10.3969/j.issn.1000-3606.2013.06.005

Objective To explore the role of miR-137 in the proliferation and migration of hepatocellular carcinoma (HCC) cells by regulating Notch1 and mediating autophagy.Methods The human SMMC7721 hepatoma cell line was transfected with miR-137 mimics,miR-137 inhibitor and Notch1 interfering RNA (siRNA),and divided into normal control group (NC group),miR-137 mimics group,miR-137 inhibitor group,Notch1 siRNA group.The expression levels of miR-137 and Notch1 mRNA after the transfection were detected by RT-PCR in SMMC7721 cells.Transwell experiments were performed to analyze the effect of miR-137 and Notch1 on the migration and invasion of SMMC7721 cells.The expression levels of β-catenin and vimentin in SMMC7721 cells were detected by immunohistochemistry.The number of autophagosomes was detected by double labeled adenovirus.Western blot was utilized to detect the expression of Notch1,E-Cadherin,N-Cadherin,vimentin,P62,and LC3.Results The results of RT-PCR showed that the relative expression level of Notch1 in miR-137 inhibitor group (5.71 ± 0.45) was significantly higher than that in miR-137 mimics group (0.21 ± 0.06) with statistical significance (P ＜ 0.05).The Transwell experiments showed that there were fewer invasive metastatic hepatoma cells in miR-137 mimics group (66.00 ± 4.55) and Notch1 siRNA group (88.00 ± 6.78) than that in the miR-137 inhibitor group (515.00 ±35.12) (P ＜0.05).The expression levels of β-catenin in miR-137 mimics group and Notch1 siRNA group were significantly increased and the expression level of vimentin was decreased (P ＜ 0.05).The results of autophagy double labeled adenovirus test showed that the number of autophagosomes in miR-137 mimics group (5.50 ± 3.70) was significantly fewer than that in miR-137 inhibitor group (32.75 ± 4.11),and the difference was statistically significant (P ＜ 0.05).The expression levels of Notch1,N-cadherin,vimentin,and LC3 protein in miR-137 mimics group were much lower than that in miR-137 inhibitor group and NC group,and the expression levels of E-Cadherin and P62 protein were greatly increased.The expression level of Notch1,N-cadherin,and LC3 protein in Notch1 siRNA group were significantly lower than that in NC group,and the expression levels of E-cadherin and P62 protein were much higher than that in NC group.Conclusion MiR-137 can inhibit the proliferation,migration and invasion of HCC cells by inhibiting the expression of Notch1 and autophagy,which may become a new target for the treatment of HCC.

Objective To investigate the effect of nucleotide-binding oligomerization domaincontaining protein 1 (NOD1) on pyroptosis in hepatic ischemia-reperfusion injury.Methods An animal model of ischemia-reperfusion injury was established.Thirty healthy,male,and clean C57 BL mice were randomly divided into sham operation group (sham group) and ischemia-reperfusion group (IR,including 2 h,6 h,12 h,24 h subgroups),6 per group.Serum ALT and AST levels in each group were measured by blood biochemistry.HE staining and TUNEL were used to observe the pathological changes of liver and hepatocyte apoptosis.Immunohistochemistry was used to detect the expression and distribution of NOD1 in each group.Western blotting was used to detect NOD1,MM2,pro-Caspase-1 and active-Caspase-1 expression.NOD1 siRNA and empty control siRNA were transfected into AML12 cells,then the hypoxia/reoxygenation model was established and cells were collected to detect the expression of NOD1,AIM2 and active-Caspase-1.Results The ALT and AST levels in IR group were significantly higher than those in sham group,and peaked at IR 12-h subgroup (P＜0.05).HE staining showed that hepatic injury was the most severe at 12 h after reperfusion.TUNEL results showed that the number of apoptotic cells was the greated at 12 h after reperfusion.Western blotting showed that NOD1 protein expression was highest at 12 h after reperfusion.With the prolongation of reperfusion time,the expression of AIM2 and active-Caspase-1 gradually increased,and that of pro-Caspase-1 gradually decreased.The expression of NOD1,AIM2 and activeCaspase-1 decreased after transfection of NOD1 siRNA into AML12 cells.Conclusions NOD1 promotes liver ischemia-reperfusion injury,which may be related to NOD1 promoting liver injury by activating pyroptosis.

Objective To explore the effect of different acupuncture stimuli on uterine micro-circulation of dysmenorrheal rats with cold stagnation syndrome. Methods Totally 32 three-month old female SD rats in diestrus were randomly divided into saline control group, model group, A stimuli group, and B stimuli group, 8 rats in each group. Model group and treatment groups were given whole body freezing combined with estradiol benzoate injection method to establish models. A stimuli group was given deep puncture with manipulation, while B stimuli group was treated by shallow puncture without manipulation. Diameter of uterine capillary,micro-vessel, TXB2, and 6-keto-PGF1αlevels were observed in each group. Results Compared with the saline group, capillary diameter in model group was significantly reduced at 5, 10, 20, 30 min time point (P<0.01);micro-vascular diameter was significantly reduced at 5, 10, 20, 30, 40 min time point (P<0.01);plasma 6-keto-PGF1α levels decreased (P<0.01);TXB2/6-keto-PGF1αincreased significantly (P<0.01). Compared with model group, capillary diameter in A stimuli group enlarged at 5, 10, 20, 30 min time point (P<0.05), micro-vascular diameter dilated at 5, 10, 20, 30, 40 min time point (P <0.01), plasma 6-keto-PGF1α level increased (P <0.05), TXB2/6-keto-PGF1αdecreased significantly (P<0.05);micro-vascular diameter in B stimuli group dilated at 20, 30 min time point (P<0.05), plasma TXB2/6-keto-PGF1α decreased significantly (P<0.05). Compared with B stimuli group, capillary diameter in A stimuli group dilated at 5, 10, 20, 30 min time point (P<0.05) and micro-vascular diameter dilated at 5, 10, 20, 30, 40 min time point significantly (P<0.01). Conclusion Dysmenorrheal rats with cold stagnation syndrome show obvious disorder of the uterus micro-circulation and circulation related substances. Both A and B acupuncture stimuli improved uterus micro-circulation of dysmenorrheal rats with cold stagnation syndrome, and its mechanism may be related to the recovery the balance between TXB2 and 6-keto-PGF1α.

Objective To observe the analgesic effect of acupuncture at Guanyuan (CV4) and its effect on vasomotor substances in rats with dysmenorrhea due to coagulated cold syndrome. Method The coagulated-cold dysmenorrhea rat model was developed by Estrodiol benzoate and Oxytocin injectin plus physical freezing. The writhing response (writhing latency, writhing frequency, and writhing score) was observed, and the contents of TXB2 and 6-keto-PGF1a were detected by using enzyme-linked immunosorbent assay (ELISA). Result Compared with the saline water group, the writhing latency was significantly shortened, the writhing frequency was significantly increased, and the writhing score was more significantly increased in the model group (P<0.01);compared with the model group, the writhing latency was significantly prolonged, the writhing frequency was decreased, and the writhing score was significantly lower in the acupuncture group (P<0.05, P<0.01). Compared with saline water group, the content of plasma 6-keto-PGF1a was significantly lower (P<0.05) and the content of plasma TXB2 showed an increasing tendency (P>0.05) in the model group. Compared with the model group, the content of plasma 6-keto-PGF1a showed an increasing tendency (P>0.05) and the content of plasma TXB2 showed a decreasing tendency (P>0.05) in the acupuncture group. Conclusion The vasomotor substances are obviously disordered in the blood of cold-coagulated dysmenorrhea rat models. Acupuncture at Guanyuan can improve the writhing response and release pain, and meanwhile positively regulate the vasomotor substances such as TXB2 and 6-keto-PGF1a. The vasomotor substances are plausibly one of the major substances in the action of acupuncture in preventing and treating dysmenorrhea.

Objective Infrared thermal imaging can be applied to the diagnosis and auxiliary diagnosis of some diseases . The aim of this study is to explore acupuncture-induced changes in skin temperature in acupoint areas and whether skin temperature in -creases or decreases in the acupoint areas along meridians . Methods Thirty two female SD rats were randomly and equally divided into four groups:saline control,cold congealing and dysmenorrhea model , Sanyinjiao (SP6), and Guanyuan( CV4).Models were es-tablished in the latter three groups by subcutaneous injection of estradiol benzoate at 0.5 mg for 10 successive days and , 1hour after the last administration , intraperitoneal injection of oxytocin at 2 U, followed by exposure of the rats to-25℃in a freezer 4 hours a day for 5 days.Meanwhile , the control rats received normal saline only and were not exposed to low temperature .Infrared thermal imaging was used to measure the skin temperature at the acupoint areas of SP6, Xuehai (SP10), and CV4 before and at 0, 5, 10, 20, 30, 40, 50 and 60 min after needling . R esults At 0 to 5 min after nee-dling, the skin temperature of the left SP6 and right SP10 was signifi-cantly decreased in both the SP6 and CV4 groups ( [ -0. 56 ± 0.22]℃and [-0.48 ±0.11]℃, P<0.01), and so was that of the right SP10 ([ -0.64 ±0.21]℃ and [ -0.45 ±0.13]℃, P<0.05).At 5 to 10min, the skin temperature of the right SP6 and SP10 was markedly increased in the SP6 group ([-0.49 ±0.35]℃and [-0 .18 ±0.20]℃, P<0.01), and so was that of the right SP6 in the SP6 group at 20 to 30 min ([ -0.14 ±0.25]℃) as compared with the model and CV4 groups (P<0.01).At 30 to 40 min, the skin temperature of the right SP10 was remarkably elevat-ed in the SP6 group ([ -0.03 ±0.11]℃) in comparison with the model group (P<0.01).No significant differences were observed in the skin temperature of the left SP10 and CV4 at different time points among the four groups (P>0.05). Conclusion The skin temperature of SP6 and SP10 can be regulated by needling both the acupoints of SP 6 and CV4.The increase in the skin temperature of the right SP6 and SP10 in the SP6 group and no change in the CV 4 group indicated dynamic temperature changes in the acupoint area along the meridian after needling.

Objective To investigate the effect of miRNA-30a-3p on the proliferation,invasion and metastasis of liver cancer cells by targeting Atg3-mediated autophagy pathway.Methods The immunohistochemical staining was used to detect content of miRNA-30a-3p and Atg3 and their correlation in human hepatocellular carcinoma.Liver cancer cells were cultured in vitro and hunger environment was used to induce autophagy.RFP-GFP-LC3 double-labeled adenovirus infected hepatoma cells were used to detect autophagosomes in hepatoma cells.The expressions of autophagy-related proteins (autophagocytosis associated protein (Atg3),polyubiquitin-binding protein p62,autophagy microtubule-associated protein light chain 3 (LC3)) and EMT-related proteins (N-cadherin,vimentin,snail,ZO-1) were detected by Western blot.Platelet cloning assay and transwell assay were carried out to detect the proliferation,invasion and metastasis of carcinoma cell.CCK-8 kit was used to detect hepatocarcinoma cells' viability.Results The expression of miRNA-30a-3p was down-regulated.The expression of Atg3,E-cadherin and N-cadherin in miRNA-30a-3p high-expressed hepatocellular carcinoma was lower than that in miRNA-30a-3p low-expressed hepatocellular carcinoma.Increasing the expression of miRNA-30a-3p in hepatocellular carcinoma cells can decrease the expression of Atg3 and LC3,increase the expression of p62 and inhibit the formation of autophagosomes;otherwise,Atg3 and LC3 were increased,p62 was decreased and the formation of autophagosomes was promoted.Inhibition of Atg3 expression could decrease the expression of EMT-related proteins.When miRNA-30a-3p was inhibited,the cell viability of HCC was increased at each time point (F1 =10.314,P ＜0.05).When miRNA-30a-3p and Atg3 were inhibitor together,the cell viability of HCC was decreased at each time point(F2 =6.599,P ＜ 0.05).Conclusion miRNA-30a-3p can inhibit Atg3-mediated autophagy pathway and reduce cell autophagy activity,thus inhibiting the proliferation,invasion and metastasis of hepatocarcinoma cells.

OBJECTIVE To explore the intervention effect and related mechanism of Tongxinluo capsule on renal fibrosis in rats with diabetic nephropathy （DN）. METHODS Eight rats were selected as control group （ordinary feed）， the remaining rats were given high-glucose and high-fat diet combined with ip injection of streptozotocin （35 mg/kg） to induce DN model. Model rats were randomly divided into model group （purified water）， irbesartan group （positive control， 14.12 mg/kg） and Tongxinluo capsule group （0.3 g/kg）， including 12 rats in the model group and 11 rats for each of the other two groups. All groups were given relevant medicine or water intragastrically， once a day， for 16 consecutive weeks. After the last medication， fasting blood glucose and 24 h urinary total protein （24 h UTP） were detected. Pathological changes in renal cortex of rats in each group were observed. Serum levels of tissue-type plasminogen activator （PA） and plasminogen activator inhibitor 1 （PAI-1） were measured. mRNA expressions of transforming growth factor-β（1 TGF-β1）， type Ⅳ collagen（COL-Ⅳ）， Wnt4 and β-catenin in renal cortex of rats were detected. The protein depositions or expressions of TGF-β1， COL-Ⅳ， focal adhesion kinase （FAK）， integrin-linked kinase （ILK）， E-cadherin， PA， PAI-1， Wnt4 and β-catenin in renal cortex of rats were observed or determined. RESULTS Compared with model group， 24 h UTP of rats in Tongxinluo capsule group were all significantly reduced （P＜0.05）； pathological damage and fibrosis of renal cortex were relieved； the expression of PA in serum and renal cortex was significantly increased， while PAI-1 level was significantly reduced （P＜0.05）； the depositions of COL-Ⅳ and TGF-β1 in renal cortex were all reduced， and corresponding mRNA expression was decreased significantly （P＜0.05）； the depositions of ILK and FAK were decreased， while the deposition of E-cadherin was increased； protein and mRNA expressions of Wnt4 and β-catenin were significantly reduced （P＜0.05）. CONCLUSIONS Tongxinluo capsule can relieve pathological damage to renal tissue and renal fibrosis of DN model rats， and reduce extracellular matrix deposition. The mechanism may be related to regulation of fibrinolytic system activity， the decrease of ILK and FAK expression， and inhibition of Wnt/β-catenin signaling pathway.

OBJECTIVE：To establish the method for content determination of 6 active ingredients in Paeonia lactiflora during different harvest periods ，and to investigate its variation rules so as to determine the optimal harvesting period. METHODS ：HPLC method was adopted to determine the contents of gallic acid ， catechin， alibiflorin， paeoniflorin， benzoic acid and benzoylpaeoniflorin，and principal component analysis was conducted . The determination was performed on Thermo C 18 column with mobile phase consisted of acetonitrile- 0.1％ phosphoric acid aqueous solution （gradient elution ）at the flow rate of 1.0 mL/min. The detection wavelength was 230 nm，and column temperature was 35 ℃. The sample size was 10 µL. RESULTS ：The linear range of the above 6 ingredients were 0.013 2-0.25 mg/mL（r＝0.999 2），0.013 2-0.25 mg/mL（r＝0.999 9），0.026 8-0.51 mg/mL（r＝0.999 7）， 0.42-8.01 mg/mL（r＝0.999 2），0.016-0.31 mg/mL（r＝0.999 4），0.02-0.38 mg/mL（r＝0.999 8），respectively. The limits of quantification were 0.009 3，0.008 5，0.016 3，0.021 7，0.011 3，0.017 4 mg/mL，and the limits of detection were 0.003 3，0.002 7， 0.005 4，0.007 3，0.003 8，0.005 9 mg/mL. RSDs of precision ，stability and repeatability tests were less than 2％. The recoveries were 96.01％-99.43％（RSD＝1.23％，n＝9），97.95％-100.45％（RSD＝0.79％，n＝9），97.98％-100.11％（RSD＝0.68％，n＝ 9），98.83％ -100.09％ （RSD＝0.65％ ，n＝9），98.58％ - 100.95％（RSD＝1.35％，n＝9），96.28％-103.26％（RSD＝ 1.76％，n＝9）. The contents of above 6 ingredients in Radix Paeoniae Rubra （roots） were 0.016％ -0.057％ ，0-0.067％ ， 0.207％-0.640％，2.350％-5.887％，0.030％- 0.245％，0.054％- 0.381％，respectively. On May 30th，the drying rate of Radix 0451-87266873。E-mail：wangzhen_yue@163.com Paeoniae Rubra was the lowest （about 33％），and on Sept. 15th，the drying rate was the highest （about 49％）. The contents of gallic acid and paeoniflorin in the leaves of P. lactiflora were higher than the root during Jul.-Oct. Results of principal component analysis showed that the variance contribution rates of the first two principal components were 71.845％ and 18.170％，respectively；cumulative variance contribution rate was 90.015％. The months with higher comprehensive scores were May to Jun. and Sept. to Oct. CONCLUSIONS ：Established method is simple ， accurate，reproducible and precise. It can be used to determine the contents of 6 active ingredients in Radix Paeoniae Rubra during different harvest periods. Sept. 30th to Oct. 15th is the optimum harvesting periods for Radix Paeoniae Rubra ，and leaves can be harvested around Jul. 15th.

OBJECTIVE To investigate the effects of Taohong siwu decoction modified granules on podocyte epithelial- mesenchymal-transition （EMT） and renal fibrosis in diabetic kidney disease （DKD） model rats. METHODS Eight rats were selected as normal group （ordinary feed）； the remaining rats were given a high-glucose and high-fat diet combined with intraperitoneal injection of streptozotocin （35 mg/kg） to induce the DKD model. Model rats were randomly divided into model group， irbesartan group [positive control， 13.5 mg/（kg·d）] and modified Taohong siwu decoction group [6.48 g/（kg·d）]， with 8 rats in each group. All groups were given relevant medicine intragastrically， once a day， for 16 consecutive weeks. Twenty-four- hour urinary total protein （24 h UTP） was detected at the end of the 4th， 8th， 12th and 16th week of administration. After the last medication， the body mass， water intake， food intake， urine output， the levels of fasting blood glucose， serum creatinine （Scr） and blood urea nitrogen （BUN） as well as mRNA and protein expressions of P-cadherin， nephrin， α -smooth muscle actin （α-SMA）， Wilms’ tumor gene 1 （WT1）， transforming growth factor-β1（ TGF-β1） and type Ⅳ collagen （Col-Ⅳ） in renal tissue were determined. The pathological and morphological changes in renal tissue were observed and the thickness of the glomerular basement membrane was determined. RESULTS Compared with the model group， 24 h UTP of rats was significantly decreased in modified Taohong siwu decoction group since the 8th weekend （P＜0.05）； the body weight of rats increased significantly， but the amount of water intake and urine decreased significantly； Scr and BUN level， mRNA expression of α-SMA， mRNA and protein expressions of TGF-β1 and Col-Ⅳ were significantly reduced， while the mRNA expressions of P-cadherin， nephrin and WT1 were increased significantly （P＜0.05）； the protein deposition of α-SMA was reduced， protein depositions of P-cadherin， nephrin and WT1 were increased； the pathological damage and fibrosis of renal tissue were relieved； the thickness of glomerular basement membrane was decreased significantly （P＜0.05）. CONCLUSIONS Taohong siwu decoction modified granules can inhibit the EMT of podocyte in DKD model rats， and alleviate renal pathological damage and podocyte damage， thus protecting renal function， and delaying the process of renal fibrosis.