Effect and mechanism of atorvastatin on cellular inflammatory response induced by calcium oxalate crystals / 中华泌尿外科杂志

Objective@#To investigate the effect and mechanism of atorvastatin (ATV) on the inflammatory response of human renal tubular epithelial cells (HK-2 cells) induced by calcium oxalate crystals.@*Methods@#HK-2 cells were divided into control group (normal medium), ATV group (after 3 h pretreatment with 40 μmol/L ATV, replaced with normal medium), calcium oxalate crystal stimulation group (4 mmol/L calcium oxalate crystal) and ATV treatment group (after 3 h pretreatment with 40 μmol/L ATV, replaced with 4 mmol/L calcium oxalate crystals). After 12 h, the cells were collected, and the expression levels of NLRP3 and Cleaved caspase-1 were detected by immunohistochemical staining and Western blotting. The expression level of NF-κB was detected by immunofluorescence and Western blotting. The cell culture supernatant was collected to detecte the concentrations of interleukin-1β (IL-1β) and interleukin-18 (IL-18) by enzyme linked immunosorbent assay (ELISA).@*Results@#Western blot analysis showed that the relative expression of NLRP3 (0.125±0.013 vs. 0.135±0.007) and Cleaved caspase-1 (0.090±0.014 vs. 0.095±0.006) was decreased in the ATV group compared with the control group, but the difference was not statistically significant (P>0.05). The relative expression of NLRP3 (0.315±0.021 vs. 0.135±0.007, P<0.001) and Cleaved caspase-1 (0.235±0.008 vs. 0.095±0.006, P<0.001) was significantly increased in the calcium oxalate crystal stimulation group compared with the control group. While the relative expression of NLRP3 (0.245±0.007 vs. 0.315±0.021, P<0.05) and Cleaved caspase-1 (0.170±0.017 vs. 0.235±0.008, P<0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group. The results of immunohistochemical staining showed that the expression trends of NLRP3 and Cleaved caspase-1 in each group were consistent with those obtained by Western blotting. The ELISA results showed that the concentration of inflammatory factors IL-1β [(162.00±21.21)pg/ml vs. (183.50±7.78) pg/ml, P>0.05] and IL-18 [(176.50±24.12)pg/ml vs.(182.50±20.51)pg/ml, P>0.05] in the ATV group was lower than that in the control group, but the difference were not statistically significant (P>0.05). The concentrations of IL-1β[(850.50±48.79)pg/ml vs. (183.50±7.78)pg/ml, P<0.001] and IL-18 [(526.00±39.61)pg/ml vs. (182.50±20.51)pg/ml, P<0.001] were significantly increased in the cell culture medium of the calcium oxalate crystal stimulation group compared with the control group, while the concentrations of IL-1β [(452.50±36.06)pg/ml vs. (850.50±48.79) pg/ml, P<0.01] and IL-18 [(403.50±23.33)pg/ml vs. (526.00±39.61)pg/ml, P<0.05] was significantly reduced in the cell culture medium of the ATV treatment group compared with the calcium oxalate crystal stimulation group. Western blot analysis showed that the relative expression of NF-κB (0.105±0.021 vs. 0.100±0.014) in the ATV group was decreased compared with the control group, but the difference was not statistically significant (P>0.05). The relative expression of NF-κB (0.295±0.035 vs. 0.100±0.014, P<0.001) in the calcium oxalate crystal stimulation group was significantly increased compared with the control group. While the relative expression of NF-κB (0.160±0.012 vs. 0.295±0.035, P<0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group. The expression of NF-κB by immunofluorescence staining was consistent with the results of Western blotting.@*Conclusions@#Calcium oxalate crystals can induce the inflammatory response of HK-2 cells, while ATV can exert anti-inflammatory effects by inhibiting the activation of NLRP3 inflammasome and decreasing the secretion of inflammatory factors IL-1β, IL-18 and the expression of NF-κB.

Effect and mechanism of atorvastatin on cellular inflammatory response induced by calcium oxalate crystals / 中华泌尿外科杂志

Objective To investigate the effect and mechanism of atorvastatin (ATV) on the inflammatory response of human renal tubular epithelial cells (HK-2 cells) induced by calcium oxalate crystals.Methods HK-2 cells were divided into control group (normal medium),ATV group (after 3 h pretreatment with 40 μmol/L ATV,replaced with normal medium),calcium oxalate crystal stimulation group (4 mmol/L calcium oxalate crystal) and ATV treatment group (after 3 h pretreatment with 40 μmol/L ATV,replaced with 4 mmol/L calcium oxalate crystals).After 12 h,the cells were collected,and the expression levels of NLRP3 and Cleaved caspase-1 were detected by immunohistochemical staining and Western blotting.The expression level of NF-κB was detected by immunofluorescence and Western blotting.The cell culture supernatant was collected to detecte the concentrations of interleukin-1 β (IL-1β) and intedeukin-18 (IL-18) by enzyme linked immunosorbent assay (ELISA).Results Western blot analysis showed that the relative expression of NLRP3 (0.125 ±0.013 vs.0.135 ±0.007) and Cleaved caspase-1 (0.090 ±0.014 vs.0.095±0.006) was decreased in the ATV group compared with the control group,but the difference was not statistically significant (P ＞ 0.05).The relative expression of NLRP3 (0.315 ±0.021 vs.0.135 ± 0.007,P ＜ 0.001) and Cleaved caspase-1 (0.235 ± 0.008 vs.0.095 ± 0.006,P ＜0.001) was significantly increased in the calcium oxalate crystal stimulation group compared with the control group.While the relative expression of NLRP3 (0.245 ±0.007 vs.0.315 ±0.021,P ＜0.05) and Cleaved caspase-1 (0.170 ±0.017 vs.0.235 ±0.008,P ＜0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group.The results of immunohistochemical staining showed that the expression trends of NLRP3 and Cleaved caspase-1 in each group were consistent with those obtained by Western blotting.The ELISA results showed that the concentration of inflammatory factors IL-1 β [(162.00±21.21)pg/ml vs.(183.50±7.78) pg/ml,P＞0.05] and IL-18 [(176.50±24.12)pg/ml vs.(182.50 ± 20.51) pg/ml,P ＞ 0.05] in the ATV group was lower than that in the control group,but the difference were not statistically significant (P ＞ 0.05).The concentrations of IL-1β [(850.50 ± 48.79)pg/ml vs.(183.50 ± 7.78) pg/ml,P ＜ 0.001] and IL-18 [(526.00 ± 39.61) pg/ml vs.(182.50 ±20.51)pg/ml,P ＜0.001] were significantly increased in the cell culture medium of the calcium oxalate crystal stimulation group compared with the control group,while the concentrations of IL-1 β [(452.50 ±36.06)pg/ml vs.(850.50±48.79) pg/ml,P＜0.01] and IL-18 [(403.50 ±23.33)pg/ml vs.(526.00 ±39.61)pg/ml,P ＜0.05] was significantly reduced in the cell culture medium of the ATV treatment group compared with the calcium oxalate crystal stimulation group.Western blot analysis showed that the relative expression of NF-κB (0.105 ±0.021 vs.0.100 ±0.014) in the ATV group was decreased compared with the control group,but the difference was not statistically significant (P ＞ 0.05).The relative expression of NF-κB (0.295 ±0.035 vs.0.100 ±0.014,P ＜0.001) in the calcium oxalate crystal stimulation group was significantly increased compared with the control group.While the relative expression of NF-κB (0.160 ± 0.012 vs.0.295 ± 0.035,P ＜ 0.05) in the ATV treatment group was significantly lower than that in the calcium oxalate crystal stimulation group.The expression of NF-κB by immunofluorescence staining was consistent with the results of Western blotting.Conclusions Calcium oxalate crystals can induce the inflammatory response of HK-2 cells,while ATV can exert anti-inflammatory effects by inhibiting the activation of NLRP3 inflammasome and decreasing the secretion of inflammatory factors IL-1β,IL-18 and the expression of NF-κB.

The role of cell-crystal reaction mediated inflammation in the formation of intrarenal calcium oxalate crystals / 中华外科杂志

Calcium oxalate nephrolithiasis is the common disease of urinary surgery, its exact pathogenesis is still unclear.It is believed that the renal inflammatory injury induced by cell-crystal reaction plays an important role in the formation of intrarenal calcium oxalate crystals. Recent studies indicated that inflammation induced by cell-crystal reaction can cause renal cell damage, stimulate intracellular expression of NADPH oxidase, trigger the massive production of reactive oxygen species, activate nuclear factor-κB signaling pathway, release a large number of inflammatory factors, and cause inflammatory cascade effect of the kidney, thus promoting the accumulation, nucleation and growth of calcium salt crystals, eventually leading to the formation of intrarenal crystals and even stones. In this process, the regulatory factors and mechanisms involved include macrophages, NLRP3-high mobility group box-1 protein inflammation network, fetuin A, autophagy activation and other factors.

Induction of osteogenic differentiation of human renal fibroblasts in vitro / 中国病理生理杂志

AIM:To investigate the effects of osteogenic induction media and the medias containing different concentration of calcium on the induction of osteogenic differentiation of human renal fibroblasts in vitro.METHODS: Culturedhuman renal fibroblasts were divided into 5 groups in this experiment: osteogenic induction group (osteogenic inductionmedia), CaⅠgroup (0.5 mmol/L Ca2 + media), CaⅡgroup (1.5 mmol/L Ca2 + media), Ca Ⅲ group (2.5 mmol/LCa2 + media) and control group (PBS).The cell activity in each groups was measured by MTT assay .At 9th day, the cellcalcium Alizarin red S staining and alkaline phosphatase (ALP) Gomori calcium cobalt staining were performed respectivelyto observe the formation of calcium nidus and the expression of ALP .In addition, the expression of Runt-related transcriptionfactor 2 (Runx2) at mRNA and protein levels was determined by real -time PCR and Western blot, respectively.RE- SULTS: The remarkable positive signs which represented the formation of calcium nidus and the deposit of calcium objectsin all experiment groups were observed .The mRNA and protein expression of Runx2 in osteogenic induction group increasedin accordance with the induction time .Compared with control group, the mRNA and protein expression of Runx2 inthe CaⅠ ~Ⅲ groups increased gradually in a calcium concentration dependent manner at the 9th induction day.CON- CLUSION: Human renal interstitial fibroblasts show the potential activity in osteogenic differentiation induced by osteogen -ic induction media or high level calcium in vitro, which may be account for the cytological formation of the Randall ’splaque in the kidney.

Synergistic effect of high mobility group protein B1 on calcium phosphate-induced release of inflammatory cytokines from macrophages / 中国组织工程研究

BACKGROUND:More and more evidence suggests that macrophages and inflammation reactions are involved in the formation and development of nephrolithiasis. Previous studies have found that calculi crystals can stimulate macrophages to release high mobility group protein B1. OBJECTIVE:To investigate the synergistic effect of high mobility group protein B1 in calcium phosphate induced release of interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 from human macrophages. METHODS:(1) The induced U937 cells were respectively stimulated with RPMI (blank), 100 mg/L calcium phosphate, 100μg/L high mobility group protein B1 and 100 mg/L calcium phosphate+100μg/L high mobility group protein B1 for 1, 2 and 4 hours to col ect cellsupernatant. (2) The induced U937 cells were respectively stimulated with 100 mg/L calcium phosphate, 100 mg/L calcium phosphate+10μg/L high mobility group protein B1, 100 mg/L calcium phosphate+50μg/L high mobility group protein B1, 100 mg/L calcium phosphate+100μg/L high mobility group protein B1 for 4 hours to col ect cellsupernatant. Levels of interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 were determined by ELISA. RESULTS AND CONCLUSION:The levels of interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 in the cellculture supernatant of 100 mg/L calcium phosphate group and 100μg/L high mobility group protein B1 group were both higher than those in the blank group in a time-dependent manner (P<0.05). The levels of interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 in the cellculture supernatant of different concentrations of high mobility group protein B1 groups were al higher than those in the 100 mg/L calcium phosphate group in a concentration-dependent manner (P<0.05). The results suggest that both calcium phosphate and high mobility group protein B1 can induce the release of interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 from human macrophages and the high mobility group protein B1 has the synergistic effect with calcium phosphate to induce interleukin-1β, interleukin-6, tumor necrosis factorαand monocyte chemotactic factor 1 from human macrophages.

Protective effect of taurine on HK-2 cells exposed to oxalate and calcium oxalate monohydrate crystal in vivo / 中华泌尿外科杂志

Objective To investigate the protective effect of taurine on HK-2 cells exposed to oxalate (Ox) and calcium oxalate monohydrate crystal (COM) in vivo.Methods HK-2 cells,a proximal tubular epithelial cell line,were cultured.Five groups were divided in this study:control group (only HK-2 cells) ; Ox and COM group (HK-2 cells + Ox + COM) ; Taurine group (HK-2 cells + Ox + COM + Taurine) ; Apocynin group (HK-2 cells + Ox + COM + Apocynin) ; Catalase group (HK-2 cells + Ox + COM +Catalase).After 6 hrs,the cultures medias from each group were tested for LDH,H2O2,8-isoprostane,and MCP-1 protein.Cellular expression of MCP-1 mRNA and P47phox mRNA were determined by reverse transcriptase-polymerase chain reaction.After 24 hrs,cells livability was investigated by MTT.Results Compared with the control,cells livability was reduced when exposed to Ox and COM (P ＜ 0.05),Treatment with Taurine,Apocynin and Catalase significantly increased the cells livability (P ＜ 0.05).Compared with the control,the expression of LDH,H2O2,8-isoprostane,and cellular expression of MCP-1 mRNA and P47phox mRNA were increased following exposure to Ox and COM (P＜0.01,P＜0.01,P＜0.01,P＜0.01,P ＜0.05).Treatment with Taurine,Apocynin and Catalase significantly reduced the expression of LDH,H2O2,8-isoprostane,as well as the cellular expression of MCP-1 mRNA.Expression of P47phox mRNA in Taurine group was not reduced significantly (P ＞ 0.05).Conclusions This study showed that Taurine protected the HK-2 cells from oxidative injury exposed to Ox and COM by the pathway that may not be in relation to the inhibition of P47phox mRNA expression.

Optimized imaging quality and radiation dose for coronary artery angiography using 128-slice,dual-source Flash Spiral CT under the natural heart rate / 中华放射学杂志

Objective To compare the quality and radiation doses of coronary artery angiography under the natural heart rate condition between Flash spiral heart mode and prospective electrocardiogramtriggering sequence mode using dual-source,in order to choose personalized low doses of coronary artery scanning mode.Methods Sixty patients who underwent coronary angiography(CTA)on a 128-slice,dualsource CT scanner were divided into 2 group i.e,group A(27cases)and group B(33 cases).Flash spiral heart scan mode was employed for group A.Inclusion criteria included:heart rate75 bpm),date acquisition was set at 30%-50%of the R-R interval. (3)At the arrhythmias,premature beat,fibrillation atrial,date acquisition was set at 20%-90%of the R-R interval.In both gronps,patients with a BMI≥25.0kg/m2 were examined with a tube voltage of 120 kV.while the other patients with a BMI<25.0 kg/m2 were examined with a tube voltage of 100 kV.The BMl was(24.6±1.0)kg/m2 in group A,while that was (24.6±0.9)kg/m2 in group B.In both groups,all images were transferred to the workstation for further processing and analysis.The imaging quality of coronary artery segments and the radiation dose were compared with t test.Results A total of 336 coronary artery segments were evaluated in group A and 412 segments were evaluated in group B.The imaging quality of coronary artery segments were scored.Excellent or good was achieved in 98.2%(330 of 336)artery segments in group A,and that was 98.1%(404 of 412)in group B.There was no statistical difference in imaging quality between the two groups(t=0.513,P=0.608).The average effective dose was(0.74±0.29)mSv in group A,whereas that was(3.67±1.37)mSv in group B.There was a significant difference between the two groups(t=-10.858,P=0.000).Conclusions The personalized low doses coronary artery scanning mode can substantially reduce radiation damage while preserving good imaging quality.

Clinical application of monoenergetic technique of dual-energy CT in the fractures fixed with metal fixer / 中华放射学杂志

Objective To analyze the clinical application of monoenergetic technique of dual-energy CT in removing metal artifacts for patients with fractures fixed with metal fixer.Methods Fofly-five patients with fractures fixed with metal fixer underwent dual-energy CT scanning for the fractures.Two different data were collected in one-time scanning using dual-energy scanning sequence.With monoenergetic technique,two different data at 100 and 140 kilovolts were used for subtraction to removing metal artifacts based on different densities.Raw data were reconstructed with monoenergetic technique(group A)and conventional simulation method(group B),respectively.And,all data were reconstructed with multiplanar reconstruction (MPR),volume rendering(VR)and maximum intensity projection(MIP),respectively.Wilcoxon signed rank test was applied for the comparison of imaging quality and artifacts between the two groups.Results There were fewer artifacts on the images due to the application of monoenergetic technique in dual-energy CT scanning.In group A,the rate of high-quality images reached to 91.9%(124/135);and,in group B,it was 59.3%(80/135).There were statistical diference between the two groups(Z=-12.541.P<0.01).The images without artifact reached to 89.6%(121/135)in group A;whereas,it was 45.2%(61/135)in group B.There was statistical difference between the two groups(Z=-11.910,P<0.01).Conclusion Using monoenergetic technique,metal artifacts were removed effectively and the fine structure of fracture was clearly displayed.

Dynamic enhanced multi-slice spiral CT in evaluation of blood flow patterns of solitary pulmonary nodules with enhancement / 中国肺癌杂志

<p><b>BACKGROUND</b>To investigate the methods of dynamic enhanced multi-slice spiral CT in evaluation of blood flow patterns of solitary pulmonary nodules (SPNs) with enhancement.</p><p><b>METHODS</b>Seventy-eight patients with SPNs (≤4 cm) with strong enhancement underwent dynamic multi-slice spiral CT (Marconi Mx8000) scan before and after contrast enhancement by injecting contrast material with a rate of 4 mL/s. For the 40 patients in protocol one, one scan was obtained every 2 seconds during 15-45 and 75-105 seconds after injection, while for the 38 patients in protocol two, one scan was obtained every 2 seconds during 11-41 and 71-101 seconds. For all the patients, one scan was obtained every 30 seconds during 2-9 minutes. The section thickness was 2.5 mm for lesions ≤3 cm and 5 mm for lesions > 3 cm. Standard algorithm was used in the image reconstruction. Precontrast and postcontrast attenuation on every scan was recorded. The perfusion, peak height, ratio of peak height of the SPN to that of the aorta and mean transit time were calculated.</p><p><b>RESULTS</b>The peak height, perfusion, ratio of peak height of the SPN to that of the aorta and mean transit time in malignant SPNs were 34.85 Hu±10.87 Hu, 30.37 ml/(min*100 g)±11.14 ml/(min*100 g), 13.78%± 3.96% , 14.19 s±6.19 s respectively in protocol one, while those in protocol two were 36.62 Hu±10.75 Hu, 30.01 ml/(min*100 g)±8.10 ml/(min*100 g), 14.70 %±4.71%, 13.91 s±4.82 s respectively. No statistically significant differences were found between the peak height (t= 0.673, P=0.503), perfusion (t= 0.152 , P=0.880), ratio of peak height of the SPN to that of the aorta (t= 0.861, P=0.393) and mean transit time (t= 0.199, P=0.843) in malignant SPNs measured in protocol one and those measured in protocol two. All mean transit time in protocol two (36/36) were obtained, but only part of them (25/32) were obtained in protocol one.</p><p><b>CONCLUSIONS</b>Dynamic enhanced multi-slice spiral CT is a non-invasive method for quantitative evaluation of blood flow patterns of SPNs with enhancement and scans beginning at 11 seconds after injection of contrast material is suggested.</p>

Preliminary study on the dynamic enhanced multi-slice spiral CT in the evaluation of blood flow patterns of malignant solitary pulmonary nodules / 中国肺癌杂志

<p><b>BACKGROUND</b>To investigate the methods of dynamic enhanced multi-slice spiral CT in the evaluation of blood flow patterns of malignant solitary pulmonary nodules (SPNs).</p><p><b>METHODS</b>Fifty-seven patients with malignant SPNs (≤4 cm) underwent dynamic multi-slice spiral CT (Marconi Mx8000) scan before and after contrast enhancement by injecting 90 ml contrast material with a rate of 4 ml/s. Twenty-nine patients in protocol one were scanned every 2 seconds during 15-45 seconds and 75-105 seconds after injection, while 28 patients in protocol two were scanned every 2 seconds during 11-41 seconds and 71-101 seconds. All patients were then scanned every 30 seconds during 2-9 minutes. The collimation was 2.5 mm for lesions of ≤3 cm and 5 mm for lesions of 3-4 cm. Standard algorithm was used in the image reconstruction. The perfusion, peak height, ratio of peak height of the SPN to that of the aorta and mean transit time were calculated.</p><p><b>RESULTS</b>The enhancement value, perfusion, ratio of peak height of the SPN to that of the aorta and mean transit time were (34.61±11.37) HU, (31.17±11.18) ml/(min*100 g), 13.90%±4.15%, (13.96±5.86) s separately in protocol one, and (36.54±10.89) HU, (29.80±8.80) ml/(min*100 g), 15.01%±4.83%, (13.34±5.12) s respectively in protocol two. No statistically significant difference was found between the two groups. In addition, mean transit time from all 28 patients in protocol two were obtained, but only part of them were measured in protocol one (22/29).</p><p><b>CONCLUSIONS</b>Dynamic enhanced multi-slice spiral CT is a kind of non-invasive method for quantitative evaluation of blood flow patterns of malignant solitary pulmonary nodules. It might have potential significance in angiogenesis research for lung cancer.</p>