Effect of Modified Da Chaihutang on FXR/FGF15/FGFR4 Signaling Pathway in Mice with Cholesterol Gallstone Differentiated into Damp-Heat Syndrome / 中国实验方剂学杂志

ObjectiveTo observe the effect of modified Da Chaihutang on cholesterol gallstone （CS） in mice due to damp-heat based on the farnesoid X receptor （FXR）/fibroblast growth factor 15 （FGF15）/fibroblast growth factor receptor 4 （FGFR4） pathway and explore the molecular biological mechanisms of CS differentiated into damp-heat syndrome from the perspective of correspondence between prescription and syndrome. MethodForty-eight six-week-old mice were randomly divided into the blank group， model group， modified Da Chaihutang （23.4 g·kg-1） group， and ursodeoxycholic acid （0.12 g·kg-1） group， with 12 mice in each group. The ones in the latter three groups were exposed to "internal dampness + external dampness + high-cholesterol diet" for 12 weeks for inducing CS due to damp-heat. Mice in the modified Da Chaihutang group and ursodeoxycholic acid group were gavaged with the corresponding drugs， while those in the model and blank groups with the same amount of normal saline for a total of four weeks. Before and after modeling， mice in each group were subjected to open field tests for determining their activities and mental states. Such general conditions as body mass， food intake， fur， and urine and stool of mice in each group were observed and recorded weekly for judging the damp-heat syndrome. After the intervention， the sampled liver and gallbladder tissues of mice in each group were stained with hematoxylin-eosin （HE） staining， and the serum γ-glutamyltransferase （GGT）， alkaline phosphatase （ALP）， and total bilirubin （TBIL） were determined. The total cholesterol （TC） and total bile acid （TBA） contents in bile were measured by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein expression levels of FXR， FGF15， FGFR4， and cholesterol 7α-hydroxylase gene （CYP7A1） were assayed by real-time fluorescence quantitative polynucleotide chain reaction （Real-time PCR） and Western blot. ResultCompared with the blank group， the model group exhibited enlarged gallbladder， brown turbid bile with flocculent precipitation visible to the naked eye， obvious damp-heat syndrome， lipoid degeneration in the liver tissue， rough and thickened gallbladder wall， elevated ALP， GGT， and TBIL in serum （P<0.01） and TC in bile （P<0.01）， reduced TBA （P<0.01）， up-regulated FXR， FGF15， and FGFR4 mRNA and protein expression in ileum （P<0.05， P<0.01）， and down-regulated CYP7A1 mRNA and protein expression （P<0.01）. Compared with the model group， the two medication groups displayed improved bile turbidity， and the bile in the modified Da Chaihutang group became clearer. After intervention， the damp-heat syndrome of mice in the modified Da Chaihutang group was significantly alleviated. The liver and gallbladder lesions of mice in the two medication groups were significantly relieved， manifested as reduced serum ALP， GGT， and TBIL （P<0.01）. The reduction in ALP and TBIL of the modified Da Chaihutang group was more significant （P<0.01）. The TC contents in the bile of mice from the two medication groups were significantly lowered， whereas the TBA contents were elevated （P<0.01）， with more significant changes present in the modified Da Chaihutang group （P<0.01）. The mRNA and protein expression levels of FXR， FGF15， and FGFR4 in the modified Da Chaihutang group were down-regulated （P<0.05， P<0.01）， while the mRNA and protein expression levels of CYP7A1 rose （P<0.05）， except that the elevation in FGF15 and FGFR4 protein expression and reduction in CYP7A1 protein expression were not significant. The mRNA and protein expression levels of FXR， FGF15， and FGFR4 in the ursodeoxycholic acid group all decreased， among which the reduction in FXR was remarkable （P<0.05）， and the mRNA and protein expression levels of CYP7A1 were significantly up-regulated （P<0.05）. ConclusionModified Da Chaihutang significantly improves the stone， liver function， bile composition， abnormal cholesterol-bile acid metabolism， and damp-heat syndrome in the model mice of CS differentiated into damp-heat syndrome， which may be related to its regulation of key factors FXR， FGF15， FGFR4， and CYP7A1 mRNA and protein expression in the cholesterol-bile acid metabolism pathway.

Tanshinone IIa attenuates vascular calcification through inhibition of NF-κB and β-catenin signaling pathways / 生理学报

Tanshinone IIa is a key ingredient extracted from the traditional Chinese medicine Salvia miltiorrhiza (Danshen), and is widely used to treat various cardiovascular diseases. Vascular calcification is a common pathological change of cardiovascular tissues in patients with chronic kidney disease, diabetes, hypertension and atherosclerosis. However, whether Tanshinone IIa inhibits vascular calcification and the underlying mechanisms remain largely unknown. This study aims to investigate whether Tanshinone IIa can inhibit vascular calcification using high phosphate-induced vascular smooth muscle cell and aortic ring calcification model, and high dose vitamin D3 (vD3)-induced mouse models of vascular calcification. Alizarin red staining and calcium quantitative assay showed that Tanshinone IIa significantly inhibited high phosphate-induced vascular smooth muscle cell and aortic ring calcification. qPCR and Western blot showed that Tanshinone IIa attenuated the osteogenic transition of vascular smooth muscle cells. In addition, Tanshinone IIa also significantly inhibited high dose vD3-induced mouse aortic calcification and aortic osteogenic transition. Mechanistically, Tanshinone IIa inhibited the activation of NF-κB and β-catenin signaling in normal vascular smooth muscle cells. Similar to Tanshinone IIa, inhibition of NF-κB and β-catenin signaling using the chemical inhibitors SC75741 and LF3 attenuated high phosphate-induced vascular smooth muscle cell calcification. These results suggest that Tanshinone IIa attenuates vascular calcification at least in part through inhibition of NF-κB and β-catenin signaling, and Tanshinone IIa may be a potential drug for the treatment of vascular calcification.

Eosinophilic vacuolated tumor of the kidney: clinicopathological and molecular characteristics / 中华病理学杂志

Objective: To study the clinicopathological features, immunophenotype, molecular changes, differential diagnosis and prognosis of eosinophilic vacuolated tumor (EVT) of the kidney. Methods: Four cases were collected retrospectively from 2014 to 2020 at Ningbo Diagnostic Pathology Center. The clinicopathologic features and immunophenotypic profile were studied by light microscopy and immunohistochemistry. Targeted next-generation sequencing (NGS) panel was used to detect cancer-associated mutation. Follow-up and literature review were also performed. Results: Among the 4 patients studied,2 were males and 2 were females. The age of the patients ranged from 44 to 63 years (the mean age: 51 years).Tumor size ranged from 1.5 to 4.2 cm (mean: 2.3 cm). Microscopically, tumors were well-circumscribed, unencapsulated. Thick-walled vessels and entrapped renal tubules were found within or at the periphery of the tumors. The tumors were predominantly composed of nest pattern, and focal tubular pattern. The tumor cells exhibited abundant, eosinophilic, granular cytoplasm and conspicuous, large nucleoli. Prominent intracytoplasmic vacuoles were seen. These cytoplasmic vacuoles varied in size and frequently coalesced into a large space. Loose fibromatous or hyaline stroma was focally noted. Immunohistochemically, the tumor cells in all cases exhibited a CD117+/CK7-phenotype. All cases were positive for CD10 and p504s. MTOR, S6 and cathepsin K were positive in 4 cases. TFE3, CA9, Melan A and HMB45 were negative in all cases. SDHB retained expression. NGS demonstrated MTOR mutations in all cases, and TSC2 mutation in 2 cases. Conclusions: EVT is a rarely oncocytic renal tumor with unique morphology, immunohistochemical phenotype, molecular profile and an indolent behavior. Recognition of the characteristics of this novel but rare entity will allow for better classification of renal tumors.

The Antifungal Mechanism of Bioactive Metabolites Produced by Penicillum sp. TS67 / 微生物学通报

The antifungal mechanism of the TS67(Penicillum sp.) producing bioactive metabolites was investigated in this paper. The test indicated that mycelia growth, spores forming and spores germination of Bipolaris maydis and Fusarium oxysporum can be inhibitied by metabolites, after 50% fermentation broth acting 120 hours, the inhibition rate of hyphal growth can reach about 77.78%, 70.30%, the inhibition rate of spores forming also arrived at 58.8%, 73.5%; under 50% fermentation broth acting 12 hours, the inhibition rate of spores germination were up to 78.3%, 62.0%, the mycelia under activity metabolites conditoins also showed surface nodulation and growing point expanding irregularly, protoplasmic condesing, etc. Based on all these results, we may draw a conclusion that activity metabolites maybe target at fungal cell wall.

Evaluation of Antagonism of Penicillium TS67 Against Soy-bean Root Rot Disease and Corn Southern Leaf Blight / 微生物学通报

Through pot experiments, the disease index and control efficiency of TS67 cell, the fermentation liquid of TS67 and the supernatant of TS67 separately act on Fusarium oxysporum and Bipolaris maydis was detected. Experiment results analysis with SPSS statistical analysis software indicated all treatments of TS67 could inhibit both of soybean root rot disease and corn southern leaf blight (P

Improving Potato Plants Oxidative Stress and Salt Tolerance by Gene Transfer Both of Cu/Zn Superoxide Dismutase and Ascorbate Peroxidase / 中国生物工程杂志

In plants, oxidative stress is one of the major causes of damage as a result of various environmental stresses and it is primarily due to the excessive accumulation of reactive oxygen species. To develop transgenic potato plants with enhanced tolerance to environmental stress, transgenic potato plants (Solanum tuberosum L. cv. Atlantic) expressing the Cu/ZnSOD and APX genes in chloroplasts were generated under the control of the oxidative stress-inducible promoter. To investigate oxidative stress tolerance, transgenic plants were evaluated at the level of leaf discs and plantlets after methyl viologen (MV) and salt treatment. Leaf discs from transgenic potato plants showed 13% less membrane damage compared to non-transgenic (NT) plants suffering 10 μmol/L MV treatment of 48 h, and showed 1.6-fold higher chlorophyll contents than those of NT plants at 1.0mol/L NaCl treatment (31% vs. 19%). In addition, transgenic potato plants maintained higher rooting rates (75%) during 100mmol/L NaCl treatment than those (12%) from NT plants. Moreover, the tolerance to salt stress in transgenic plants was consistent to increased transcript levels and higher activities of SOD and APX compared to NT plants. These results suggest that expression of Cu/ZnSOD and APX in chloroplasts could be used in plants to enhance the tolerance to environmental stresses.

Improving Potato Plants Oxidative Stress and Salt Tolerance by Gene Transfer Both of Cu/Zn Superoxide Dismutase and Ascorbate Peroxidase / 中国生物工程杂志

In plants, oxidative stress is one of the major causes of damage as a result of various environmental stresses and it is primarily due to the excessive accumulation of reactive oxygen species. To develop transgenic potato plants with enhanced tolerance to environmental stress, transgenic potato plants (Solanum tuberosum L. cv. Atlantic) expressing the Cu/ZnSOD and APX genes in chloroplasts were generated under the control of the oxidative stress-inducible promoter. To investigate oxidative stress tolerance, transgenic plants were evaluated at the level of leaf discs and plantlets after methyl viologen (MV) and salt treatment. Leaf discs from transgenic potato plants showed 13% less membrane damage compared to non-transgenic (NT) plants suffering 10 μmol/L MV treatment of 48 h, and showed 1.6-fold higher chlorophyll contents than those of NT plants at 1.0mol/L NaCl treatment (31% vs. 19%). In addition, transgenic potato plants maintained higher rooting rates (75%) during 100mmol/L NaCl treatment than those (12%) from NT plants. Moreover, the tolerance to salt stress in transgenic plants was consistent to increased transcript levels and higher activities of SOD and APX compared to NT plants. These results suggest that expression of Cu/ZnSOD and APX in chloroplasts could be used in plants to enhance the tolerance to environmental stresses.

Agrobacterium-mediated Transformation of an Economically Important Potato Cultivar Using Internodal Stem Explants / 中国生物工程杂志

Potato cultivar Atlantic is widely grown for potato chips in the world. However, this economically important potato cultivar exhibits very poor yields and traits under severe environmental stress. To develop an efficient plant transformation system that could be used to produce large scale transgenic potato plants with enhanced tolerance to environmental stress and therefore would be beneficial for potato processing industry, Agrobacterium-mediated transformation of internodal stem explants using both superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible SWPA2 promoter was performed. Comparing to leaf explants, stem internodal explants were less liable to damage during manipulation, more amenable to in vitro conditions. The addition of silver thiosulfate to the selection medium considerably promoted the shoot induction from explant-derived callus. Seven to nine shoots per stem explant were obtained. By combining the best treatments, this system yielded shoot induction frequency of 94.2% and transformation frequency of 80% of internodal stem explants. Stable integration of the transgenes was confirmed by PCR and Southern blot analyses. In conclusion, short duration (7～8 weeks), high efficiency and easy process make this system well suited for wider commercial applications of transgenic Atlantic potato plants.

Agrobacterium-mediated Transformation of an Economically Important Potato Cultivar Using Internodal Stem Explants / 中国生物工程杂志

Potato cultivar Atlantic is widely grown for potato chips in the world. However, this economically important potato cultivar exhibits very poor yields and traits under severe environmental stress. To develop an efficient plant transformation system that could be used to produce large scale transgenic potato plants with enhanced tolerance to environmental stress and therefore would be beneficial for potato processing industry, Agrobacterium-mediated transformation of internodal stem explants using both superoxide dismutase (SOD) and ascorbate peroxidase (APX) genes under the control of an oxidative stress-inducible SWPA2 promoter was performed. Comparing to leaf explants, stem internodal explants were less liable to damage during manipulation, more amenable to in vitro conditions. The addition of silver thiosulfate to the selection medium considerably promoted the shoot induction from explant-derived callus. Seven to nine shoots per stem explant were obtained. By combining the best treatments, this system yielded shoot induction frequency of 94.2% and transformation frequency of 80% of internodal stem explants. Stable integration of the transgenes was confirmed by PCR and Southern blot analyses. In conclusion, short duration (7～8 weeks), high efficiency and easy process make this system well suited for wider commercial applications of transgenic Atlantic potato plants.

The Effects of Spirulina on Intestinal Microflora of Diarrhea Model Mice / 微生物学通报

The effects of Spirulina on intestinal microflora of diarrhea model mice were observed.Diarrhea model mice were geted by gastric perfusion ampicillin,then these mice were given different doses spirulina by gavage.Selective culture media were used to culture bifidobacteria,bacterium lacticum,enteric bacilli,enterococci,bacteroid,clostridium perfringens in feces at different times.In order to find the difference of collecting samples from feces and intestine,the quantity of detection flora in different segment of intestine were compared too.The result showed that in middle dose group and high dose group spirulina can regulate and improve intestinal flora of diarrhea model mice,and effectively shorten time from disturbance to balance.Fecal sample and intestinal contents sample both manifest a same trend,that is the quantity of bifidobacteria,bacterium lacticum in treatment recovery group is significantly higher than saline group,and enteric bacilli,enterococci,bacteroid,clostridium perfringens is significantly lower than saline group,but each index flora in intestinal contents sample has a significant difference in quantity.

Deliberation and Practice of Innovative System for Microbiological Experiment Teaching / 微生物学通报

According to the practice and understanding in the process of microbiological experiment teaching,organizational thinking and form,the teaching contents and methods of this course are induced and summarized.It is put forward that the experimental teaching is organized as the form of research project,the students are principal part and the teachers play a leading role.Meanwhile the main problems of the teaching module and resolved measures are analyzed.