A Hypoxia-Induced SCFFBXL1 E3 Ligase Ubiquitinates and Degrades the MEN1 Tumor Suppressor to Promote Colorectal Cancer Tumorigenesis / Journal of the Korean Cancer Association, 대한암학회지

Purpose@#Emerging evidence has shown that SKP1-cullin-1-F-box-protein (SCF) E3 ligases contribute to the pathogenesis of different cancers by mediating the ubiquitination and degradation of tumor suppressors. However, the functions of SCF E3 ligases in the pathogenesis of colorectal cancer (CRC) remain obscure. @*Materials and Methods@#The cancerous and adjacent noncancerous tissues from CRC patients were collected, and protein levels were analyzed. Lentiviral short hairpin RNA (shRNA) and plasmid transfection were used to knock down and overexpress gene expression in CRC cell lines. Immunoprecipitation (IP), mass spectrometry, and co-IP analyses were used to determine protein interactions and the assembly of the SCF complex. Cell proliferation, migration, and tumor xenograft assays were performed to examine the effects of SCF members on CRC cell growth in vitro and in vivo. @*Results@#Hypoxia activated the docking of hypoxia-inducible factor 1α (HIF1α) onto the CUL1 promoter and induced CUL1 expression in CRC cells. CUL1 coupled with RBX1, SKP1, and FBXL1 to assemble the SCFFBXL1 complex in CRC biopsies and cells. The SCFFBXL1 E3 ligase specifically ubiquitinated and degraded the MEN1 tumor suppressor. Knockdown of HIF1α or SCFFBXL1 members, or blockage of SCFFBXL1 by two inhibitors (DT204 and SZLP1-41) caused the accumulation of MEN1 protein and led to a significant decrease in cell proliferation and migration in vitro and tumor growth in vivo. @*Conclusion@#The SCFFBXL1 E3 ligase is required for the ubiquitination of MEN1, and disruption of this complex may represent a new therapeutic strategy for the treatment of CRC.

Chikusetsu saponin Ⅳa ameliorates myocardial hypertrophy of rats through regulating expression of miR199a-5p/Atg5 / 中国中药杂志

The present study investigated the effects of chikusetsu saponin Ⅳa(CHS Ⅳa) on isoproterenol(ISO)-induced myocardial hypertrophy in rats and explored the underlying molecular mechanism. ISO was applied to establish a rat model of myocardial hypertrophy, and CHS Ⅳa(5 and 15 mg·kg~(-1)·d~(-1)) was used for intervention. The tail artery blood pressure was measured. Cardiac ultrasound examination was performed. The ratio of heart weight to body weight(HW/BW) was calculated. Morphological changes in the myocardial tissue were observed by HE staining. Collagen deposition in the myocardial tissue was observed by Masson staining. The mRNA expression of myocardial hypertrophy indicators(ANP and BNP), autophagy-related genes(Atg5, P62 and beclin1), and miR199 a-5 p was detected by qRT-PCR. Atg5 protein expression was detected by Western blot. The results showed that the model group exhibited increased tail artery blood pressure and HW/BW ratio, thickened left ventricular myocardium, enlarged myocardial cells, disordered myocardial fibers with widened interstitium, and a large amount of collagen aggregating around the extracellular matrix and blood vessels. ANP and BNP were largely expressed. Moreover, P62 expression was up-regulated, while beclin1 expression was down-regulated. After intervention by CHS Ⅳa at different doses, myocardial hypertrophy was ameliorated and autophagy activity in the myocardial tissue was enhanced. Meanwhile, miR199 a-5 p expression declined and Atg5 expression increased. As predicted by bioinformatics, Atg5 was a target gene of miR199 a-5 p. CHS Ⅳa was capable of preventing myocardial hypertrophy by regulating autophagy of myocardial cells through the miR-199 a-5 p/Atg5 signaling pathway.

Improved effects of saponins from Panax japonicus on decline of cognitive function in natural aging rats via NLRP3 inflammasome pathway / 中国中药杂志

The aim of this paper was to investigate the effect of total saponins from Panax japonicus( SPJ) on cognitive decline of natural aging rats and its mechanism. Thirty male SD rats of eighteen month old were randomly divided into three groups: aged group,10 mg·kg~(-1) SPJ-treated group and 30 mg·kg~(-1) SPJ-treated group. The SPJ-treated groups were given SPJ at the dosages of 10 mg·kg~(-1) and 30 mg·kg~(-1),respectively,from the age of 18 to 24 months. Aged group were lavaged the same amount of saline,10 six-month-old rats were used as control group,with 10 rats in each group. The open field test,novel object recognition and Morris water maze were performed to detect the changes of cognitive function in each group. The changes of synaptic transmission of long-term potentiation( LTP) in hippocampal CA1 region were detected by field potential recording. Western blot was used to detect the protein levels of NLRP3,ASC,caspase-1 and the changes of Glu A1,Glu A2,CAMKⅡ,CREB and phosphorylation of CAMKⅡ,CREB in each group.The results showed that SPJ could improve the decline of cognitive function in aging rats,reduce the damage of LTP in the hippocampal CA1 region of aged rats,and decrease the expression of NLRP3,ASC,caspase-1 in aging rats. At the same time,SPJ could enhance the membrane expression of AMPA receptor( Glu A1 and Glu A2),and increase the expression of p-CAMKⅡand p-CREB in aging rats.SPJ could improve cognitive decline of natural aging rats,and its mechanism may be related to regulating NLRP3 inflammasome,thus regulating the membrane expression of AMPA receptor,and enhancing the expression phosphorylation of CAMKⅡ and CREB.

Abirritation of Discogenic Pain Model in SD Rats by Paravertebral Ozone Injection / 中山大学学报(医学科学版)

@#【Objective】To investigate the analgesic and degenerated regularity of paravertebral ozone injection in the discogenic pain model of SD rats ，and to reveal the mechanism of analgesic effect of ozone preliminarily.【Methods】 Male SD rats（n = 65）were randomly divided into control group（n = 15），model group（n = 25）and ozone group（n = 25）. The L5- 6 intervertebral discs of SD rats in model group and ozone group were punctured to establish discogenic pain models. Ozone was injected paravertebrally in ozone group rats on the 22nd day after modeling. The rats in control group were normal. A quantitative allodynia assessment technique and MRI were used to detect the 50% mechanical withdrawal threshold（50%MWT）and Pfirrmann grade of L5-6 intervertebral discs at different time intervals. The expression of tumor necrosis factor-α（TNF- α）and calcitonin gene-related peptide（CGRP）in left dorsal root ganglion and sciatic nerve were detected by western blot.【Results】The 50% MWT of both hind paws were different from each other in three groups at each time after the 22nd day after modeling（P < 0.05）. In the ozone group，the 50% MWT rose on the 22nd day after modeling（left 7.6±6.8，right 3.6±1.0，P < 0.05 vs pre-ozone injection），and reached the peak on the 24th day after modeling（left 10.6±8.2，right 7.9±6.7，P < 0.05 vs pre-ozone injection），and maintained this level until the 56th day after molding. In the ozone group，the L5-6 intervertebral disc degeneration was apparently visible compared with model group（P < 0.05）. The expression of TNF- α and CGRP in dorsal root ganglion and sciatic nerve were different from each other in three groups（model>ozone>control，P < 0.05）.【conclusions】Paravertebral ozone injection can alleviate the pain of discogenic pain model rats，but aggravates the degeneration of the lumbar disc. Paravertebral ozone injection can reduce the expression of TNF-α and CGRP in the sciatic nerve and dorsal root ganglia of discogenic pain model rats.

Effect of saponins extracted from Panax japonicas on inhibiting myocardial fibrosis by TGF-β1/Smad3 signaling pathway in aging rats / 中国中药杂志

To investigate the amelioration effect of saponins extracted from Panax japonicas (SPJ) on myocardial fibrosis in natural aging rats and its mechanisms, male SD rats aged 18 months were randomly divided into 3 groups (aging model group, low-dose SPJ group and high-dose SPJ group), with 10 rats in each group. SPJ groups were given SPJ at different doses (10, 60 mg·kg⁻¹·d⁻¹) consecutively for 6 months, meanwhile, aging model group was treated with the equal volume of saline for 6 months until 24 months old. Another 10 rats aged 6 month were used as young control group. The changes of myocardial morphological were observed by haematoxylin-eosin (HE) staining. Masson staining was used to observe the changes of collagen deposition in rat hearts. RT-PCR was used to detect the mRNA expression levels of myofibroblast marker α-SMA, collagen-related protein COL1α2, COL3α1 and matrix metalloproteinase MMP2, MMP9. Western blot was used to test the changes of the protein expressions of TGF-β1, p-Smad3, IL-1β and TNF-α in heart tissues. SPJ can effectively improve the arrangement of myocardial fibers, decrease inflammatory infiltration and reduce collagen deposition in aging rats. SPJ can effectively down-regulate the mRNA expression levels of COL1α2, COL3α1, α-SMA, MMP9, MMP2 and inhibit the protein expressions of TGF-β1, p-Smad3, TNF-α, IL-1β in the natural aging heart tissues. SPJ can effectively alleviate myocardial fibrosis in natural aging rats, and its mechanisms was related to the inhibition of the protein expressions of TGF-β1, p-Smad3 and the reduction of myocardial inflammation in rat hearts.

Total saponins of Panax japonicus improve cancer cachexia in mice through inhibiting inflammatory response mediated by NF-κB / 中国药理学通报

Aim To investigate the therapeutic effect of total saponins of Panax japonicus(SPJ)on cancer cach-exia in mice with colon adenocarcinoma. Methods BALB/c mice were subcutaneously inoculated with mu-rine colon adenocarcinoma CT26 cells to induce ca-chexia. The model animals were randomly divided into three groups: model group, SPJ low dose group and high dose group. Gavage started on the 4th day after inoculation, and the dosage regimen was as follows:the normal and model groups were given 10 mL·kg-1 saline, qd ×27; the low dose and high dose groups were treated with 20 and 60 mg·kg-1SPJ respective-ly, qd ×27. After treatment, the effects of SPJ on body weight, tibialis anterior muscle, gastrocnemius muscle,spleen and epididymal fat changes of cachexia mice were observed. HE and Western blot were used to measure the changes of cross section of gastrocnemius muscle fibers and the expression of NF-κB,PAX7 and MuRF1 protein level in the gastrocnemius and tibialis anterior muscle. Results Compared with model group, the administration of SPJ could effectively re-duce the weight loss (P <0.05), increase muscle mass (P<0.05) and decrease muscle tissue degrada-tion in cachexia mice. Meanwhile,SPJ significantly re-duced the levels of IL-1β and TNF-α in serum (P <0.05) and decreased the expression of NF-κB. Con-clusion SPJ can improve cancer cachexia in mice in a dose-dependent manner. The potential mechanism may be associated with the inhibition of NF-κB mediated in-flammatory factor expression.

Effect of saponins extracted from Panax japonicus on inhibiting cardiomyocyte apoptosis by AMPK/Sirt1/NF-κB signaling pathway in aging rats / 中国中药杂志

To investigate the effects of saponins extracted from Panax japonicus(SPJ) on cardiomyocyte apoptosis in natural aging rats and explore its underlying mechanisms. SD male rats were randomly divided into four groups: young control group, natural aging group, SPJ low dose group and SPJ high dose group, with 10 rats in each group. The rats in natural aging group, SPJ low and high dose groups were respectively treated with normal saline, SPJ 10 and 60 mg•kg-1•d-1 from the beginning of 18 month-old, 6 days per week for 6 months till 24 month-old. Then the animals were sacrificed. Their myocardial morphology changes were observed by using haematoxylin-eoin(HE) staining; cardiomyocyte apoptosis was tested by using Tunel assays; and the protein expression levels of Bcl-2, Bax, IL-1β, TNF-α, AMPK, p-AMPK, Sirt1, and Ac-NF-κB p65 in myocardial tissues of rats were detected by Western blot. The results showed that SPJ could effectively improve the arrangement disorder of myocardial fibers, reduce the infiltration of inflammatory cells and inhibit cardiomyocyte apoptosis in natural aging rats. At the same time, SPJ could significantly inhibit the protein expression of Bax, IL-1β, TNF-α and Ac-NF-κB p65, and increase the expression of Bcl-2, Bcl-2/Bax, p-AMPK/AMPK and Sirt1 in the heart tissues of natural aging rats. SPJ can effectively inhibit cardiomyocyte apoptosis in natural aging rats, and its mechanisms may be related with the regulation of inflammatory reaction by AMPK/Sirt1/NF-κB signaling pathway.

Protective effect of total saponins of Panax notoginseng combined with total flavonoids of epimedium on D-galactose-incuced senescence of H9c2 cell / 中国中药杂志

To investigate the protective effect of Panax notoginseng saponins combined with total flavonoids of epimedium on D-gal-induced senescence of H9c2 cells and explore its underlying mechanisms. The 50 mol•L⁻¹ D-gal was used to induce H9c2 cells senescence. Different concentrations of TPNS, TFE, and TPNS combined with TFE were used for 4 hours for pre-treatment. D-gal was used to stimulate H9c2 cardiac muscle cells for 24 h. Then in order to determine the best combined scheme, MTT was used to detect cell viability. Cell senescence was identified by β-galactosidase staining. Levels of reactive oxygen species(ROS) was observed by DCFH-DA detection. The changes of mitochondrial membrane potential were identified by JC-1 detection. Protein levels of silentmating type information regulation 2 Homolog-1(SIRT1), peroxisomal proliferator-activated receptor-coactivator 1α(PGC-1α) and silentmating type information regulation 2 Homolog-3(SIRT3) were detected by western blot analysis. The results showed that TPNS(5 mg•L⁻¹) combined with TFE(5 mg•L⁻¹) had significant synergistic effect on H9c2 myocardial cell proliferation(Q=1.154), so 5 mg•L-1TPNS combined with 5 mg•L⁻¹ TFE was determined as the best scheme. The quantity of β-galactosidase staining and the fluorescence intensity of ROS were apparently decreased in 5 mg•L⁻¹ TPNS combined with 5 mg•L⁻¹ TFE scheme. Meanwhile, it markedly increased the florescence intensity of mitochondrial membrane potential and enhanced the protein expression of SIRT1, PGC-1α and SIRT3. TPNS combined with TFE could protect H9c2 cells from D-gal-induced senescence. The mechanism might be related to adjusting the signal pathways of SIRT1/PGC-1α, SIRT3, adjusting the structure and function of mitochondria and reducing oxidative stress injury.

A novel missense mutation of the TYR gene in a pedigree with oculocutaneous albinism type 1 from China / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The mutation of the tyrosinase (TYR) gene results in oculocutaneous albinism type 1 (OCA1), an autosomal recessive genetic disorder. OCA1 is the most common type of OCA in the Chinese population. Hence, the TYR gene was tested in this study. We also delineated the genetic analysis of OCA1 in a Chinese family.</p><p><b>METHODS</b>Genomic DNA was isolated from the blood leukocytes of a proband and his family. Mutational analysis at the TYR locus by DNA sequencing was used to screen five exons, including the intron/exon junctions. A pedigree chart was drawn and the fundus of the eyes of the proband was also examined.</p><p><b>RESULTS</b>A novel missense mutation p.I151S on exon 1, and homozygous TYR mutant alleles were identified in the proband. None of the mutants was identified among the 100 normal control subjects. Genetic analysis of the proband's wife showed normal alleles in the TYR gene. Thus, the fetus was predicated a carrier of OCA1 with a normal appearance.</p><p><b>CONCLUSION</b>This study provided new information about a novel mutation, p.I151S, in the TYR gene in a Chinese family with OCA1. Further investigation of the proband would be helpful to determine the effects of this mutation on TYR activity.</p>

Clinicopathologic features and proliferative status of colorectal serrated lesions: a study of 104 cases / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the clinicopathologic features and proliferative status of colorectal hyperplastic polyp (HP), sessile serrated adenoma (SSA) and traditional serrated adenoma (TSA).</p><p><b>METHODS</b>One hundred and four cases colorectal serrated lesions were collected from 2628 cases of colorectal polyps during the period from November, 2002 to December, 2007. The clinicopathologic features and expression of proliferation marker Ki-67 were studied.</p><p><b>RESULTS</b>On the basis of morphologic examination, 60 cases were classified as HP, 20 cases as TSA, 11 cases as SSA, 7 cases as mixed HP/SSA/TSA, and 6 cases as mixed serrated polyp/adenoma and tubular adenoma. Immunohistochemical study for Ki-67 showed that 40 cases (78%) of the 51 cases of HP were either mostly negative or rarely (<25% cells) positive. Most of the positive cells were located at crypt bases. Among the 15 cases of TSA, 11 of them revealed positive cryptal cells (25% to 50% or>50% positivity). Most of the positive cells were located in mid portion of crypts. The number and distribution of Ki-67 positive cells in SSA were similar to those in TSA but were significantly different from those in tubular adenoma and adenocarcinoma (chi2=34.601, P=0.000; chi2=63.077, P=0.000, respectively).</p><p><b>CONCLUSIONS</b>HP, SSA and TSA have their morphologic characteristics, with some overlapping features noted. The distinction between SSA and HP can be difficult. Diagnosis of SSA relies mostly on architectural rather than cytologic features. The distinction between TSA and SSA depends mainly on the presence of dysplasia. Ectopic crypt formation is almost exclusively seen in TSA. The distribution and percentage of Ki-67-positive cells are also helpful in subtyping of various colorectal serrated lesions. In general, the proliferative index is lower in serrated adenoma (TSA or SSA) than in tubular adenoma.</p>

Clinicopathological and immunohistochemical features of colorectal sessile serrated adenoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the clinicopathological characteristics and expression status of Ki67, p53, CEA, CDX, CK7 in colorectal sessile serrated adenoma (SSA).</p><p><b>METHODS</b>The clinicopathological data of 11 cases of SSA, 51 cases of hyperplastic polyp (HP) and one case with mixed HP/SSA were reviewed and analyzed retrospectively. The expression of Ki67, p53, CEA, CDX and CK7 were detected by immunohistochemistry.</p><p><b>RESULTS</b>The major histological features in SSA were architectural abnormality in crypts, dilatation of serrated crypt bases like an inverted "T" or "L" shape adjacent to muscularis mucosa. Atypical cells containing round to oval nuclei and nucleoli were also observed. The immunohistochemical staining showed that the expression of p53 increased gradually from HP to TA: 11.8% in HP, 20.0% in SSA, 41.2% in VTA and 75.0% in TA, with a significant difference among the groups (chi(2) = 17.996, P = 0.000). However, no significant difference in the expression of CDX and CK7 was observed between HP and SSA. Of the 10 SSA cases, positive expression of Ki67 was found in cells located in the base or middle part of crypt in 6 cases, positive cells index was 26% - 50% in 5 cases, and > 50% in 3. Compared with the expression of Ki67 in the HP, VTA and VA, SSA showed a significant difference in both the positive cell number and in the positive regions. (positive number: chi(2) = 34.601, P = 0.000; positive regions: chi(2) = 63.077, P = 0.000).</p><p><b>CONCLUSION</b>Morphological diagnosis of SSA was mainly based on crypt architectural and cellular abnormalities, and the crypt architectural abnormality may be more important than cellular features. Detection of p53 and Ki67 expression may be helpful in differential diagnosis and understanding the nature of SSA.</p>