In this study, liquid chromatography-mass spectrometry(LC-MS) and high performance liquid chromatography(HPLC) were employed for qualitative and quantitative analysis of the steroidal saponins in rhizomes of Paris polyphylla var. yunnanensis from three different habitats cultured in vitro, in an attempt to explore whether the rhizomes of the medicinal herb cultured in vitro can synthesize the steroidal saponins, including polyphyllinsⅠ, Ⅱ, and Ⅶ, the quality markers specified in Chinese Pharmacopoeia(2020 edition). A total of 20 steroidal saponins were identified in the rhizomes from Changxin, Yunlong(S1), Fengyi, Dali(S2), and Niujie, Eryuan(S3): parisyunnanoside A and parisyunnanoside D or E, proto-polyphyllin Ⅱ, polyphyllins G and H, polyphyllinsⅠ, Ⅱ, Ⅴ, Ⅵ, and Ⅶ, dioscin, gracillin, prosapogenin A, Tg, isomer of Th, saponin Th, reclinatoside, proto-pairs D, pseudoproto-dioscin, and 23-O-glc-(23S,25R)-spirost-5-en-3β,23α,27-triol-3-O-rha-(1→2)-[ara(1→4)]-glc or 27-O-glc-(23S,25R)-spirost-5-en-3β,27α-diol-3-O-rha-(1→2)-[ara(1→4)]-glc. Among them, polyphyllinsⅠ, Ⅱ, and Ⅶ were detected in the rhizomes from S1, with the mass fraction of 0.109 1%, 0.165 2%, and 0.051 03%, respectively(total 0.325 3%). Polyphyllins Ⅱ and Ⅶ were identified in the rhizomes from S2 with the respective mass fraction of 0.192 2% and 0.074 23% and total content of 0.266 5%. Moreover, polyphyllins Ⅱ and Ⅶ were also found in the rhizomes from S3, which had the mass fraction of 0.207 7% and 0.186 9%, separately, with the total content of 0.394 6%. Thus, steroidal saponins, including the quality makers polyphyllins Ⅰ, Ⅱ, and Ⅶ recorded in Chinese Pharmacopoeia(2020 edition) can be synthesized in rhizomes of Paris polyphylla var. yunnanensis cultured in vitro, but their total content fails to meet the standard(0.60% in Chinese Pharmacopoeia). Therefore, in vitro culture of the Paris polyphylla var. yunnanensis is feasible, but the culture conditions need to be further improved.
Chromatography, High Pressure Liquid ; Liliaceae ; Melanthiaceae ; Rhizome ; Saponins

Objective:To construct the recombinant eukaryotic expression vector plasmids with mutant C-kit cDNA and to study the effect of the mutant C-kit gene on cell proliferation and cell cycle in gastrointestinal stromal tumor (GIST). Methods: Wild-type C-kit cDNA was cloned from human embryonic brain tissue by RT-PCR technique.Site-directed mutagenesis of the wild type C-kit cDNA was performed according to the C-kit mutations we cloned. Recombinant plasmids were stably transfected into human embryonic kidney cell line and the cells expressing mutant C-kit were selected by special cell culture medium containing G418.Expressions of C-kit protein of the transfectants were detected by Western blot.Cell proliferation and cell cycle of the transfectants were detected by MTT clolorimetic assay and flow cytometry,respectively.Whether HEK cell with mutated C-kit cDNA could grow autonomously in nude mice or not was also detected. pcDNA3 vector transfected and recombinant plasmids with wild-type C-kit cDNA transfected HEK cell were used as the control groups. Results: The mutant C-kit cDNA was obtained by site-directed mutagenesis of the wild type C-kit cDNA. Compared with the 2 control groups ,the growth rate and proliferative activity of the HEK cells with mutant C-kit cDNA were increased significantly.The analysis of cell cycle showed that more HEK cells with mutanted C-kit cDNA remained in proliferation phase (S+G 2-M)than the groups without mutated C-kit cDNA.HEK cells with the mutated C-kit also grew autonomously in nude mice.Conclusion: Mutation of C-kit gene can increase proliferation of human cells,causing malignant transformation of human normal cells,which may play an important role in the malignant transformation of GIST.

OBJECTIVE: To discuss the metabolism rule of the active steroidal saponins in the roots, stems and leaves of Paris polyphylla var. yunnanensis Franch belonging to different populations and different plants in the same population. METHODS: UPLC was used to determine the contents of the active ingredients including polyphyllin I, polyphyllin II, polyphyllin VI, and polyphyllin VII from the roots, stems, and leaves, the UPLC map was established, the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine software (Version 2004 A) developed by the Chinese Pharmacopoeia Committee was used to analyze the number of peaks in the UPLC map, and One-way ANOVA was used to analyze their metabolism rule. RESULTS: There was significant difference in the contents of polyphyllin I, polyphyllin II, polyphyllin VI, polyphyllin VII, and their total contents in the roots, stems, and leaves among different populations (P < 0.01). The number of peaks in the UPLC map ranked in the folloing order: leaf > root > stem. The difference among different plants of the same Paris polyphyUa var. yunnanensis Franch population was smaller. No. 1, 2, 4, 5 peaks all existed in the UPLC maps of the roots, stems, and leaves, but there were 11 and 16 more peaks in the leaves than the roots and stems, respectively. CONCLUSION: There are fewer kinds of main active ingredients with low contents in the stems of Paris polyphyUa var. yunnanensis Franch. The population genetic capabilities of the metabolism of the steroidal saponins active ingredients are stable in the roots, stems and leaves of different plants from the same population, but there are large differences between different populations. Further pharmacological, toxicological, and clinical studies need to be done to determine whether the leaves and stems could lake the place of root as an alternative medicine.

Objective : To study the effect of EFHD2 protein deletion in Sertoli cells on Occludin，a component of tight junction protein and the localization and expression of EF-hand domain family member D2 (EFHD2) in mouse testis． Methods : Total RNA and protein were extracted from adult mice's heart ，liver ，spleen ，lung ，kidney， brain and testis tissues．The mRNA and protein levels of EFHD2 in each organ tissue were detected by qRT-PCR and Western blot．Immunofluorescence and immunohistochemistry detected the localization and expression of EF- HD2 in testicular tissues．SiRNA interference was used to reduce EFHD2 in Sertoli cells to detect Occludin protein expression. Results : qRT-PCR showed that the expression of EFHD2 was the highest in the testis．Western blot results showed that the expression level increased in testis tissue．Indirect immunofluorescence and immunohisto- chemistry results showed that the protein was mainly distributed in Sertoli cells and co-localized with cytoskeletal Vimentin，indicating that the protein was expressed in Sertoli cells．After the decrease of EFHD2 protein expres- sion，Occludin protein expression also decreased． Conclusion The expression of EFHD2 protein in the testis is relatively high，mainly distributed in Sertoli cells of the testis，co-localized with Vimentin，and can affect the nor- mal expression of tight junction protein Occludin．It is suggested that EFHD2 can promote and maintain the junction structure of Sertoli cells and provide a stable microenvironment for spermatogenesis.

The study aims to analyze the varieties and standards of compositae medicinal plants used in Dai medicine. The results showed that there were 78 species (including varieties) compositae plants recorded in literatures, which belongs to 63 medicinal materials varieties. And 47 original plants (60.25%) were recorded in Chinese medicinal material standards. In those standards and literatures of Dai medicine, there are great differences in translated Chinese names, original plants, medicinal parts, and efficacy of medicinal plants. Therefore, the variety systematization and the quality standards of Dai medicine should be strengthened.

OBJECTIVE： To investigate the acute toxicity， long-term toxicity， skin irritation and anaphylaxis of Bee venom （BV） plastics， and to evaluate its preclinical safety. METHODS： The acute toxicity of BV plastics to rats was investigated after administration of high-dose， medium-dose and low-dose （144， 96， 48 mg/kg） of BV plastics. The long-term toxicity of BV plastics was investigated by continuous administration of high-dose， medium-dose and low-dose （72， 48， 24 mg/kg） of BV plastics for 28 days. The irritation of intact and damaged skin in rabbits with 8 mg/kg BV plastics was investigated by using the self-control method of left and right homologous body. The skin anaphylaxis of guinea pig were investigated after sensitized with 15 mg/kg BV plastics on the left back （on 0， 7th， 14th day） and stimulated with 15 mg/kg BV plastics on the right back. RESULTS： During the acute toxicity experiment with BV plastic，the weight of rats and the changes of viscera were normal，and there was no relevant toxic reaction. Long-term toxicity test results showed that no significant pathological changes were observed at 24 h after the last administration； the spleen index  of rats in BV low-dose group， testicular index in middle-dose group and epididymis index in high-dose and middle-dose groups were significantly increased， while PT in plasma of rats in BV medium-dose and low-dose groups was significantly prolonged （P＜0.05）. There were no abnormal changes in organ appearance， other organ index， coagulation index and blood biochemical index. All above indexes became normal at the end of 2-week recovery period. Skin irritation test showed that BV plastics could cause slight erythema and obvious scab on the skin of rabbits which along with little irritation on intact or damaged skin. Skin anaphylaxis test showed that BV plastics produced mild erythema in the skin of guinea pigs， belonging to light allergy. CONCLUSIONS： No acute or long-term toxicity is observed after transdermal administration of BV plastics， which is safe and only causes mild irritation and irritability to skin， indicating there is good safety of the plastic under experiment doses.

Objective To master the Oncomelania hupensis distribution and infection status in the national schistosomiasis surveillance sites of Yunnan Province, so as to provide the evidence for making the control and prevention measures. Methods The data of O. hupensis surveillance in the 18 national surveillance sites of Yunnan Province from 2015 to 2017 were collected and analyzed with the descriptive analysis method according to the national schistosomiasis surveillance programme. Results The total surveillance area was 5 710.94 hm², the area with O. hupensis snails was 205.69 hm². The number of surveillance frames was 2 094 553, the occurrence rate of frames with snails was 0.62%, and the density of living snails was 0.025 4 snails/0.1 m². In the schistosomiasis epidemic controlled areas, the area with snails, the occurrence rate of frames with snails and the density of living snails were all the highest. The snail concentrated distribution areas were the small reservoir, bottomland, paddy field, ditch, and dry land, and the snails mainly distributed in the rice, dry crop, weed and wood vegetation. The number of frames with snails, occurrence rate of frames with snails, total number of snails, number of living snails, and repetition areas with snails presented increasing trends, and however, no schistosome-infected snails were found during the three years. Conclusions The O. hupensis snail status is obviously serious in the national schistosomiasis surveillance sites of Yunnan Province. The comprehensive snail control measures should continue to be strengthened, so as to effectively control the spread of the snails and reduce the risk of schistosomiasis outbreaks.

OBJECTIVETo explore the possibility of injury to the striated urethral sphincter by incision to the anterior lobe region in transurethral prostatectomy.

METHODSWe incised the anterior lobe region of 60 patients with benign prostatic hyperplasia (BPH) undergoing transurethral prostatectomy. The patients were divided into four groups according to the incision fields: proximate superficial (group 1), proximate deep (group 2), distal superficial (group 3) and distal deep (group 4). The tissues taken from the anterior lobe region were subjected to HE staining, and the smooth and striated muscles were detected by immunohistochemical identification of smooth muscle actin (SMA) and myoglobin (MYO) in the tissues. The prostate volume, age, and PSA level of the patients were analyzed against their positive or negative results. The relative contents of the striated muscle were compared among groups 2, 3 and 4. The independent-sample between-group t-test was used for statistic analysis.

RESULTSThe urethral rhabdosphincter was found in the anterior lobe region, with the smooth muscle intermixed with the striated muscle. The incision injury of the urethral rhabdosphincter was associated with the prostate volume. Increased urethral rhabdosphincter was observed in the anterior lobe region, approaching the apex of the prostate and extending to the urethral lumen.

CONCLUSIONThe anterior lobe region should not be excessively incised in transurethral prostatectomy so as to avoid direct injury of the striated urethral sphincter, which is especially important for prostates of smaller volume or operation near the apex of the prostate.

Aged ; Histological Techniques ; Humans ; Male ; Prostate ; anatomy & histology ; pathology ; Prostatic Hyperplasia ; pathology ; surgery ; Transurethral Resection of Prostate ; Urethra ; anatomy & histology ; pathology