1.Analysis of Intestinal Flora and Serum Metabolomics in Patients with Pre-diabetic Sputum Syndrome
Zhishan WANG ; Liangliang HUANG ; Wei ZHANG ; Mingyi CHEN ; Shaojian XIAO ; Shujiao CHEN
Chinese Journal of Experimental Traditional Medical Formulae 2024;30(18):146-156
Objectiveto investigate the differential expression of intestinal flora and serum metabolites and potential biomarkers in patients with pre-diabetic sputum syndrome. MethodA total of 34 patients with pre-diabetic sputum syndrome were included as the phlegm syndrome group,and 37 healthy people were selected as the normal group. Serum and fecal samples of the two groups were collected,and liquid chromatography-mass spectrometry (LC-MS) non-targeted metabolomics and 16S rRNA high-throughput sequencing technology were used to detect serum metabolites and different intestinal flora of the two groups and explore the relationship among pre-diabetic sputum syndrome,serum metabolites,and intestinal flora. ResultIn the distribution of disease syndrome elements in the phlegm syndrome group,the first five disease syndrome elements in terms of frequency and proportion were dampness (73.53%),Qi stagnation (58.82%),Yin deficiency (50.00%),blood stasis (41.18%),and heat (35.29%). According to the frequency and proportion of disease location syndrome elements,the first three main disease location syndrome elements were spleen (100.00%),liver (41.18%),and kidney (23.53%). The results of 16S rRNA high-throughput sequencing showed that there were 44 different intestinal flora between the two groups. In order genus,there were significant differences in Bifidobacterium,Veillonococcus,and Roseococcus between the two groups (P<0.05). The diversity,abundance,and evenness of intestinal flora in the phlegm syndrome group were lower than those in the normal group,with the difference not statistically significant. There was no significant difference in the community structure between two groups. The results of serum metabolomics showed that there were 13 differential metabolites in the two groups,which were mainly concentrated in amino acid metabolism,bile secretion,bile acid biosynthesis,and lipid metabolism (P<0.05). The correlation among differential metabolites,intestinal flora,and syndrome elements was analyzed,and the results showed that ① lysine was positively correlated with spleen,Yin deficiency,and blood stasis,while taurocholic acid was positively correlated with liver,kidney,blood stasis,and dampness,and there was a positive correlation between taurocholic acid and yin deficiency and heat. The taurochenodeoxycholic acid was positively correlated with liver and dampness,and there was a negative correlation between arachidonic acid and dampness,as well as a positive correlation between glucose and spleen and blood stasis. ② Clostridium was positively correlated with spleen,kidney,Yin deficiency,and Qi stagnation. Rosepiella was negatively correlated with spleen,and Sutterella was negatively correlated with dampness. Bacteroides was negatively correlated with the spleen and kidney,and Bifidobacterium was negatively correlated with the spleen and dampness. ③ Bifidobacterium was positively correlated with glycine,threonine,lysine,and deoxycholic acid significantly,negatively correlated with cholic acid significantly,and positively correlated with taurochenodeoxycholic acid and pyruvic acid. Clostridium was positively correlated with glycine significantly and positively correlated with threonine and lysine. Lachnospira was negatively correlated with glycine,threonine,and pyruvic acid. Lysine was also negatively correlated with Faecalibacterium and Eubacterium ventriosum and positively correlated with Megamonas. There was a positive correlation between taurocholic acid and glycine bile acid and Campylobacter,between taurochenodeoxycholic acid and Veillonococcus,and between glucose and Rosepiella and Eubacterium ventriosum. There was a negative correlation between pyruvic acid and Escheria-Shigella and between taurochenodeoxycholic acid and Prevotella. Conclusionthere are differences in intestinal flora and serum metabolites between patients with pre-diabetic sputum syndrome and healthy people. The intestinal flora and metabolites have been disturbed in the stage of pre-diabetes,Bifidobacterium,Clostridium,Lachnospira,glycine,threonine,and lysine may be the breakthrough to explore the development of pre-diabetic sputum syndrome.
2.Application and prospects of erythropoietin in bone tissue engineering
Yufang YANG ; Zhishan YANG ; Mianmian DUAN ; Yiheng LIU ; Zhenglong TANG ; Yu WANG
Chinese Journal of Tissue Engineering Research 2024;28(9):1443-1449
BACKGROUND:Bone defects are caused by many factors,such as inflammation,tumor,trauma or bone diseases.Erythropoietin can promote the differentiation of mesenchymal stem cells into osteoblasts and osteoclasts and act on vascular endothelial cells to induce angiogenesis and accelerate the repair of bone and cartilage defects.Erythropoietin is a growth factor with potential application in bone tissue engineering construction. OBJECTIVE:To expound the application and potential mechanism of erythropoietin in bone tissue engineering. METHODS:The first author searched the related articles published in CNKI,WanFang,VIP,and PubMed databases from 2004 to 2022 by computer.Search terms were"erythropoietin,bone defect,bone regeneration,angiogenesis,osteogenesis,osteoblast,osteoclast,bone tissue engineering"in Chinese and English.Finally,64 articles were included for review. RESULTS AND CONCLUSION:(1)Erythropoietin can directly act on osteoblasts and osteoclasts in the bone marrow microenvironment by promoting the differentiation of mesenchymal stem cells into osteoblasts,osteoclasts,adipocytes,nerve cells and stromal cells.The activation of Wnt/β-catenin,hypoxia-inducible factor 1α/vascular endothelial growth factor,p38 MAPK and EphrinB2/EphB4 signaling pathways mediates the osteogenic differentiation of mesenchymal stem cells.(2)Erythropoietin can not only regulate the production of erythrocytes to alter the oxygen-carrying capacity of blood but also stimulate vascular endothelial cells to promote angiogenesis.The new blood vessels can carry oxygen,nutrients,growth factors,and bone progenitor cells necessary for osteogenesis to the osteogenic site,thereby promoting bone formation and fracture healing.(3)Currently,erythropoietin is being used as a growth factor with osteogenic and angiogenic effects in various types of scaffold materials such as chitosan,polycaprolactone,bioceramics,and nanofibers through various drug delivery methods.Erythropoietin,along with other growth factors such as bone morphogenetic protein-2 and bone morphogenetic protein-9,has been applied to the surface of scaffold materials to participate in the repair of bone defects.Erythropoietin has demonstrated excellent practicality in the construction of new tissue-engineered bone and has potential clinical application value.
3.Clinicopathologic characteristics,gene mutation profile and prognostic analysis of thyroid diffuse large B-cell lymphoma
Zhishan DU ; Yue WANG ; Ziyang SHI ; Qing SHI ; Hongmei YI ; Lei DONG ; Li WANG ; Shu CHENG ; Pengpeng XU ; Weili ZHAO
Journal of Shanghai Jiaotong University(Medical Science) 2024;44(1):64-71
Objective·To analyze the clinicopathologic characteristics,gene mutation profile,and prognostic factors of thyroid diffuse large B-cell lymphoma(DLBCL).Methods·From November 2003 to December 2021,a total of 66 patients with thyroid DLBCL[23 cases(34.8%)with primary thyroid DLBCL,and 43 cases(65.2%)with secondary thyroid DLBCL]admitted to Ruijin Hospital,Shanghai Jiao Tong University School of Medicine were retrospectively analyzed for their clinicopathological data,survival and prognostic factors.Gene mutation profiles were evaluated by targeted sequencing(55 lymphoma-related genes)in 40 patients.Results·Compared to primary thyroid DLBCL,secondary thyroid DLBCL had advanced ratio of Ann Arbor stage Ⅲ?Ⅳ(P=0.000),elevated serum lactate dehydrogenase(LDH)(P=0.043),number of affected extranodal involvement≥2(P=0.000),non-germinal center B cell(non-GCB)(P=0.030),BCL-2/MYC double expression(DE)(P=0.026),and international prognostic index(IPI)3?5-scores(P=0.000).The proportion of patients who underwent thyroid surgery(P=0.012)was lower than that of patients with primary thyroid DLBCL.The complete remission(CR)rate in primary thyroid DLBCL patients was higher than that in secondary thyroid DLBCL patients(P=0.039).Fifty-five patients(83%)received rituximab combined with cyclophosphamide,doxorubicin,vincristine,and prednisone(R-CHOP)-based first-line regimen.The estimated 5-year progression free survival(PFS)rate of primary thyroid DLBCL patients was 95.0%,higher than the 49.7%of the secondary patients(P=0.010).Univariate analysis showed that Ann Arbor Ⅲ?Ⅳ(HR=4.411,95%CI 1.373?14.170),elevated LDH(HR=5.500,95%CI 1.519?19.911),non-GCB(HR= 5.291,95%CI 1.667?16.788),and DE(HR=6.178,95%CI 1.813?21.058)were adverse prognostic factors of PFS in patients with thyroid DLBCL.Ann Arbor Ⅲ?Ⅳ(HR=7.088,95%CI 0.827?60.717),elevated LDH(HR=6.982,95%CI 0.809?60.266),and DE(HR=18.079,95%CI 1.837?177.923)were adverse prognostic factors of overall survival(OS).Multivariate analysis showed that Ann Arbor Ⅲ?Ⅳ(HR=4.693,95%CI 1.218?18.081)and elevated LDH(HR=5.058,95%CI 1.166?21.941)were independent adverse prognostic factors of PFS in patients with thyroid DLBCL.Targeted sequencing data showed mutation frequency>20%in TET2(n=14,35%),KMT2D(n=13,32%),TP53(n=11,28%),GNA13(n=10,25%),KMT2C(n=9,22%),and TP53 were adverse prognostic factors of PFS in patients with thyroid DLBCL(P=0.000).Conclusion·Patients with primary thyroid DLBCL have better PFS and OS than those with secondary thyroid DLBCL.Ann Arbor Ⅲ?Ⅳ,elevated LDH,non-GCB,and DE(MYC and BCL2)are adverse prognostic factors in thyroid DLBCL.TET2,KMT2D,TP53,GNA13,and KMT2C are commonly highly mutated genes in thyroid DLBCL,and the prognosis of patients with TP53 mutations is poor.
4.Effects of endoscope assisted temporoparietal fascia flap harvest for the second-stage operation in auricular reconstruction of Nagata’s technique
Zhenyu WANG ; Hongyi WANG ; Yuan JIN ; Tinghui ZHANG ; Zhishan XU ; Jiulong LIANG
Chinese Journal of Plastic Surgery 2023;39(11):1222-1228
Objective:To observe the efficacy the application of endoscope assisted temporoparietal fascia flap harvest for the second-stage operation in auricular reconstruction of Nagata’s technique for microtia.Method:In this retrospective study, the clinical data were collected from the patients who received microtia reconstruction with autologous rib cartilage at the Department of Burns and Plastic Surgery, General Hospital of Northern Theater Command from January 2015 to January 2022. According to the surgical procedure, patients were divided into endoscopic group and open surgery group. In endoscopic group, endoscope-assisted temporoparietal fascia harvest were performed for the second-stage operation in auricular reconstruction of Nagata’s technique. In open surgery group, temporoparietal fascia flaps were harvested in open surgery for the second-stage operation in auricular reconstruction of Nagata’s technique. Regular follow-up was conducted to observe the survival of the fascia flaps, complications, patient satisfaction, and surgical scars. The patient satisfaction questionnaire for auricular reconstruction was used to assess patient satisfaction, and the patient and observer scar assessment scale (POSAS) was used to evaluate scar formation in the surgical area. Data analysis was performed using SPSS 26.0 statistical software. The measurement data were expressed by Mean ± SD, and the counting data were expressed as cases (%). The T-test was used to compare the age difference, length of hospital stay, intraoperative blood loss, scar length, patient satisfaction, and POSAS scores between the two groups. Chi-square test was used to compare the gender composition and incidence of complications between the two groups. P<0.05 was considered statistically significant. Results:A total of 51 patients were included, with 26 in the endoscopic group (14 men and 12 women) and 25 in the open surgery group (12 men and 13 women). The age of the patients in the endoscopic group was (9.8±2.9) years (ranging from 7 to 17 years), while in the open surgery group was (10.3±3.8) years (ranging from 7 to 17 years). The postoperative follow-up period was (15.4±3.4) months (1 to 2 years), and all fascia flaps survived without any severe complications. There were no statistically significant differences between the two groups in terms of age difference, length of hospital stay, intraoperative blood loss, postoperative satisfaction, sex composition ratio, and postoperative complications ( P>0.05). The scar quality in the endoscopy group was superior to that in the open surgery group, and POSAS scores of endoscopic group were lower than those in the open surgery group, and the difference was statistically significant ( P<0.05). Conclusion:Endoscope assisted temporoparietal fascia flap harvest for the second-stage operation in auricular reconstruction of Nagata’s technique for microtia can minimize scarring, improve the postoperative appearance and is not statistically associated with the appearance of reconstructed auricles or complications.
5.Effects of endoscope assisted temporoparietal fascia flap harvest for the second-stage operation in auricular reconstruction of Nagata’s technique
Zhenyu WANG ; Hongyi WANG ; Yuan JIN ; Tinghui ZHANG ; Zhishan XU ; Jiulong LIANG
Chinese Journal of Plastic Surgery 2023;39(11):1222-1228
Objective:To observe the efficacy the application of endoscope assisted temporoparietal fascia flap harvest for the second-stage operation in auricular reconstruction of Nagata’s technique for microtia.Method:In this retrospective study, the clinical data were collected from the patients who received microtia reconstruction with autologous rib cartilage at the Department of Burns and Plastic Surgery, General Hospital of Northern Theater Command from January 2015 to January 2022. According to the surgical procedure, patients were divided into endoscopic group and open surgery group. In endoscopic group, endoscope-assisted temporoparietal fascia harvest were performed for the second-stage operation in auricular reconstruction of Nagata’s technique. In open surgery group, temporoparietal fascia flaps were harvested in open surgery for the second-stage operation in auricular reconstruction of Nagata’s technique. Regular follow-up was conducted to observe the survival of the fascia flaps, complications, patient satisfaction, and surgical scars. The patient satisfaction questionnaire for auricular reconstruction was used to assess patient satisfaction, and the patient and observer scar assessment scale (POSAS) was used to evaluate scar formation in the surgical area. Data analysis was performed using SPSS 26.0 statistical software. The measurement data were expressed by Mean ± SD, and the counting data were expressed as cases (%). The T-test was used to compare the age difference, length of hospital stay, intraoperative blood loss, scar length, patient satisfaction, and POSAS scores between the two groups. Chi-square test was used to compare the gender composition and incidence of complications between the two groups. P<0.05 was considered statistically significant. Results:A total of 51 patients were included, with 26 in the endoscopic group (14 men and 12 women) and 25 in the open surgery group (12 men and 13 women). The age of the patients in the endoscopic group was (9.8±2.9) years (ranging from 7 to 17 years), while in the open surgery group was (10.3±3.8) years (ranging from 7 to 17 years). The postoperative follow-up period was (15.4±3.4) months (1 to 2 years), and all fascia flaps survived without any severe complications. There were no statistically significant differences between the two groups in terms of age difference, length of hospital stay, intraoperative blood loss, postoperative satisfaction, sex composition ratio, and postoperative complications ( P>0.05). The scar quality in the endoscopy group was superior to that in the open surgery group, and POSAS scores of endoscopic group were lower than those in the open surgery group, and the difference was statistically significant ( P<0.05). Conclusion:Endoscope assisted temporoparietal fascia flap harvest for the second-stage operation in auricular reconstruction of Nagata’s technique for microtia can minimize scarring, improve the postoperative appearance and is not statistically associated with the appearance of reconstructed auricles or complications.
6.A case of severe hookworm infection.
Linghui MA ; Dibo NOUHOUM ; Wanni WANG ; Zhishan ZHANG ; Xunya HOU ; Xinling YU ; Weicheng DENG ; Jie GUAN ; Shuaiqin HUANG ; Xiang WU
Journal of Central South University(Medical Sciences) 2022;47(6):814-822
With the improvement of sanitation, the infection rate of hookworm is greatly reduced and the severe infected case is rarely reported. Combined morphological and molecular biological examinations, a severe hookworm infection patient was diagnosed in Department of Laboratorial Examination, Quanzhou First Affiliated Hospital of Fujian Medical University. The morphological methods such as direct fecal smear microscopy, saturated brine flotation and hookworm larvae culture methods were used to identify the eggs and larvae from stool samples of the patient. There were a large number of hookworm eggs in patient's stool samples, and the average count was 60 840 per gram by modified Kato method, which belonged to severe hookworm infection. Meanwhile, to distinguish the hookworm species, the semi-nested RT-PCR assay was employed to detect hookworm internal transcribed spacer series from eggs in patient's stool samples, and the result showed that the hookworm species was confirmed to be Necator americanus.
Ancylostomatoidea/genetics*
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Animals
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Feces
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Hookworm Infections/diagnosis*
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Humans
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Necator americanus/genetics*
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Polymerase Chain Reaction
7.Expression changes of prostacyclin and thromboxane in blood of rats with tail vibration
Ziyu CHEN ; Nuoyan WEI ; Zhishan LIANG ; Huimin HUANG ; Xiuwen HU ; Junyi WANG ; Qingsong CHEN ; Hongyu YANG
Journal of Environmental and Occupational Medicine 2022;39(11):1226-1230
Background Long-term exposure to hand-transmitted vibration can lead to hand-arm vibration syndrome, one manifestation of which is impaired peripheral blood circulation in the arms. Altered expressions of prostacyclin I2 (PGI2) and thromboxane A2 (TXA2) in blood may be one of the important mechanisms of vibration-induced hand-arm vibration syndrome. Objective To reveal the effects of rat tail vibration on the expressions of PGI2 and TXA2 in plasma, and to establish the correlation between the change of rat plasma PGI2 to TXA2 ratio and rat tail vibration. Methods Fifty SPF-grade male SD rats were randomly divided into five groups: control group, 1 d exposure group, 3 d exposure group, 7 d exposure group, and 14 d exposure group, with 10 rats in each group. The rats were placed in rat immobilizes on a immobilization table, and the rats' tails were connected to a shaker and fixed with medical tape. There was no overlap between the immobilizes and between the rats' tails by no contact between the immobilization table and the shaker. The exposure dose was 125 Hz, 5.9 m·s−2, 4 h·d−1, and the vibration direction was linear vertical vibration. Abdominal aortic blood was taken at the end of vibration exposure, and the expressions of PGI2, TXA2, and their hydrolysates 6-keto-prostaglandin F1α (6-keto-PGF1α) and thromboxane B2 (TXB2) were measured by enzyme-linked immunosorbent assay, and the 6-keto-PGF1α/TXB2 values were calculated. Spearman rank correlation was used to analyze whether the expression of vascular factors correlated with the accumulated time of vibration. Results The expression levels of plasma 6-keto-PGF1α were (896.12±124.37), (1068.13±119.41), (1215.26±122.64), and (1317.94±106.54) ng·L−1 in the 1 d, 3 d, 7 d, and 14 d groups of rats, respectively, which were higher than that in the control group, (830.60±109.47) ng·L−1 (P<0.001). The PGI2 expression levels were (86.49±2.40), (107.90±2.65), (114.02±2.16), and (126.95±1.94) ng·L−1 in the 1 d, 3 d, 7 d, and 14 d groups of rats, respectively, all higher than (60.09±2.11) ng·L−1 in the control group (P<0.001). The expression levels of TXB2 were (132.14±4.10), (145.52±4.09), (179.91±4.98), and (204.10±3.22) ng·L−1 in the 1 d, 3 d, 7 d, and 14 d groups of rats, respectively, which were higher than that in the control group, (106.08±3.26) ng·L−1 (P<0.001). The expression levels of plasma TXA2 were (211.99±3.24), (236.33±3.88), and (245.45±4.23) ng·L−1 in rats in the 3 d, 7 d, and 14 d groups, respectively, which were all elevated compared with (174.79±4.19) ng·L−1 in the control group (P<0.001). Compared with the control group, the 6-keto-PGF1α/TXB2 values were decreased in the 7 d and 14 d groups (P<0.05). The 6-keto-PGF1α, PGI2, TXB2, and TXA2 expressions were positively correlated with vibration accumulation time (r=0.84, 0.84, 0.80, 0.84, P<0.001) and the 6-keto-PGF1α/TXB2 values were negatively correlated with vibration accumulation time (r=-0.24, P=0.003). Conclusion Local exposure of rat tail to vibration could increase the expressions of PGI2 and TXA2 in blood, and the elevated expressions show a dose-effect relationship with the duration of vibration exposure, but the PGI2/TXA2 tends to decrease with the accumulation of vibration exposure.
8.Metabolomics study of rat tail vibration model
Zhishan LIANG ; Hongyu YANG ; Chunguang DING ; Ziyu CHEN ; Huimin HUANG ; Xiuwen HU ; Junyi WANG ; Nuoyan WEI ; Qingsong CHEN
Journal of Environmental and Occupational Medicine 2022;39(11):1231-1236
Background The metabolites and metabolic pathways of hand-arm vibration syndrome have not yet been elucidated. Objective To investigate the effect of local vibration on endogenous metabolites in rat serum by metabolomic analysis, to preliminarily explore the potential metabolic pathway of endogenous metabolites, so as to provide evidence for further research on the mechanism of hand-arm vibration syndrome. Methods Thirty-two SPF male SD rats, (211.3±11.1) g, 7−8 weeks of age, were selected and randomly divided into three groups: control group (14 rats, without vibration), 7 d vibration group (9 rats, continuously vibration for 7 d), and 14 d vibration group (9 rats, continuous vibration for 14 d). The vibration rats were vibrated every day for 4 h, the frequency weighted acceleration was 4.9 m·s−2, the vibration frequency was 125 Hz, and the vibration direction was one-way vertical vibration. The control group had the same conditions except not contacting vibration. After the vibration exposure, the blood samples taken from the abnormal aorta of rats were collected, and the changes of rat serum metabolome were analyzed by ultra-performance liquid chromatography-tandem time-of-flight mass spectrometry. Principal components analysis (PCA) was used to explore changes in rat serum metabolic profile, and orthogonal partial least squares-discriminant analysis (OPLS-DA) was used to screen out differential metabolites. Combined with online databases, a metabolic pathway enrichment analysis of differential metabolites was performed. Results The PCA analysis showed that compared with the control group, the rat serum metabolic profiles in the 7 d group and the 14 d group were clearly differentiated, and the rat serum metabolic profiles in the 7 d group and the 14 d group partially overlapped. The OPLS-DA analysis showed significant differences between groups. The main parameters were: model interpretation rate R2Y=0.914, model predictive ability Q2=0.58. The OPLS-DA analysis screened out 26 and 119 differential metabolites from the 7 d group and the 14 d group respectively, and there were 24 common differential metabolites between the 7 d group and the 14 d group. The metabolomic pathway analysis showed that local vibration-induced changes in rat serum metabolism were mainly related to arachidonic acid metabolism in the 14 d group, among which the metabolites with significant effects were arachidonic acid, prostaglandin E2, and prostaglandin D2. Conclusion Local vibration could affect the normal metabolism in rats, and the metabolic pathway with significant influence is arachidonic acid metabolism after a 14 d exposure and the involved metabolites are arachidonic acid, prostaglandin E2, and prostaglandin D2.
9.Effect of oleanolic acid on biological function of keloid fibroblasts
Zhishan XU ; Hongyi WANG ; Shixiu LIN ; Jiulong LIANG ; Quan ZHANG ; Kai TAO
Chinese Journal of Plastic Surgery 2021;37(4):430-437
Objective:To investigate the effects of oleanolic acid on the growth and migration of keloid fibroblasts.Methods:Keloid tissue samples from 9 patients in the Department of Plastic Surgery of General Hospital of Northern Theater were collected and fibroblasts were cultured in vitro. Fibroblasts were treated with different concentrations of oleanolic acid and divided into three groups: control group added 0.9% NaCl; 5 μmol/L oleanolic acid group added 5 μmol/L oleanolic acid; 10 μmol/L oleanolic acid group added 10 μmol/L oleanolic acid. MTT assay was used to detect cell proliferation; flow cytometry was used to detect cell cycle. Annexin V propidium iodide (AV-PI) staining was used to detect cell apoptosis. Transwell assay was used to detect the migration of oleanolic acid. Western blotting and real-time PCR were used to detect the expression of related proteins and mRNA activity. Each group was made in triplicate. Analysis of variance was used to compare the data among the three groups. LSD- t test was used for pairwise comparison, and P<0.05 was considered to be statistically significant. Results:MTT result showed that oleanolic acid could inhibit the proliferation of cells. After 24 hours, the proliferation of cells in 5 μmol/L oleanolic acid group and 10 μmol/L oleanolic acid group were 0.660±0.020 and 0.460±0.020, respectively, compared with 0.780±0.001 in the control group, F=114.4, P<0.001. Compared with the control group, the difference was statistically significant ( t=5.94, P<0.001, t=15.60, P<0.001); flow cytometry showed that the cell cycle G1/S phase transduction was blocked, 5 μmol/L oleanolic acid group and 10 μmol/L oleanolic acid group were significantly inhibited. The percentage of G1 phase cells in the 5 μmol/L oleanolic acid group was significantly higher than that in the control group ( t=3.14, P=0.030, t=6.38, P< 0.001). AⅤ-PI staining showed that the number of apoptotic cells in the 5 μmol/L oleanolic acid group (0.9%) and 10 μmol/L oleanolic acid group (3.4%) was significantly higher than that in the control group (0.4%), and the difference among the three groups was F=119.6, P<0.001. Transwell assay showed that the migration number of cells in 5 μmol/L oleanolic acid group (57.13 ± 2.65) and 10 μmol/L oleanolic acid group (42.15 ± 2.55) was significantly lower than that in control group (72.27± 3.32), F=101.3, P<0.001. Compared with the control group, the difference was statistically significant ( t=6.50, P<0.001, t=14.41, P<0.001). Western blotting showed that oleanolic acid could inhibit the expression of Cyclin D1, Bcl-2, Bax and MMP2. Compared with the control group, 5 μmol/L oleanolic acid t=8.70, P<0.001, t=5.00, P=0.040, t=12.41, P<0.001, t=10.46, P<0.001; compared with the control group, 10 μmol/L oleanolic acid t=31.61, P<0.001, t=23.17, P<0.001, t=12.11, P<0.001, t=44.52, P<0.001. Real-time PCR reaction showed that the mRNA activity levels of Cyclin D1, Bcl-2, Bax, MMP2 were also inhibited. Compared with the control group, 5 μmol/L oleanolic acid t=5.42, P< 0.001, t=3.11, P=0.040, t=16.11, P<0.001, t=11.71, P<0.001; compared with the control group, 10 μmol/L oleanolic acid t=51.78, P<0.001, t=30.89, P<0.001, t=10.64, P<0.001, t=17.10, P< 0.001. Conclusions:Oleanolic acid (5 μmol/L and 10 μmol/L) can inhibit the proliferation and migration of keloid fibroblasts and induce apoptosis of keloid fibroblasts after treating keloid fibroblasts for 24 hours, which can inhibit the growth of keloid and be used for the prevention and treatment of keloid.
10.Effects of Enterococcus faecalis supernatants on inflammatory responses of human periodontal ligament cells under pressure
Lei MENG ; Xue LIU ; Lei ZHANG ; Facheng WANG ; Liping YAO ; Xiaoning LI ; Yao LU ; Zhishan LU
Chinese Journal of Stomatology 2021;56(4):335-341
Objective:To study the effect of various concentrations of Enterococcus faecalis (Ef) supernatants on human periodontal ligament cell (hPDLC) and the inflammatory response of hPDLC under static pressure. Methods:The method of methyl thiazolyl tetrazolium (MTT) was used to detect the effect of various concentrations of Ef supernatants on the proliferation of hPDLCs and the flow cytometry was used to detect the expression of Toll-like receptor 2 (TLR-2) on the surface of hPDLC after 24-hour-stimulation of Ef supernatant. Furthermore, the hPDLCs were divided into non inducing group without Ef supernatant and inducing group with 5% Ef supernatant, and hPDLCs in each group were loaded with 0, 49 and 196 Pa static pressures respectively. The expressions of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) mRNA and protein were detected by reverse transcription-PCR (RT-PCR) and enzyme linked immunosorbent assay (ELISA) after 24 hours.Results:MTT results showed that the supernatant of Ef with concentratio n≥5% could significantly inhibit the proliferation activity of hPDLCs at 48 hours of cell culture ( P<0.05). Flow cytometry showed that the positive cell rates of TLR-2 increased with increasing volume fractions of the Ef supernatants. The values were (2.12±0.07)%, (2.41±0.32)%, (2.65±0.27)%, (4.76±0.46)%, (9.91±0.92)% and (12.01±1.35)%, respectively. The differences were statistically significant when the concentrations≥5% ( P<0.05). There were no significant differences in the expressions of IL-1β and TNF-α mRNA between the non inducing group and the control group under the pressure of 49 Pa ( P>0.05). However, there were significant differences in the expressions of IL-1β and TNF-α mRNA between the non inducing group and the control group under the pressure of 196 Pa ( P<0.05), while the expressions of IL-1β and TNF-α in the inducing group were significantly lower than that in the control group under the pressures of 49 and 196 Pa ( P<0.05). Compared with the control group, the mRNA expression was significantly increased ( P<0.05). The result of ELISA was consistent with that of PCR. Conclusions:High concentration of Ef supernatant could inhibit the proliferation of hPDLC. Ef supernatant might promote the expression of TLR-2 on the surface of hPDLC. Excessive mechanical pressure induced the inflammatory response of hPDLC. The presence of inflammatory mediators could lead to the intolerance of hPDLC to pressures and small pressure could aggravate the inflammatory response.

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