OBJECTIVE To establish a method for simultaneous determination of 7 anti-tumor drugs （irinotecan， capecitabine， paclitaxel， docetaxel， tamoxifen， letrozole and methotrexate） in human plasma and apply it to the clinic. METHODS After precipitating with a methanol-acetonitrile mixture （1∶ 1， V/V） containing 0.1% formic acid， liquid chromatography-tandem mass spectrometry （LC-MS/MS） was used to determine the plasma concentration， using deuterium isotopes of each analyte as internal standards. The chromatography was performed on the Agilent Eclipse Plus C18 column with a gradient elution of water （containing 0.1% formic acid+0.04% 5 mmol/L ammonium formate） as mobile phase A and acetonitrile （containing 0.1% formic acid） as mobile phase B. The flow rate was 0.6 mL/min， and the column temperature was set at 40 ℃ . The sample size was 10 μL， and the analysis lasted for 5.5 min. Electrospray ionization was used in positive and negative ion mode， and multiple reaction monitoring mode was used. The ion pairs used for quantitative analysis were m/z 587.1→167.1 （irinotecan）， m/z 360.1→244.1 （capecitabine）， m/z 876.4→308.0 （paclitaxel）， m/z 830.3→304.2 （docetaxel）， m/z 372.1→129.1 （tamoxifen）， m/z 284.1→242.1 （letrozole）， and m/z 455.0→ 308.0 （methotrexate）. A total of 97 patients with malignant tumors in our hospital were selected to measure the plasma concentrations of 7 anti-tumor drugs using the above method. RESULTS The linear ranges of irinotecan， capecitabine， paclitaxel， docetaxel， tamoxifen， letrozole and methotrexate were 2-1 000 ng/mL （r＝0.994 3）， 20-10 000 ng/mL （r＝0.997 5）， 2-1 000 ng/mL （r＝0.997 9）， 1-500 ng/mL （r＝0.995 8）， 1-500 ng/mL （r＝0.995 2）， 1-500 ng/mL （r＝0.996 4）， 10-5 000 （r＝0.997 7）， respectively. The quantitative lower limits were 2， 20， 2， 1， 1， 1 and 10 ng/mL； RSDs of intra-assay precision were 0.08%-14.86% （n＝6）. RSDs of inter-batch precision were 1.51%-11.55% （n＝3）， and the accuracies were 89.17%-114.93% （n＝6）. The matrix effects ranged from 89.89%-119.74% （n＝6）. RSDs of the stability tests were 1.98%-14.88% （n＝6）. The results of E-mail：hangpengzhou@163.com clinical application showed， the average plasma concentrations of irinotecan， capecitabine， paclitaxel and docetaxel were 704.09， 909.40， 36.45， 150.43 ng/mL， respectively. The values of the coefficient of variation were 25.24%， 62.65%， 122.69%， and 92.27%. CONCLUSIONS The established LC-MS/MS method is simple and rapid， and can be used for the simultaneous determination of 7 commonly used anti-tumor drugs in the plasma of patients with malignancy.

Objective:To evaluate the 10-year protective effect and immunogenicity of quadrivalent human papillomavirus (HPV) vaccine in Chinese women aged 20 to 45 years.Methods:From October 2019 to April 2020, a long-term follow-up study was conducted on the subjects of the Phase III clinical trial of the quadrivalent HPV vaccine (NCT00834106). Participants underwent a questionnaire survey, venous blood sampling, gynecological examination, cervical exfoliated cell pathology examination, and serum neutralizing antibody titers for HPV-6, 11, 16, and 18 were measured using a pseudovirus neutralization assay. The results of the cytological examination and the positive rate and titers of serum antibodies of different cervical exfoliated cells were compared.Results:A total of 889 subjects were followed up, including 240 in the control group, 453 in the vaccination group and 196 in the post-trial vaccination group. The age of the control group was (40±7) years old, which was higher than that of the supplementary vaccination group and the vaccination group [(38±4) and (38±6) years old, respectively] ( P<0.05). There were no statistically significant differences in condom use and sexual frequency among all groups (all P values>0.05). The abnormal proportion of cervical exfoliation cytopathology in the vaccination group was 3.7% (17/453), which was significantly lower than that in the control group [9.6% (23/240)] and post-trial vaccination group [5.6% (11/196)] ( P<0.05). There were two cases of cervical intraepithelial neoplasia (CIN) grade 1 in the vaccination group, two cases of CIN grade 1 and three cases of CIN grade 2 and above in the control group, and no CIN grade 1 and above cases in the post-trial vaccination group. The positive rate of HPV-18 antibody was 35.5% (161/453) in the vaccination group and 76.0% (149/196) in the post-trial vaccination group, which was significantly lower than that of other types ( P<0.05). The neutralizing antibody GMT ratio between the vaccination group and the control group ranged from 2.62 to 25.33 (9.05 to 83.08). Conclusion:Protective neutralizing antibodies are sustained in Chinese women aged 20 to 45 years after ten years of vaccination with quadrivalent HPV vaccine.

Objective:To evaluate the 10-year protective effect and immunogenicity of quadrivalent human papillomavirus (HPV) vaccine in Chinese women aged 20 to 45 years.Methods:From October 2019 to April 2020, a long-term follow-up study was conducted on the subjects of the Phase III clinical trial of the quadrivalent HPV vaccine (NCT00834106). Participants underwent a questionnaire survey, venous blood sampling, gynecological examination, cervical exfoliated cell pathology examination, and serum neutralizing antibody titers for HPV-6, 11, 16, and 18 were measured using a pseudovirus neutralization assay. The results of the cytological examination and the positive rate and titers of serum antibodies of different cervical exfoliated cells were compared.Results:A total of 889 subjects were followed up, including 240 in the control group, 453 in the vaccination group and 196 in the post-trial vaccination group. The age of the control group was (40±7) years old, which was higher than that of the supplementary vaccination group and the vaccination group [(38±4) and (38±6) years old, respectively] ( P<0.05). There were no statistically significant differences in condom use and sexual frequency among all groups (all P values>0.05). The abnormal proportion of cervical exfoliation cytopathology in the vaccination group was 3.7% (17/453), which was significantly lower than that in the control group [9.6% (23/240)] and post-trial vaccination group [5.6% (11/196)] ( P<0.05). There were two cases of cervical intraepithelial neoplasia (CIN) grade 1 in the vaccination group, two cases of CIN grade 1 and three cases of CIN grade 2 and above in the control group, and no CIN grade 1 and above cases in the post-trial vaccination group. The positive rate of HPV-18 antibody was 35.5% (161/453) in the vaccination group and 76.0% (149/196) in the post-trial vaccination group, which was significantly lower than that of other types ( P<0.05). The neutralizing antibody GMT ratio between the vaccination group and the control group ranged from 2.62 to 25.33 (9.05 to 83.08). Conclusion:Protective neutralizing antibodies are sustained in Chinese women aged 20 to 45 years after ten years of vaccination with quadrivalent HPV vaccine.

Objective:To investigate the clinical effects of free transplantation of inguinal flap in repairing electrical burn wounds of mice in the limbs.Methods:A retrospective observational study was conducted. From January 2012 to May 2023, 24 male patients with electrical burns in the limbs meeting the inclusion criteria, aged 20 to 69 years, were admitted to the Department of Burns of Tongren Hospital of Wuhan University & Wuhan Third Hospital. Totally 27 limbs were involved, including 18 upper limbs and 9 lower limbs. After debridement, the wound area in the limbs proposed to be repaired with the inguinal flap was 5.0 cm×1.5 cm-20.0 cm×9.0 cm. A total of 26 operations were performed in 24 patients, and 27 free inguinal flaps with area being 5.0 cm×3.5 cm-22.0 cm×12.0 cm were resected, including 6 for lobed transplantation, 2 for chimeric transplantation with iliac bone graft, 3 for conjoined transplantation with pedicled latissimus dorsi myocutaneous flap, and 1 for conjoined transplantation with free paraumbilical perforator flap. Indocyanine green angiography was used to evaluate the blood supply of the inguinal flap during operation, to guide arterial supercharge during transplantation. Among the transplanted inguinal flaps, 5 were pedicled with the common trunk of the superficial circumflex iliac artery and the superficial abdominal wall artery, 13 were pedicled with the superficial circumflex iliac artery alone, 6 were pedicled with the superficial abdominal wall artery alone, and the remaining 3 were simultaneously anastomosed with the superficial circumflex iliac artery and the superficial abdominal wall artery for arterial supercharge. In another patient, the deep branch of the superficial circumflex iliac artery was used as a bridging vessel to recanalize the proper artery of the index finger in the distal segment. The wound in the donor area were sutured directly. The blood supply status of the inguinal flap immediately after resection but before cutting off blood vessels and after transplantation and vascular anastomosis was recorded. The survival of the tissue flap, the fixation of the iliac bone graft, and the healing of secondary defect in the donor area were observed after surgery. The appearance of the inguinal flap and the functional recovery of the recipient area were followed up. At the last follow-up, the function of the affected upper limb was scored using the Arm, Shoulder, and Hand Disability (DASH) scoring scale, and the satisfaction of patients with the efficacy of each surgical treatment was investigated by referring to the 5-level Likert scale.Results:Totally 27 inguinal flaps showed good arterial blood perfusion and venous return immediately after resection but before cutting off blood vessels and after transplantation and vascular anastomosis. Except for one inguinal flap that developed necrosis 12 days after operation and was repaired by split-thickness skin graft from the head, the remaining 26 inguinal flaps and conjointly transplanted tissue flaps survived completely without vascular crisis. The iliac bone graft was fixed securely after operation. All secondary defects in donor area healed well after operation. After 6 to 36 months of follow-up, the inguinal flap was soft in texture, without obvious swelling, the function of the recipient area was significantly improved as compared with that before surgery, and the protective feeling of the inguinal flap was restored. At the last follow-up, the functional scores of DASH scoring scale of the 18 affected upper limbs ranged from 0 to 100 (with a mean of 27). The patients were very satisfied with the efficacy of 17 surgical treatments and relatively satisfied with the efficacy of 9 surgical treatments.Conclusions:The donor area of the inguinal flap is concealed, and the damage resulted from flap resection in this area is small. This flap has a rich blood supply. In the electrical burn wounds of mice in the limbs repaired with lobed transplantation, chimeric transplantation, and conjoined transplantation with other flaps, the appearance and function of the recipient area are well restored, and the patients' satisfaction with the efficacy of surgical treatment is high, which is worthy of clinical promotion.

Objective:To observe the clinical efficacy of Qingzhenfang for plasmoby （chronic urticaria）， and to investigate its effect on cellular immune function. Method:One hundred and thirty-two cases patients were divided into control group and observation group evenly according to random number table. The 60 patients in control group finished the study because of 6 cases of dropout， loss of follow-up and withdrawal， and 62 patients in observation group finished the study. Patients in both groups got Yiebastine tablets， 10-20 mg/time， 1 time/day. Patients in control group additionally got Piminxiao capsule， 4 grains/time， 3 times/day， while patients in observation additionally got Qingzhenfang， 1 dose/day. The treatment continued for 8 weeks in both groups. Before the treatment， and at the second， fourth， and eighth week after treatment， scores of urticaria activity for 7 days （USA7） and total symptom score （TSS） were graded. Before and after treatment， scores of chronic urticaria quality of life scale （CU-Q2oL） and syndrome of rheumatic fever were graded. A follow-up of 3 months was conducted for the patients whose score of USA7 was less than 7 to record the recurrence. Complement 3 and 4 （C3， C4）， CD4⁺， CD8⁺ cells were detected， and Th17/ CD4⁺ and Treg/ CD4⁺， CD4⁺/CD8⁺ and Th17/Treg were calculated. Levels of peripheral blood interleukin-10 （IL-10）， IL-17 and IL-23 were detected， and safety was evaluated after the treatment. Result:At the second， fourth and eighth week after the treatment， scores of USA7， TSS， CU-Q2oL and syndrome of rheumatic fever in observation group were lower than those in the control group （P<0.01）. Levels of C3， C4， CD4⁺， Treg， CD4⁺/CD8⁺and IL-35 in observation group were higher than those levels detected in control group （P<0.01）， while levels of CD8⁺， Th17， Th17/Treg， IL-10， IL-17 and IL-23 were lower than those in the control group （P<0.01）. Recurrence rate was 25.58% （11/43） in observation group， lower than 48.48% （16/33） in control group （χ2=4.276， P<0.05）， and the clinical efficacy in observation group was superior to that in control group （Z=2.021， P<0.05）. Conclusion:Yaoyi Qingzhenfang can control the degree of disease and improve the quality of life for patients with chronic urticaria， with superior clinical efficacy. In addition， it can reduce recurrence rate， increase the levels of C3， C4， regulate cellular immune function， and reduce immune inflammatory response， so it is worthy of further clinical research and use.

Objective:To evaluate the diagnostic value of the Caprini risk assessment model combined with D-dimer in perioperative venous thromboembolic (VTE) of hysterectomy.Methods:The clinical data of 160 patients who had underwent hysterectomy in Lianjiang City Maternal and Child Health Hospital from February 2017 to February 2019 were retrospectively analyzed. During perioperative period, VTE occurred in 80 patients (VTE group), and 80 patients had no VTE (control group). The sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and Youden index of Caprini risk assessment model, D-dimer level detection and Caprini risk assessment model combined with D-dimer in the diagnosis of VTE were analyzed and compared, and the diagnostic efficacy was evaluated by receiver operating characteristic (ROC) curve.Results:The positive rates of Caprini risk assessment model and D-dimer in VTE group were significantly higher than those in control group: 87.50% (70/80) vs. 17.50% (14/80) and 90.00% (72/80) vs. 41.25% (33/80), and there was statistical difference ( P<0.05). The sensitivity, specificity, negative predictive value, positive predictive value, negative likelihood ratio, positive likelihood ratio and Youden index of the Caprini risk assessment model were 87.50% (70/80), 82.50% (66/80), 86.84% (66/76), 83.33% (70/84), 0.15, 0.50 and 0.70, respectively; the indexes of D-dimer were 90.00% (72/80), 58.75% (47/80), 85.45% (47/55), 68.57% (72/105), 0.17, 2.18 and 0.49, respectively; the indexes of Caprini risk assessment model combined with D-dimer were 97.50% (78/80), 52.50% (42/80), 95.45% (42/44), 67.24% (78/116), 0.05, 2.05 and 0.50, respectively. The areas under curve of Caprini risk assessment model, D-dimer and Caprini risk assessment model combined with D-dimer were 0.888, 0.877 and 0.945 (95% CI 0.833 to 0.943, 0.820 to 0.933 and 0.908 to 0.983, P < 0.01). Conclusions:Caprini risk assessment model and D-dimer have good results in the diagnosis of perioperative VTE of hysterectomy, and Caprini risk assessment model combined with D-dimer has the highest diagnostic value.

Objective To study the underlying mechanisms through which uric acid upregulates local renin-angiotensin system in adipocytes. Methods The primary cultured rat adipocytes were administered with 0, 1, 5, 10, and 15 mg/dl uric acid for 0, 12, 24, 48, and 72 h. Some of the pre-adipocytes were infected with siRNA-TLR2 or its negative control before differentiation. Then infected mature adipocytes were treated with 10 mg/dl uric acid for 48 h. After that procedure, mRNA levels of TLR2, TNF-α, IL-6, MCP-1, AGT, ACE1, AT1R, and AT2R were detected with real-time PCR method. The protein levels of TLR2 and NF-κB were detected by Western blotting. The concentrations of angiotensinⅡ( Ang Ⅱ) in the conditioned medium or cell lysate were measured using ELISA method. Results The mRNA levels mRNA of TLR2 increased in parallel with uric acid concentration. Moreover, it also increased with the time. By contrast, TLR2 mRNA expression decreased at 72 h. Uric acid increased levels of TNF-α, IL-6, and MCP-1 in adipocytes. It was also found that uric acid upregulated RAS components, including AGT, ACE1, AT1R, AT2R, and AngⅡ. However, siRNA-TLR2 infection significantly reduced the levels of TLR2 and NF-κB. As a result, both inflammatory cytokines and RAS components were significantly decreased in adipocytes. Conclusion Uric acid up-regulates RAS expression partially via TLR2 inflammatory signaling pathway in adipocytes.

Objective To investigate the effect of complement C3a on mouse podocytes phenotype transformation.Methods Purified C3a recombinant protein was used to stimulate mature mouse podocytes.The expression of the mature podocyte markers synaptopodin,podocin,nephrin,CD2-associated protein (CD2AP) and the mesenchymal cell markers fibroblast specific protein 1 (FSP-1),α-smooth muscle actin (α-SMA) were detected by RT-PCR,Western blotting,immunochemistry and immunofluorescence,respectively.Some podocytes were transfected with integrin-linked kinase (ILK) siRNA before the administration of C3a,the expression of nephrin and α-SMA were accessed by Western blotting,and the expression of Snail and α-actinin 4 were accessed by Western blotting and immunochemical method.The migration ability of podocytes was observed by scratch test.Results Immunocytochemistry and immunofluorescence analysis showed that synaptopodin,podocin,nephrin,CD2AP were highly expressed by mature mouse podocytes.The expression of these podocyte markers could be markedly inhibited after 24 h of C3a (0.1 μmol/L) treatment,and accompanied by the induction of mesenchymal markers FSP-1 and α-SMA.Compared with control group,the mRNA levels of synaptopodin,podocin,CD2AP and nephrin were significantly repressed by the administration of C3a in a dose-dependent manner,whereas the transcription of FSP-1 and α-SMA were remarkably up-regulated by C3a treatment (P ＜ 0.05,respectively).Western blotting analysis also confirmed the decrease of synaptopodin,podocin,nephrin and CD2AP protein and the increase of FSP-1 and α-SMA protein were closely depend on the C3a concentration (P ＜ 0.05,respectively).To further assess the downstream of C3a,some podocytes were transfected with ILK siRNA before the administration of C3a.Compared with C3a group,the protein levels of nephrin and α-SMA were significantly changed by the administration of ILK siRNA (P ＜ 0.05,respectively).The expression of α-actinin 4 and Snail induced by C3a were inhibited by ILK knockdown (P ＜ 0.05,respectively),accompanied by a decline of cell migration potency.Conclusion Complement fragment C3a can induce transformation of mouse podocytes to mesenehymal cells,and ILK signaling pathway is involved in this cell type transformation.

OBJECTIVE:To investigate the effects of puerarin on bone mineral density around the femoral prosthesis of el-derly women after osteoporotic fracture artificial hip joint replacement. METHODS:99 elderly women after osteoporotic fracture artificial hip joint replacement were divided into control group(49 cases)and test group(50 cases)according to random number table. Control group received conventional treatment:calcium carbonate and vitamin D3+alendronate sodium+salmon calcitonin;test group was additionally given Puerarin injection 200-400 mg dissolved in Glucose injection 500 ml intravenously,qd,on the basis of control group. A treatment course lasted for 20 d,and both groups received 2 courses of treatment. The hip joint function score and bone mineral density around the femoral prosthesis of 2 groups were observed and compared after surgery,and the oc-currence of ADR was also observed. RESULTS:3 and 2 patients withdrew from control group and test group,respectively. 18 months after surgery,the patients with hip joint function score ranged 70-79 in test group was significantly less than in control group;the rate of excellent hip joint function score in test group was significantly higher than in control group,with statistical significance (P<0.05). 15 and 18 months after surgery,bone mineral density in R1-R5 range of Gruen range in test group was slightly higher than in control group,without statistical significance(P>0.05);bone mineral density in R6-R7 range was signifi-cantly higher than control group(89.58% vs. 69.57%),with statistical significance(P<0.05). The prosthesis loosening was not found in both groups,and ADR was also not found as fever,erythra,nausea,vomiting,headache,dizziness,etc. CONCLU-SIONS:For the use of puerarin in elderly women after osteoporotic fracture artificial hip joint replacement,puerarin can increase the periprosthetic femur bone mineral density with good safety.

AIM:To investigate the effect of activation of retinoid X receptor (RXR) on transforming growth factor β1 (TGF-β1) induced collagen synthesis under hypoxic environment in rat cardiac fibroblasts (CFs) and underlying molecular mechanisms .METHODS: CFs were cultured using myocardial tissue with dry method .Hypoxic environment was established for CFs by continuous nitrogen supplement .Type I and type III collagens in supernatants were detected by ELISA.Nuclear and cytoplasmic extractions were prepared using NE-PER nuclear and cytoplasmic extraction reagents .The protein levels of Smad2 and p-Smad2 were determined by Western blot and immunocytochemical staining .RESULTS:Un-der hypoxic condition , TGF-β1 (0.01~10 μg/L) increased the synthesis of type I and type III collagens in a dose-de-pendent manner in the CFs .At the concentration of 5μg/L, the synthesis of collagen I and III was significantly increased as compared with control group (P<0.01).RXR agonist 9-cis-retinoic acid (9-cis-RA;10 -9 ~10 -6 mol/L) decreased TGF-β1 (5μg/L)-induced synthesis of type I and III collagens in a dose-dependent manner in the CFs under hypoxic con-dition.The synthesis of type I and type III collagens was significantly inhibited by 9-cis-RA (P<0.01).Smad2 inhibitor ( 20 nmol/L) showed similar inhibitory effect on the synthesis of type I and III collagens induced by TGF -β1 under hypoxic condition.Compared with TGF-β1 intervention group, the cytoplasmic level of p-Smad2 in the CFs was significantly in-creased in TGF-β1+9-cis-RA group, but the nuclear p-Smad2 level was significantly decreased (P<0.05).CONCLU-SION:Retinoid X receptor agonist 9-cis-RA inhibits TGF-β1-induced synthesis of type I and type III collagens in the CFs by repressing p-Smad2 nuclear translocation under hypoxic condition .