Role of succinate dehydrogenase in hypoxic postconditioning-induced reduction of hypoxia-reoxygenation injury in myocardial cells of rats and the relationship with mito-K ATP / 中华麻醉学杂志

Objective:To evaluate the role of succinate dehydrogenase (SDH) in hypoxic postconditioning (HPC)-induced reduction of hypoxia-reoxygenation (H/R) injury in myocardial cells of rats and the relationship with mitochondrial ATP-sensitive potassium channels (mito-K ATP). Methods:Myocardial cells isolated from adult male Sprague-Dawley rats were cultured for 48 h and then divided into 7 groups ( n=24 each) using a random number table method: blank control group (Nor group), H/R group, SDHA-siRNA adenovirus+ H/R group (siRNA+ H/R group), HPC group, SDHA-siRNA adenovirus+ HPC group (siRNA+ HPC group), 5-HD+ HPC group, and SDHA-siRNA adenovirus+ 5-HD+ HPC group (siRNA+ 5-HD+ HPC group). Nor group was continuously cultured for 195 min under normoxic conditions. The H/R injury model was prepared by exposing the cells to hypoxia for 45 min in 5% CO 2 + 1% O 2 + 94% N 2, followed by reoxygenation for 150 min. The HPC method involved three cycles of 5 min reoxygenation/5 min hypoxia at the end of 45 min ischemia before 120 min reoxygenation. The mito-K ATP blocker 5-HD administration method involved adding 5-HD at a final concentration of 100 μmol/L at 30 min of hypoxia. The myocardial cells in each siRNA group were successfully transfected with SDHA-siRNA adenovirus to silence SDHA expression. The cell viability, calcium ion level, SDH activity, ATP content, degree of mitochondrial permeability transition pore (mPTP) opening, and mitochondrial membrane potential (MMP) were measured at the end of reoxygenation. Results:Compared with Nor group, the cell viability, ATP content and MMP were significantly decreased, and the degree of mPTP opening, level of calcium ion and activity of SDH were increased in H/R group ( P<0.05). Compared with H/R group, the cell viability, ATP content and MMP were significantly increased, and the degree of mPTP opening, calcium ion level and SDH activity were decreased in siRNA+ H/R group and HPC group ( P<0.05). Compared with HPC group, the cell viability, ATP content and MMP were significantly decreased, and the degree of mPTP opening, calcium ion level and SDH activity were increased in 5-HD+ HPC group ( P<0.05), and the cell viability, ATP content and MMP were significantly increased, and the degree of mPTP opening, calcium ion level and SDH activity were decreased in siRNA+ HPC group ( P<0.05). Compared with siRNA+ HPC group, the cell viability, ATP content and MMP were significantly decreased, the opening degree of mPTP and calcium ion level were increased ( P<0.05), and no significant change was found in the SDH activity in siRNA+ 5-HD+ HPC group ( P>0.05). Compared with 5-HD+ HPC group, the SDH activity was significantly decreased, and no significant change was found in the other parameters in siRNA+ 5-HD+ HPC group ( P>0.05). Conclusions:HPC alleviates H/R injury probably by reducing SDH activity and opening mito-K ATP in myocardial cells of rats.

Succinate dehydrogenase deficiency renal cell carcinoma: a case report / 中华泌尿外科杂志

Succinate dehydrogenase (SDH) defective renal cell carcinoma (RCC) is a new subtype of renal carcinoma newly identified by WHO(2016). Until now, only a few samples and a few cases have been reported retrospectively. This article reported a young female patient who was found to have a small tumor in the left kidney by physical examination and underwent left partial nephrectomy. The postoperative pathological result was SDH-RCC. There was no recurrence and metastasis of the tumor 3 months after operation.

Anti-aging effects of chlorpropamide depend on mitochondrial complex-II and the production of mitochondrial reactive oxygen species

Sulfonylureas are widely used oral anti-diabetic drugs. However, its long-term usage effects on patients' lifespan remain controversial, with no reports of influence on animal longevity. Hence, the anti-aging effects of chlorpropamide along with glimepiride, glibenclamide, and tolbutamide were studied with special emphasis on the interaction of chlorpropamide with mitochondrial ATP-sensitive K+ (mitoK-ATP) channels and mitochondrial complex II. Chlorpropamide delayed aging in Caenorhabditis elegans, human lung fibroblast MRC-5 cells and reduced doxorubicin-induced senescence in both MRC-5 cells and mice. In addition, the mitochondrial membrane potential and ATP levels were significantly increased in chlorpropamide-treated worms, which is consistent with the function of its reported targets, mitoK-ATP channels. Increased levels of mitochondrial reactive oxygen species (mtROS) were observed in chlorpropamide-treated worms. Moreover, the lifespan extension by chlorpropamide required complex II and increased mtROS levels, indicating that chlorpropamide acts on complex II directly or indirectly via mitoK-ATP to increase the production of mtROS as a pro-longevity signal. This study provides mechanistic insight into the anti-aging effects of sulfonylureas in C. elegans.

Mitochondrial mechanism of diabetic mellitus-caused abolition of cardioprotection induced by ischemia postconditioning in rats: succinate dehydrogenase / 中华麻醉学杂志

Objective:To evaluate the relationship between the mitochondrial mechanism of diabetic mellitus-caused abolition of cardioprotection induced by ischemia postconditioning (IPO) and succinate dehydrogenase (SDH) in rats.Methods:Thirty-six SPF male non-diabetic Sprague-Dawley rats, aged 16-20 weeks, weighing about 300 g, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group (ND+ Sham group), ischemia-reperfusion (I/R) group (ND+ I/R group) and IPO group (ND+ IPO group). Seventy-two rats with diabetes mellitus were divided into 6 groups ( n=12 each) using a random number table method: sham operation group (DM+ Sham group), I/R group (DM+ I/R group), DM+ IPO group, sham operation+ dimethyl malonate group (group DM+ Sham+ Dme), I/R+ dimethyl malonate group (group DM+ I/R + Dme) and IPO+ dimethyl malonate group (group DM+ IPO+ Dme). The model of cardiopulmonary bypass (CPB) was established, and the model of total I/R injury was induced by ligating the ascending aorta for 30 min followed by 60 min of reperfusion.The animals underwent 3 cycles of 30-s reperfusion followed by 30-s ischemia starting from the onset of reperfusion in each IPO group.In each Dme group, dimethyl malonate was infused through the tail vein at a rate of 4 mg· kg -1·min -1 for 40 min starting from the beginning of CPB.At the end of reperfusion, the myocardial tissues were taken for measurement of mitochondrial respiratory control ratio (RCR) (by the Lufthansa electrode method), mitochondrial membrane potential (MMP) (by the JC-1 method) and the opening of mitochondrial permeability transition pore (mPTP) (by absorptiometry) and for determination of the activity of reactive oxygen species (ROS) (with the fluorescent probe), succinate dehydrogenase (SDH) (using spectrophotometric method) and the contents of succinic acid and fumarate. Results:Compared with ND+ Sham group, the activities of SDH and ROS, opening of mPTP and content of fumarate were significantly increased, and MMP, RCR and succinic acid content were decreased in ND+ I/R ( P<0.05). Compared with group ND+ I/R, the activities of SDH and ROS, opening of mPTP and content of fumarate were significantly decreased, and MMP, RCR and succinic acid content were increased in ND+ IPO ( P<0.05). Compared with group DM+ Sham, the activities of SDH and ROS, opening of mPTP and content of fumarate were significantly increased, and MMP, RCR and succinic acid content were decreased in group DM+ I/R ( P<0.05). Compared with group DM+ I/R, no significant change was found in the parameters mentioned above in group DM+ IPO ( P>0.05). Compared with group DM+ IPO, the activities of SDH and ROS, opening of mPTP and content of fumarate were significantly decreased, and MMP, RCR and succinic acid content were increased in group DM+ IPO+ Dme ( P<0.05). Compared with group DM+ I/R+ Dme, the activities of SDH and ROS, opening of mPTP and content of fumarate were significantly decreased, and MMP, RCR and succinic acid content were increased in group DM+ IPO+ Dme ( P<0.05). Conclusion:The mitochondrial mechanism of diabetic mellitus-caused abolition of cardioprotection induced by IPO may be related to the enhancement of SDH activity in rats.

Colorectal Cancer Modeling and Difference Analysis in Cold and Heat Conditions / 中国实验方剂学杂志

Objective:To explore the correlation between the occurrence of colorectal cancer and different internal environment （cold and heat）. Method:The 70 Wistar rats （male and female） were randomly divided into blank group （10 cases）， cold model group （30 cases） and heat model group （30 cases）. The cold syndrome model was made by intragastric infusion of cold water （0 ℃） and soaking in cold water （10 ℃）. The heat model was made by ethanol （30%） and capsaicin solution （0.9 g·L-1）. The blank group was given normal saline by gavage， 10 mL·kg-1·d-1， for 5 consecutive weeks. The colorectal cancer model was made by subcutaneous injection of DMH solution in the back of neck in the cold model group and heat model group at the 6th week， 25 mg·kg-1，once a week，for 12 consecutive times. During the carcinogenesis， only 30% ethanol solution was given to the heat model group， and the modeling was maintained in cold model group， 10 mL·kg-1·d-1， for 38 weeks. The general state of the rats in each group was observed， and the changes of food intake and body weight were measured. At the 27th， 29th， 32th， 35th and 38th weeks， samples were collected in batches. Intestinal tissues were subjected to hematoxylin-eosin staining （HE） and detection of sodium， potassium-adenosine triphosphate （Na+ K+-ATP）， calcium， magnesium-adenosine triphosphate （Ca2+ Mg2+-ATP） activity， lactate dehydrogenase （LDH） activity and succinate dehydrogenase （SDH） activity. Result:The symptoms of hematochezia and ascites in cold model group were earlier than those in heat model group. As compared with the blank group， the food intake and body weight were decreased in cold model group and heat model group. As compared with the blank group， the length of the large intestine was shorter in cold model group at the 32nd and 35th week （P<0.05）， the activities of Na+ K+-ATP， Ca2+ Mg2+-ATP increased significantly in heat model group at the 27th， 29th and 38th week （P<0.05， P<0.01）， the SDH enzyme activity was decreased in the cold model group at the 29th and 35th week （P<0.05， P<0.01）， the SDH enzyme activity was significantly increased in the heat model group at the 38th week （P<0.01）. At the 27th week， LDH enzyme activity was significantly reduced in cold model group （P<0.01）. The LDH activity was increased significantly in heat model group at 29th and 32nd week （P<0.05， P<0.01）. As compared with the heat model group， the large intestine texture of the cold model rats showed greater brittleness， aggravated fibrosis， more obvious fibroproliferative characteristics， higher tumor incidence， and more serious tumor differentiation. The Na+ K+-ATP， Ca2+ Mg2+-ATP enzyme activities of the cold model rats were significantly reduced at 27th， 29th， and 38th weeks （P<0.01）， the SDH enzyme activity was significantly reduced in the cold model rats at 29th and 38th weeks （P<0.01）， the LDH activity was reduced in the cold model group at 32nd week （P<0.05）. Conclusion:Cold environment for colorectal cancer promotes tumorigenesis， and the hot environment can also promote tumorigenesis in a later stage.

Pheochromocytoma and paraganglioma: implications of germline mutation investigation for treatment, screening, and surveillance

ABSTRACT Objective Paraganglioma (PGL) and pheochromocytoma (PCC) are rare neuroendocrine tumors that were considered to be predominantly sporadic. However, with the identification of novel susceptibility genes over the last decade, it is currently estimated that up to 40% of cases can occur in the context of a hereditary syndrome. We aimed to characterize PGL/PCC families to exemplify the different scenarios in which hereditary syndromes can be suspected and to emphasize the importance for patients and their families of making an opportune genetic diagnosis. Materials and methods Retrospective analysis of patients diagnosed with PGL/PCC. Germline mutations were studied using next-generation sequencing panels including SDHA, SDHB, SDHC and SDHD. Clinical data were collected from clinical records, and all patients received genetic counseling. Results We describe 4 families with PGL/PCC and germline mutations in SDH complex genes. 2 families have SDHB mutations and 2 SDHD mutations. The clinical presentation of the patients and their families was heterogeneous, with some being atypical according to the literature. Conclusions PGL/PCC are more commonly associated with a germline mutation than any other cancer type, therefore, all individuals with these types of tumors should undergo genetic risk evaluation. NGS multigene panel testing is a cost-effective approach given the overlapping phenotypes. Individuals with germline mutations associated with PGL/PCC should undergo lifelong clinical, biochemical and imaging surveillance and their families should undergo genetic counseling. For all these reasons, it is critical that all medical staff can suspect and diagnose these inherited cancer predisposition syndromes.

Enzyme histochemical detection of cell viability in ablation area of thyroid nodules at 6 months after microwave ablation / 第二军医大学学报

Objective To explore the cell viability in the ablation area of thyroid nodules at 6 months after microwave ablation by enzyme histochemical staining. Methods Twenty-four ablation areas of thyroid nodules were selected from 20 patients who underwent histopathological assessment of the ablation area by core needle biopsy at 6 months after microwave ablation between Dec. 2017 and Sep. 2018. Core needle biopsy was performed at the central and marginal regions of the ablation area with a cutting biopsy needle. The specimens were obtained and placed in liquid nitrogen to make frozen sections. Enzyme histochemical staining was used to detect the activities of succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), and the difference of cell morphology and histological structure was compared with H-E staining results. Results The specimens of the central and marginal regions of 24 ablation areas were successfully obtained. The histochemical staining of SDH and NADPH-d in the central region of ablation area had good consistency, and the negative rates were both 95.83% (23/24). The histochemical staining of SDH and NADPH-d in the marginal region of ablation area also had good consistency, and the negative rates were both 91.67% (22/24). H-E staining of 23 central regions and 22 marginal regions showed pink amorphous mass of necrosis. H-E staining of 1 central region and 2 marginal regions showed partly necrotic and fibrous tissue hyperplasia. The location of fibrous tissue hyperplasia was consistent with the location of the positive region of enzyme histochemical staining. Conclusion At 6 months after microwave ablation, the tissue in the ablation area of thyroid nodules is consistent with coagulative necrosis, and is still inactivated. SDH or NADPH-d enzyme histochemical staining combined with H-E staining can objectively evaluate the old ablation area.

Effects of dexmedetomidine pretreatment on expression of TFAM and SDHA during lung ischemia-reperfusion in mice / 中华麻醉学杂志

Objective To evaluate the effect of dexmedetomidine pretreatment on the expression of mitochondrial transcription factor A ( TFAM) and succinate dehydrogenase ( SDHA) during lung ischemia-reperfusion ( I∕R) in mice. Methods Twenty-four clean-grade healthy male C57BL∕6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 3 groups ( n=8 each) using a random number table method:sham operation group ( group S ) , lung I∕R group ( group I∕R ) and dexmedetomidine pretreatment group ( group D) . In I∕R and D groups, lung I∕R was induced by clamping the left pulmonary hilum for 60 min followed by 120-min reperfusion in anesthetized mice. The chest was only opened, but the left pulmonary hilum was not occluded in group S. Dexmedetomidine 25μg∕kg was intraperitoneally injected at 30 min be-fore ischemia in group D, while the equal volume of normal saline was given instead of dexmedetomidine in I∕R and S groups. The mice were sacrificed at 120 min of reperfusion, and lungs were removed for determi-nation of wet∕dry weight ratio ( W∕D ratio) , cell apoptosis ( by TUNEL) and expression of TFAM and SDHA mRNA in lung tissues ( by real-time polymerase chain reaction ) and for examination of the pathological changes of lung tissues. The apoptosis index was calculated. Results Compared with group S, the W∕D ratio, apoptosis index and lung injury scores were significantly increased, and the expression of TFAM and SDHA mRNA was down-regulated in I∕R and D groups ( P<0. 05) . Compared with group I∕R, the W∕D ra-tio, apoptosis index and lung injury scores were significantly decreased, and the expression of TFAM and SDHA mRNA was up-regulated in group D ( P<0. 05) . Conclusion The mechanism by which dexmedeto-midine pretreatment attenuates lung I∕R injury is related to up-regulating the expression of TFAM and SDHA in mice.

Quantitation of muscle pathology abnormalities in 18 patients with mitochondrial disorders / Quantificação das anormalidades patológicas musculares em 18 pacientes com desordens mitocondriais

ABSTRACT INTRODUCTION: Mitochondrial disorders can lead to the accumulation of mitochondria in muscle fibers, as indicated by ragged red (RRF) or ragged blue fibers when stained with modified Gomori trichrome or succinate dehydrogenase (SDH+), respectively, and, absence of activity of cytochrome c oxidase, COX negative fibers (COX-). The combined COX-SDH stain (COMBO+) can reveal even more COX-deficient fibers. OBJECTIVE: To quantify RRFs, SDH+, COX-, and COMBO+ fibers in muscle biopsies with mitochondrial findings. MATERIAL AND METHODS: We retrospectively selected 18 muscle biopsies with mitochondrial abnormalities based on the Walker criteria (percentage of RRFs/COX- fibers, and clinical picture), and/or the Sleigh criteria (percentage of RRFs, SDH+, and COX- fibers). RESULTS: Females represented 83.3%, with a mean age of 38.6 years (5 months-70 years). Patients were diagnosed with chronic progressive external ophthalmoplegia (CPEO, 66.7%), proximal myopathy (22.2%), idiopathic hyperCKemia (11.1%), Kearns-Sayre syndrome (5.6%), mitochondrial encephalomyopathy with ragged red fibers and stroke-like episodes (5.6%), and a dystrophic pattern (5.6%). Some cases of CPEO were combined with proximal myopathy. The quantitative pathologic findings were: RRFs, 3.95% ± 3.17%; SDH+, 7.55% ± 6.1%; COX-, 10.9% ± 7.2%; COMBO+, 14.22% ± 12.79%. We found a slight variation in the diameter of muscle fibers, no necrosis or proliferative connective tissue, few fibers with internal nuclei, and some cases with fiber type grouping. CONCLUSION: Pathologic events, grouped in ascending order of frequency, were RRFs, SDH+ fibers, COX- fibers, and COMBO+ fibers. These data emphasize the importance of the COMBO technique in revealing occult COX deficiency in muscle fibers.

RESUMO INTRODUÇÃO: Desordens mitocondriais são usualmente caracterizadas por: 1. acúmulo de mitocôndria nas fibras musculares que aparecem como fibras vermelhas rasgadas (FVR) ou azuis rasgadas quando coradas, respectivamente, pelo tricrômio modificado de Gomori ou pelo succinato desidrogenase (SDH+); 2. ausência de atividade da citocromo c oxidase (COX), originando fibras COX negativa (COX-). A combinação de colorações COX e SDH pode revelar ainda mais fibras COX deficiente (COMBO+). Objetivos: Quantificar FVR, SDH+, COX- e COMBO+ em biópsias musculares com anormalidades mitocondriais. MATERIAL E MÉTODOS: Foram analisadas retrospectivamente 18 biópsias com anormalidades mitocondriais com base no critério de Walker (percentagem de FVR/ COX- e quadro clínico) e/ou critério de Sleigh (percentagem de FVR, SDH+ e COX-). RESULTADOS: Sexo feminino representou 83, 3% e média de idade 38, 6 anos (5 meses a 70 anos). Oftalmoplegia externa progressiva crônica (OEPC) representou 66, 7%; miopatia proximal, 22, 2%; hiperCKemia idiopática, 11, 1%; síndrome de Kearns-Sayre, 5, 6%; encefalopatia mitocondrial com FVR e episódios semelhantes a acidente vascular cerebral, 5, 6%; e padrão distrófico, 5, 6%. Alguns casos de OEPC estavam associados à miopatia proximal. Achados patológicos quantitativos: FVR, 3, 95% ± 3, 17%; SDH+, 7, 55% ± 6, 1%; COX-, 10, 9% ± 7, 2%; COMBO+, 14, 22% ± 12, 79%. Encontramos leve variação de calibre das fibras musculares sem necrose ou proliferação de tecido conjuntivo, poucas fibras com núcleos internos e alguns casos com agrupamento de fibras. CONCLUSÃO: As anormalidades patológicas nas fibras musculares em ordem ascendente de frequência foram: FVR, SDH+, COX- e COMBO+. Nossos achados enfatizam a importância da técnica COMBO (COX + SDH) para aumento na frequência de fibras musculares COX deficiente ocultas.

Expression of succinate dehydrogenase subunit protein in succinate dehydrogenase-deficient gastrointestinal stromal tumors / 中华病理学杂志

Objective@#To investigate the expression of succinate dehydrogenase complex subunit protein in succinate dehydrogenase-deficient gastrointestinal stromal tumors (SDH-deficient GISTs).@*Methods@#Three hundred fifty-two cases of GISTs were collected from January 2003 to January 2017 at the Affiliated Hospital of Guizhou Medical University and West China Hospital of Sichuan University.The expression of succinate dehydrogenase subunit protein was detected by immunohistochemical EnVision technique in 352 cases of GISTs, and the negative cases were analyzed for clinicopathologic features and outcome. The gene segments of CKIT exons 9, 11, 13 and 17 and PDGFRA exons 12 and 18 were amplified and detected in SDH-deficient (negative) cases.@*Results@#A total of 15 SDHB-deficient (negative) GISTs (4.3%, 15/352) were found among 352 cases of GISTs. Six patients were male and nine were female. The age of initial diagnosis ranged from 15 to 84 years (median=53 years, mean=47 years). The tumor involved stomach (14 cases) and mesentery (1 case). The tumor sizes varied from 0.5 cm to 15.0 cm (mean=6.9 cm). There were six, six and three cases of epithelioid, mixed and spindle cell types respectively. Eight cases showed multi-nodularity in the wall of stomach. Metastasis to lymph node was noted in four cases, one case showed intraperitoneal implantation metastasis. Metastases to liver, pancreas and lymph node were found in one case, and one case showed vascular invasion. Among SDHB-deficient GISTs, two SDHA-deficient (negative) cases were found (0.6%, 2/352), but there were no SDHC and SDHD deficient (negative) cases. Five of the fifteen SDH-deficient GISTs had follow-up data: one patient died 8 months after surgery from unknown cause, four had no recurrences or metastases, and there was no history of paraganglioma and pulmonary chondroma found in patients and their families. No mutation in CKIT and PDGFRA gene was identified in 15 cases of SDH-deficient GISTs.@*Conclusion@#SDH-deficient GISTs have unique clinicopathologic features and a favorable prognosis, and a small proportion of cases are SDHA-deficient.

Application of SDHB and SDHC immunohistochemistry in the differentiation of malignant and benign pheochromocytoma and paraganglioma / 中华内分泌代谢杂志

Objective To evaluate the expression profile of succinate dehydrogenase (SDH)B and SDHC in pheochromocytoma (PCC) and paraganglioma(PGL) (collectively abbreviated as PPGL), and their value in the early diagnosis of malignancy. Methods SDHB and SDHC immunohistochemistry were performed on 140 tumor specimens from 126 PPGL patients (PCC n＝62, PGL n＝61, PCC+PGL n＝3). Results (1) Germline mutation status of 67 patients were determined, of which, identifying 37(55.2%) patients with germline mutation: 2 (3.0%) SDHA, 18 ( 26. 9%) SDHB, 2 ( 3. 0%) SDHC, 5 ( 7. 5%) SDHD, 2 ( 3. 0%) VHL, 7 ( 10. 4%) RET, and 1(1.5%) NF1; and 30 (44.8%) individuals without known mutation. (2) Among 30 PPGLs from 27 patients with SDH-related (SDHx) mutations, 96.7%(29/30) stained negative for SDHB, 76.7%(23/30) stained negative for SDHC, while only 28.6%(14/49) and 18.4%(9/49) stained negative for SDHB and SDHC respectively in the 49 PPGLs without SDHx mutation (P<0.05). (3) The sensitivity of the SDH immunostaining in detecting the presence of germline SDHx mutation was 96.7%for SDHB and 76.7%for SDHC, while the specificity was 71.4%for SDHB and 81.6% for SDHC. ( 4 ) Among PPGLs without SDHB expression, 22. 9% were malignant. This percentage is significantly higher than that in PPGLs with preserved SDHB expression (3.8%, P<0.05). Conclusion SDHB and SDHC immunohistochemistry may serve as post-surgical screening tools to predict the presence of germline SDHx mutation in PPGLs. Negative SDHB expression calls for intense follow-up to rule out malignancy.

Study on the overexpression of succinate dehydrogenase B and MIB-1 in pheochromocytoma/paraganglioma tissue / 中华泌尿外科杂志

Objective We aimed to investigate the overexpression of succinic dehydrogenase (SDH) B and MIB-1 in patients with pheochromocytoma/paraganglioma(PHEO/PGLs) and its significance for predicting the clinical malignant behavior.Methods From August 2008 to April 2016,the clinical characteristics of 93 patients with PHEO/PGLs were analyzed retrospectively.There were 57 males and 36 females,with an average of 34 years,ranging 8-73 years old.There were 68 cases of adrenal pheochromocytoma and 25 cases of paraganglioma.There were 79 cases with hypertension and 14 cases of adrenal accidental tumors.Sixty-six cases with typical hyper-catecholamine secretion symptoms and 27 cases with non-functional PHEO/PGL.Benign PHEO/PGLs were 77 cases and malignant 16 cases.The tumor was located on the left side in 39 cases,on the right side in 32 cases and multiple lesions in 22 cases.The diameter of the PHEO/PGL tumor was (6.8 ± 2.7) cm.The 24 h urine catecholamine was measured before operation,which showed epinephrine was (42.6 ± 5.1) μg/24 h,norepinephrine was (167.5 ± 13.5) μg/24h and dopamine was (246.4 ± 71.2)μg/24h.Six cases wihtout hereditary diseases of urinary system were selected as normal control group.SDHB,SDHAF2,SDHC,SDHD,VHL and RET gene mutations were detected in all patients.Immunohistochemical panel has been performed to detect the expression of SDHB,MIB-1,EPAS1,VEGF-1 receptor (VEGF-1 R),and chromain A (CgA) in 93 specimens of PHEO/PGL tissue.The positive granular cytoplasm staining ＞ 50％ was strongly positive (+ + +),11％ to 50％ was moderately positive (+ +),1％ to 10％ was weak positive (+) and the negative was compared with the known positive internal reference,that is,there was less than 1％ or no stain completely.Results SDHB,SDHAF2,SDHC,SDHD,VHL and RET gene mutations in 27 cases (29.5％).Nine patients with SDHB gene mutation (9.7％).RET proto-oncogene mutations in 8 cases (8.6％).3 cases had VHL mutation (3.2％).Immunohistochemical staining showed that MIB-1 positive expression was found in 7 of 9 patients with SDHB gene mutation.Six cases in the control group were negative for gene detection and MIB-1,EPAS1,CgA and VEGF-1R immunohistochemical results.EPAS1 showed moderately positive in patients with PHEO/PGL and strong positive in patients with malignant PHEO/PGL.In 9 cases with SDHM mutation,EPAS1 was noticed positive in seven cases,which showed the relationship with CgA,MIB-1 and VEGF-1R.Conclusions The SDHB gene mutation is usually shown as a paraganglioma focus outside the adrenal gland.And 9.8％ of the paragangliomas were associated with a mutation of the SDHB gene with an increase in malignant risk.The SDHB mutation caused over-expression of MIB-1 and the positive expression of EPAS1 and VEGF-1R in PHEO/PGL tissues,which was associated with invasion and metastasis of malignant PHEO/PGL.

Loss of expression of succinate dehydrogenase subunit A and B in renal cell carcinoma / 实用医学杂志

Objective To investigate the expression of succinate dehydrogenase subunit A(SDHA)and subunit B (SDHB) in patients with renal cell carcinoma (RCC),and its relationship with the clinicopathologic characteristics. Methods The expression of SDHA and SDHB was detected in 179 cases of RCC by immunohisto-chemistry and the relationship between the SDHA ,SDHB expression and the clinicopathologic characteristics of RCC were analyzed. Results In 179 cases of RCC,18 cases(10.1%)were shown with negative or suspicious-negative expression of SDHA and SDHB,including 2 cases(1.1%)with suspicious-negative expression of SDHA and SDHB. 12 cases(66.7%)had tumor located in left kidney and 6 cases(33.3%)had tumor located in right kidney. The well-differentiation group contained 15 cases(83.3%),moderate-differentiation group contained 1 case (5.6%)and poor-differentiation group contained 2 cases(11.1%). Most of SDH negative and suspicious negative RCC had the following characteristics that the normal renal tubule could be seen within the tumor. The tumor was composed of polygonal cells arranged in nest ,and the polygonal cells were separated by a fine fibrovascular stroma. The nuclei were shown around with vesicular chromatin. The eosinophilic flocculet material ,vacuole and eosino-philic inclusion body could be seen in the cytoplasm of tumor cells. Conclusion The RCC cases with negative expression of SDHA and SDHB protein have unique histological features ,with significantly higher incidence in the left kidney than that in the right kidney.

ACN9 Regulates the Inflammatory Responses in Human Bronchial Epithelial Cells / 결핵및호흡기질환

BACKGROUND: Airway epithelial cells are the first line of defense, against pathogens and environmental pollutants, in the lungs. Cellular stress by cadmium (Cd), resulting in airway inflammation, is assumed to be directly involved in tissue injury, linked to the development of lung cancer, and chronic obstructive pulmonary disease (COPD). We had earlier shown that ACN9 (chromosome 7q21), is a potential candidate gene for COPD, and identified significant interaction with smoking, based on genetic studies. However, the role of ACN9 in the inflammatory response, in the airway cells, has not yet been reported. METHODS: We first checked the anatomical distribution of ACN9 in lung tissues, using mRNA in situ hybridization, and immunohistochemistry. Gene expression profiling in bronchial epithelial cells (BEAS-2B), was performed, after silencing ACN9. We further tested the roles of ACN9, in the intracellular mechanism, leading to Cd-induced production, of proinflammatory cytokines in BEAS-2B. RESULTS: ACN9 was localized in lymphoid, and epithelial cells, of human lung tissues. ACN9 silencing, led to differential expression of 216 genes. Pathways of sensory perception to chemical stimuli, and cell surface receptor-linked signal transduction, were significantly enriched. ACN9 silencing, further increased the expression of proinflammatory cytokines, in BEAS-2B after Cd exposure. CONCLUSION: Our findings suggest, that ACN9 may have a role, in the inflammatory response in the airway.

Effect of Heat Reinforcing Acupuncture Manipulation on Articular Synovium LDH, SDH and CCO in Rheumatoid Arthritis ;Rabbits / 上海针灸杂志

Objective To investigate the regulating effect of heat reinforcing acupuncture manipulation on body energy metabolism enzymes in rheumatoid arthritis (RA) and preliminarily explain the mechanism of heat-producing action of heat reinforcing acupuncture manipulation. Method Forty chinchilla rabbits were randomized into normal, model, equal reinforcement and reduction, twirling reinforcement, and heat reinforcing acupuncture manipulation groups. A model of cold syndrome-type RA was made by ovalbumin induction and exposure to low temperature in the other four groups not including the normal group. From two days after successful model making, the normal and model groups were grabbed and fastened (bound) by the same way as for the acupuncture groups, 30 min once daily. The equal reinforcement and reduction group received even reinforcing-reducing method;the twirling reinforcement group, twirling reinforcement method;the heat reinforcing group, heat reinforcing acupuncture manipulation. The needle was manipulated for 1 min and retained for 30 min once daily, for a total of seven days. The RA rabbit knee joint circumference was measured and the inflammation score was recorded according to synovial histopathological sections before and after treatment. After the completion of intervention, the rabbits were sacrificed and the articular synovium was rapidly separated for frozen sections. Articular synovium lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and cytochrome oxidase (CCO) activities were measured by histochemical staining. Result After acupuncture intervention, the RA rabbit knee joint circumference was shortened in all the equal reinforcement and reduction, twirling reinforcement, and heat reinforcing acupuncture manipulation groups, but the shortening effect on the RA rabbit knee joint circumference was better in the heat reinforcing acupuncture manipulation group than in the equal reinforcement and reduction and twirling reinforcement method groups (P<0.05);the inflammation score recorded according to the RA rabbit synovial histopathological sections was also decreased more in the heat reinforcing acupuncture manipulation group than in the equal reinforcement and reduction and twirling reinforcement method groups (P<0.05). Synovial LDH integral optical density, total positive area and area percentage were significantly higher in the model group than in the normal group (P<0.05), and significantly lower in the equal reinforcement and reduction, twirling reinforcement, and heat reinforcing acupuncture manipulation groups than in the model group(P<0.05) and also in the heat reinforcing acupuncture manipulation group than in the equal reinforcement and reduction and twirling reinforcement method groups (P<0.05). Rabbit synovial SDH and CCO activities were significantly higher in the model group than in the normal group (P<0.05). Rabbit synovial SDH and CCO integral optical density, total positive area and area percentage were significantly higher in the equal reinforcement and reduction, twirling reinforcement, and heat reinforcing acupuncture manipulation groups than in the model group (P<0.05) and also in the heat reinforcing acupuncture manipulation group than in the equal reinforcement and reduction and twirling reinforcement method groups (P<0.05). Conclusion Heat reinforcing acupuncture manipulation has a definite therapeutic effect on RA and can increase SDH and CCO activities to enhance aerobic metabolism and produce more local energy in a rabbit model of RA. That may be the mechanism by which heat reinforcing acupuncture manipulation produces heat.

Effect of succinate dehydrogenase complex subunit A gene knockdown on cell proliferation,cell cycle and apoptosis of mouse hepatic cell line BNL CL.2 cells / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of succinate dehydrogenase complex subunit A (sdha)gene on cell proliferation,cell cycle and apoptosis of mouse hepatic cell line BNL CL.2 cells. METHODS The BNL CL.2 cells were transfected by two kinds of sdha-shRNA lentivirus to knockdown sdha gene. The infection efficiency of BNL CL.2 cells infected with lentiviral vectors was analyzed by flow cytometry. The expression of sdha gene and SDHA protein was detected by real-time PCR and Western blotting,respectively. The effect of sdha gene on cell proliferation of BNL CL.2 cells was examined by growth curve,while cell cycle and apoptosis were analyzed by flow cytometry. RESULTS The infection efficiency of BNL CL.2 cells in sh-control group and in sdha-shRNA group was above 80%. Compared with sh-control group,the expression of sdha gene in BNL CL.2 cells infected with sdha-shRNA lentivirus was decreased by about 20 times(P<0.01),the expression of SDHA protein was decreased by about 10 times(P<0.01),and the growth rate was about 70%that of sh-control group(P<0.05). The cells were arrested in S phase,and the percentage of cells in S phase was 0.74 times that of sh-control group(P<0.01). The percentage of cells in G0/GI phase was 1.17 times that of sh-control group(P<0.01). The percentage of cells in G2/M was 1.37 times that of sh-control group(P<0.01). But there was no obvious difference in the apoptosis rate. CONCLUSION The reduced expression of SDHA protein can inhibit the proliferation of mouse hepatic cells,and the inhibitory mechanism may be cell cycle arrest. There is possibly no relationship between inhibition and cell apoptosis.

Succinate dehydrogenase gene mutations and DNA methylation analysis in hereditary pheochromocytoma paraganglioma syndrome / 中华泌尿外科杂志

Objective To detect the genetic mutations of succinate dehydrogenase B (SDHB),SDHC,SDHD,SDHAF2 and RET,VHL in hereditary pheochromocytoma (PHEO) paraganglioma (PGL) syndrome in order to analyze the relationship between the pathogenesis and SDHx mutations and DNA methylation.Methods SDH genes,VHL and RET were analyzed for germline mutations in 97 PHEOs/PGLs patients.Correlations were analyzed between the results and the clinical characteristics including age,tumor localization,multifocality,24 h urine CA,IGF and NSE.Direct DNA sequence analysis was carried out for SDHB (1q36.1-1q35,exons1-8),SDHC (1q21,exons 1-5),SDHD (11q23,exons 1-4),SDHAF2 (11q12.2,exons 1-4),RET (10q11.2,exons10,11,13,14&15,and 16) and VHL (3p25.3,exons 1-3) genes,and promoter region methylation of SDHB was detected in PHEO/PGL peripheral blood samples.Results Germinal mutations were found in 17 patients (17.5％),with 8 cases in RET proto-oncogene (8.2％),7 cases in SDHB genes (7.2％) and 2 cases in VHL gene (2.1％).The comparison of some of the clinical features in two groups (with and without promoter region methylation of SDHB) showed significant differences (P＜0.01).Conclusions Genetic predisposition is frequent in chromaffin tissue tumors,indicating that DNA analysis is necessary.The mutation of SDHB is highly associated with abdominal PGL and the following distant metastasis (malignant PGL).

Over-representation of the G12S polymorphism of the SDHD gene in patients with MEN2A syndrome

OBJECTIVE: To evaluate whether germline variants of the succinate dehydrogenase genes might be phenotypic modifiers in patients with multiple endocrine neoplasia type 2. Mutations of genes encoding subunits of the succinate dehydrogenase are associated with hereditary paraganglioma/pheochromocytoma syndrome. Pheochromocytoma is one of the main manifestations of multiple endocrine neoplasia type 2 caused by germline mutation of the rearranged during transfection proto-oncogene. METHODS: Polymorphisms of the succinate dehydrogenase genes were analyzed in 77 rearranged during transfection mutation carriers, 47 patients with sporadic medullary thyroid cancer, 48 patients with sporadic Pheo, and 100 healthy individuals. Exons 10-16 of the rearranged during transfection proto-oncogene were analyzed by direct DNA sequencing, and all exons of the von Hippel-Lindau, succinate dehydrogenase B, and succinate dehydrogenase subunit D genes were tested by direct DNA sequencing and multiple ligation probe analysis. The G12S polymorphism of the succinate dehydrogenase subunit D gene was determined by restriction fragment length polymorphism. RESULTS: Of the 77 rearranged during transfection mutation carriers, 55 from 16 families had multiple endocrine neoplasia type 2A, three from three families had multiple endocrine neoplasia type 2B, and 19 from two families had familial medullary thyroid carcinoma. Eight of 55 (14.5%) patients with multiple endocrine neoplasia type 2A had this variant whereas it was absent in multiple endocrine neoplasia type 2B, familial medullary thyroid carcinoma, sporadic medullary thyroid carcinoma, and sporadic pheochromocytoma groups, and its prevalence in controls was 1% (p<0.002 multiple endocrine neoplasia type 2A versus controls). No associations between G12S and age of manifestation, incidence of pheochromocytoma or hyperparathyroidism, or level of serum calcitonin were observed. CONCLUSION: The high prevalence of the G12S variant in patients with multiple endocrine neoplasia type 2A raises questions about its role as a genetic modifier, but this proposal remains to be established.

Paragangliomas múltiples asociados a mutación del gen SDHB: Caso clínico / Multiple paragangliomas associated to a SDHB gene mutation: Report of one case

Paragangliomas are tumors arising from sympathetic and parasympathetic tissues. The classic associated syndromes are neurofibromatosis type 1, multiple endocrine neoplasia type 2 and von Hippel-Lindau. Germline mutations of succinate dehydroge-nase subunits genes, are associated with familial paraganglioma syndromes 1,2,3 and 4. We report a 29-year-old woman with a family background of pheochromocytoma and history of paroxysmal headache, nausea, sweating, palpitations, associated with severe hypertension. The patient had elevated plasma noradrenalin and urinary normetanephrines. Imaging studies revealed three retroperitoneal extra-adrenal masses. The clinical and laboratory study of classic syndromes associated with para-ganglioma was negative. The patient was operated and the pathological study of the surgical specimen was consistent with paragangliomas. The genetic study showed a mutation in the SDHB succinate dehydrogenase gen, Exon 2 of CCTCA c.300_304 (p.P56delYfsX5).

Biochemical and Mitochondrial Changes Induced by Cd, Fe and Zn in Limnodrillus hoffmeisteri / Cambios Bioquímicos y Mitocondriales Inducidos por Cd, Fe y Zn en Limnodrillus hoffmeisteri

The effects of sublethal concentrations of cadmium (0.64 µg/L), iron (0.043 mg/L) and zinc (0.31 mg/L) and a mixture of these metals on succinate dehydrogenase (SD) and alkaline phosphatase (AP) activity and on structural changes in the mitochondria of epithelium cells of the digestive tract were examined in the oligochaete Limnodrillus hoffmeisteri after 96 h of exposure in artificial sediments. SD activity was significantly inhibited, particularly in treatments with Cd alone (92.57 percent), while AP increased its activity with Cd alone (73.23 percent). However, when this metal was mixed with Fe and Zn, the inhibition of SD activity was lower (67.82 percent) than with Cd alone, showing an antagonistic effect and AP increased its activity (73.26 percent). Mitochondria were structurally damaged by exposure to Cd alone. However, in the metal mixtures, the toxic effects may exert interactive effects eliciting a less structural damage in the mitochondria of epithelium cells of the digestive tract than when Cd is alone.

Se estudió el efecto de las concentraciones subletales de Cd (0,64 µg/L), Fe (0,043 mg/L) y Zn (0,31 mg/L) en forma aislada y en mezcla sobre la actividad de la succinato deshidrogenasa (SD) y la fosfatasa alcalina (AP) en las mitocondrias de las células epiteliales del tracto digestivo en el oligoqueto Limnodrillus hoffmeisteri después de 96 h de exposición en sedimentos artificiales. La SD se inhibió significativamente, particularmente en los tratamientos con Cd en forma aislada (92,57 por ciento), mientras que la AP se incrementó con Cd en forma aislada (73,23 por ciento). Sin embargo, cuando este metal se mezcló con Fe y Zn, la inhibición de la SD fue menor (67,82 por ciento) que con Cd en forma aislada, lo que mostró un efecto antagonístico y la AP incrementó su actividad (73,23 por ciento). Sin embargo, cuando este metal estaba en mezcla con Fe y Zn, la inhibición de la SD fue menor (67,82 por ciento) que con Cd en forma aislada, mostrando un efecto antagonístico y un incremento en la actividad de la AP (73,26 por ciento). Las mitocondrias fueron dañadas estructuralmente por exposición al Cd en forma aislada. Sin embargo, con los metales en mezcla, los efectos tóxicos pudieron ejercer efectos interactivos provocando un menor daño estructural en la mitocondria de las células del epitelio del tracto digestivo que cuando el Cd estaba en forma aislada.