190-0.223， 0.594-0.782， 0.080-0.152 mg/g； the contents of them determined by external standard method were 0.235-0.523， 1.798-2.265， 2.137-2.599， 0.190-0.224， 0.597-0.786， 0.077-0.151 mg/g， respectively. The percentage difference between the results measured by the two methods should not exceed 4.00%. CONCLUSIONS QAMS has been constructed for the simultaneous determination of 6 components in Jinlian qingre granules based on a variety of internal reference substances. The results obtained by this method are not significantly different from those obtained by the external standard method， and can be used for the quality control of Jinlian qingre granules.
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Objective To investigate the effects of cardiopulmonary bypass (CPB) on the differentiation of T lymphocyte subsets and the expression of specific transcription regulator T-bet/GATA binding protein 3 (GATA3). Methods A prospective double-blind study was conducted. Patients with CPB pulmonary repair of ventricular septal defect (observation group) or off-pump ligation of ductus arteriosus (control group) with 20 cases each in the 150th Military Hospital from February 2015 to February 2016 were enrolled. The blood sampled was collected on the time of before operation, at the end of CPB or operation, 4 hours after operation, and 24 hours after operation. T lymphocytes were isolated, the helper T cell 1 (Th1) specific transcription factor T-bet mRNA, helper T cell 2 (Th2) specific transcription factor GATA3 mRNA expression and cytokine γ-interferon (IFN-γ) mRNA, interleukin-4 (IL-4) mRNA expression were measured by Northern Blot. Results Compared with before operation, expression levels of T-bet mRNA [integral gray values: (1.39±0.52)×105vs. (2.92±0.88)×105], IFN-γ mRNA [integral gray values: (3.68±0.65)×105vs. (6.10±0.93)×105] were decreased transiently at the end of CPB in the observation group (both P < 0.05), returned to preoperative levels at 24 hours after operation [integral gray values: (2.77±0.74)×105, (6.22±1.25)×105, respectively, both P > 0.05]; expression levels of GATA3 mRNA [integral gray values:(4.96±0.88)×105vs. (3.21±0.68)×105], IL-4 mRNA [integral gray values: (3.52±1.13)×105vs. (1.85±0.63)×105] were increased (both P < 0.05), recovered to the preoperative levels at 24 hours after operation [integral gray values: (3.11±0.51)×105, (1.93±0.84)×105, respectively, both P > 0.05]. There were no significant differences in the expressions of T-bet, GATA3, IFN-γ and IL-4 mRNA in the control group at each time points (all P >0.05). Conclusions CPB causes the imbalance of Th1, Tc1/Th2, Tc2 and pro-inflammatory and anti-inflammatory reactions specially, which participate the complication occurrence after CPB. The changing of T-bet/GATA3 may be the internal mechanism for these changes.
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Objective To investigate the potential role of CXC chemokine ligand 16 (CXCL16)/CXC chemokine receptor 6 (CXCR6) pathway in the progression of diabetic nephropathy (DN). Methods 8?week old male db/db mice were randomly divided into DN group and DN inflamed group. 10% casein was subcutaneously injected to induce the DN mouse model with inflammation. In vitro, HK?2 cells were treated with high glucose (HG), and IL?1β+HG to investigate the effect of inflammatory stress on HK?2 cells. Further knockdown CXCL16 was mediated by RNA interference to determine the effects of CXCl16, then cells were divided into HG+IL?1βgroup, HG+IL?1β + siCXCL16 group and HG + IL?1β + vehicle group. Changes of renal function in mice were assessed by 24 h proteinuria and N?acetyl?β?D?glucosaminidase (NAG) during 8 weeks. The ultra?microstructure was checked by electron microscopy at 8th week. Lipid accumulation in kidneys and HK?2 were observed by Filipin staining and quantitative assay of intracellular free cholesterol. The protein expressions of CXCl16, CXCR6, a disintegrin and metalloproteinase?10 (ADAM10), fibronectin and α smooth muscle actin (α?SMA) in renal tissue were detected by immunohistochemistry and Western blotting. The mRNA and protein expressions of CXCl16, CXCR6, ADAM10, fibronectin andα?SMA in HK?2 cells were detected by real?time PCR and Western blotting, and protein expressions of CXCl16, CXCR6 and ADAM10 in HK?2 cells were also tested by cell immunofluorescence. Results Mice in DN inflamed group had higher 24 h proteinuria and NAG than those in DN group, and the differences between two groups shown statistical significance at 8th week (all P<0.05). Compared with DN mice, DN inflamed mice had more vacuoles within renal tubular cells, with mitochondrial swelling, deformation and decrease. Lipid accumulation and protein expressions of fibronectin and α?SMA were increased in DN inflamed group when compared with DN group (all P<0.05). Further, the expressions of CXCL16, CXCR6, ADAM10 were significantly increased in DN inflamed group (all P<0.05). In vitro, the mRNA and protein expressions of CXCL16, CXCR6, ADAM10, fibronectin and α?SMA, and lipid accumulation were increased in high glucose plus IL?1βgroup when compared with high glucose group (all P<0.05). However, after siRNA of CXCL16 transfection, the mRNA and protein expressions of CXCL16, CXCR6, ADAM10, fibronectin andα?SMA were down?regulated in HG+IL?1β+siCXCL16 group as compared with high glucose+IL?1βgroup (all P<0.05). Furthermore, lipid accumulation was decreased (P<0.05). Conclusion Inflammation accelerates tubulointerstitial injury in DN partly through the activation of CXCL16 pathway, which may facilitate the lipid accumulation in tubular epithelial cells.
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Objective To investigate the effect of Cordyceps sinensis on renal fibrosis and its possible mechanism.Methods Thirty Sprague-Dawley rats were randomly divided into three groups:Sham operation group (Sham,n =10),5/6 subtotal uephrectomy group (SNx,n =10),and 5/6subtotal nephrectomy treated with Cordyceps sinensis group (CS,n =10).Body weights were assessed and 24-hour urine excretion was collected before and every four weeks after surgery.Rats were sacrificed at 12 weeks after surgery.Blood samples were taken for biochemical study,and kidney tissues were used for HE and Masson stains to assess histological changes.Immunohistochemical staining was used to detect the expression of transforming growth factor β1 (TGF-β1) and its receptors of type Ⅰ (TβR Ⅰ),type Ⅱ (TβR Ⅱ).Immunofluorescence was used to detect E-cadherin and α-SMA.The relative protein level of TGF-β1,TβR Ⅰ,TβR Ⅱ,p-Smad2/3,Smad7,E-cadherin,α-SMA were examined by Western blotting.Results CS group had higher body weights and lower urinary protein,BUN and Scr level compared with SNx group.Glomerulosclerosis index and tubulointerstitial injury score were significantly reduced in CS group compared with those in SNx group (all P ＜ 0.05).The protein expressions of TGF-β1,TβR Ⅰ,TβR Ⅱ,p-Smad2/3 were decreased in CS group compared with those in SNx group (all P ＜ 0.05).CS treatment up-regulated the expression of E-cadherin,Smad7 and down-regulated the expression of α-SMA compared with that in SNx group (all P ＜ 0.05).Conclusion Cordyceps sinensis has inhibitory effect on renal fibrosis in 5/6 subtotal nephrectomy rat model,which might be related with the suppression of TGF-β 1 signal pathway.