Background Studies confirmed that hydroxycamptothecin cause the apoptosis of human Tenon capsule fibroblasts (HTFs) by protein kinase R-like endoplasmic reticulum stress kinase (PERK) single pathway.Autophagy and apoptosis are programmed cell death following stress reaction,so they remain a close association.However,the effect of hydroxycamptothecin on the autophagy of HTFs and its mechanism are still unclear.Objective This study was to explore the promoting effect of PERK signal pathway on hydroxycamptothecin inducing the autophagy of HTFs.Methods This study procedure was approval by Ethic Committee of Nanjing Medical University.Human Tenon capsule tissue was obtained from fresh adult donors.HTFs were cultured and passaged by explant-culture method and identified by immunofluorescence for vimentin and keratin.pLVX-PERK lentiviral packed by 293T cells was transfected into HTFs to obtain stable PERK-knockout cell line by puromycin selection.Then the HTFs were treated with 0.10 g/L of hydroxycamptothecin for 5 minutes and consecutively cultivated for 24 hours,and the untreated cells were used as the control group.Western blot assay was used to detect the expressions of autophagy specific proteins in the cells,including autophagy related gene 5 (ATG-5),Beclin-1,light chain 3 (LC-3).Cyto-ID staining was used to identify the autophagosome in the cells.The experimental results were analyzed and compared between different treating groups.Results The gray scales for the expressions of Beclin 1,ATG-5,LC-3-Ⅰ and LC-3-Ⅱ proteins in HTFs were 0.365:±0.045,0.765 ±0.055,0.120±0.030 and 0.215 ±0.035 in the control group,and those in the hydroxycamptothecin treated group were 0.980±0.070,1.495±0.095,0.585±0.025 and 0.785±0.055,showing a significant decline in the hydroxycamptothecin treated group(P=0.018,0.022,0.007,0.013).The green fluorescence of the autophagosome was stronger in the hydroxycamptothecin treated group compared with the control group.Western blot revealed that the gray scale of PERK expression in the cells was 0.130±0.030 in the PERK-knockout group,with a significant reduce in comparison with 0.765 ±0.055 of the control group (P =0.010).However,no obvious distinctions were seen in the band intensities of the expressions of Beclin-1,ATG-5 and LC-3 proteins between the two groups.Western blot indicated that the grey scale of the PERK expression in the cells was 1.790± 0.060 in the 0.10 g/L hydroxycamptothecin group,which was significantly higher than 0.880 ± 0.070 of the control group (P =0.010).Expression levels (gray scales) of Beclin-1,ATG-5,LC-3-Ⅰand LC-3-Ⅱ in the PERK-knockout+ 0.10 g/L hydroxycamptothecin group were 0.475 ± 0.045,0.390 ± 0.040,0.055 ± 0.015 and 0.075 ± 0.025,which were significantly lowed in comparison with 0.955 ± 0.065,0.765 ± 0.055,0.155 ± 0.015 and 0.280 ± 0.030 of the control+ 0.10 g/L hydroxycamptothecin group (P =0.026,0.031,0.042,0.034).In addition,the fluorescence intensity of autophagosomes was weaker in the PERK-knockout+0.10 g/L hydroxycamptothecin group compared with the control+0.10 g/L hydroxycamptothecin group.Conclusions Hydroxycamptothecin induces the autophagy of HTFs by PERK signal pathway.

Background Previous study showed that both hydroxycamptothecin (HCPT) and etoposide (VP-16) can induce the apoptosis of human Tenon capsule fibroblasts (HTFs).However,whether the combination of HCPT with VP-16 enhance the efficacy of drugs is unknown.Olbjeetive This study was to investigate the synergistic effect and its mechanism of HCPT combined with VP-16 on apoptosis of HTFs.Methods Human Tenon capsule tissue was obtained from the eye bank of Jiangsu Province People's Hospital.HTFs were cultured in vitro using explant method and identified by immunofluorescence with vimentin.The fourth generation of cells were incubated in 96-well plate,and different concentrations of HCPT (1,5,10,50,100 mg/L),VP-16 (0.6,2.5,5.0,25.0,50.0 mg/L) and HCPT+VP-16 (2:1,final concentrations 0.80,3.75,7.50,37.50,75.00 mg/L) were added for 24 hours.The inhibiting rate of drugs to HTFs growth was detected using CCK-8 kit.The HTFs were divided into blank control group,HCPT (50 ng/L) treated group,VP-16 (25 mg/L) treated group and HCPT+ VP-16 (37.5 mg/L) treated group,and the apoptosis rates of HTFs in various groups were assayed by flow cytometry.The expressions of caspase-3,cleaved caspase-3,bax,bcl-2,JNK,p-JNK,Akt,p-Akt in the cells were detected by Western blot assay.Results Cultured cells grew well with the polygon shape and positive response for vimentin.The inhibiting rate was elevated with the increase of drug dosage 24 hours after addition of drugs (HCPT:F=41.34,P=0.00 ; VP-16:F =62.60,P =0.00 ; HCPT+VP-16:F =46.77,P =0.00).The half maximal inhibitory concentrations (IC50) of HCPT,VP-16,HCPT+VP-16 were 80.99,27.93,19.81 mg/L,respectively,and the combined index (CI) of HCPT with VP-16 was 0.399,showing a stronger synergistic action.The apoptotic rates of HTFs were (4.87±0.78) ％,(11.20± 1.94)％,(12.67±1.51)％ and (19.77±2.01)％ in the blank control group,HCPT treated group,VP-16 treated group and HCPT+VP-16 treated group,respectively,with a significant difference among them (F=18.23,P ＜ 0.01),and the apoptotic rate was significantly raised in the HCPT + VP-16 treated group,HCPT treated group and VP-16 treated group compared with the blank control group (q'=15.67,16.32,26.88,all at P＜0.01).Compared with the blank control group,the grey scale values of cleaved caspase-3,bax,p-JNK in the cells of HCPT+VP-16 treated group,HCPT treated group and VP-16 treated group were significantly increased (all at P＜0.01),and those in the HCPT+VP-16 treated group significant ascent in comparison with the HCPT treated group and VP-16 treated group (all at P＜0.01).However,the changes of caspase-3,JNK and Akt expression were insignificant.The grey scale values of bcl-2 and p-Akt in the HTFs of the HCPT,VP-16 and HCPT+VP-16 treated groups were significantly lower than those of the blank control group,with a dominant reducing in the HCPT+VP-16 treated group (all at P＜0.01).Conclusions HCPT and VP-16 induce the apoptosis of HTFs in vitro at a dose-dependent manner.The combination of HCPT with VP-16 has a stronger synergistic efficacy.The up-regulation of p-JNK and bax as well as the down-regulation of p-Akt and bcl-2 in HTFs are involved in the coaction of HCPT and VP-16.

BACKGROUND:The principle of lyophilization is to sublimate the solvent of frozen materials in vacuum and retain the solute, thus making a pore structure. OBJECTIVE: To produce a chitosan tubular scaffold by lyophilization, and to test its physicochemical properties. METHODS: The chitosan tubular material was prepared by lyophilization method, folowed by gross observation and electron microscopic observation. The chitosan tubular material samples were placed into PBS solution and pure water for 50 days, respectively, and then immersed in trypsin liquid for 1 day folowed by embedded into the muscle and dorsal skin of neonatal Sprague-Dawley rats for 30 days. The degradation rate and porosity of the material were observed and calculated. The breaking strength and compressive strength of the material were determined both under drying and soaking conditions using tensile instrument and pressure meter, respectively. RESULTS AND CONCLUSION:The external form of the chitosan tubular material was normaly tubular. Under the electron microscope, it was composited by different size pores, and the pore size was 50-200μm. The degradation rates of the material were (5.33±0.12)% in PBS, (11.26±0.15) in water, 0.012% in the trypsin liquid and (35.2±3.7)in vivo. The porosity rate was (97.5±1.5)%. The breaking strength and compressive strength of the material was higher under the drying state than under the soaking state (P < 0.05). These findings indicate that the lyophilization method can produce the chitosan tubular material with good porosity rate and degradation rate as wel as good tensile ability and compressive capability.

OBJECTIVETo observe the effect of different concentrations of zoledronic acid on human gingival fibroblast (HGF) cells proliferation and apoptosis, and to investigate the mechanism of bisphosphonate-related osteonecrosis of the jaw (BRONJ) caused by zoledronic acid.

METHODSDifferent concentrations (0, 0.5, l.0, 5.0, 10.0 µmol/L) of zoledronic acid acted on the HGF. After 24 hours, flow cytometry was used to detect the rate of apoptosis and methyl thiazolyl terazolium (MTT) assay used to observe the proliferation of HGF. The effect of different concentrations of zoledronic acid on cell proliferation was examined by proliferation test on day 2, 4 and 7.

RESULTSFlow cytometry showed that when the concentration of zoledronic acid was 0, 0.5, 1.0, 5.0, 10.0 µmol/L, the apoptosis rate was (0.67 ± 0.50)%, (2.13 ± 0.21)%, (3.27 ± 0.23)%, (4.17 ± 0.35)%, (9.87 ± 1.79)% respectively. The difference in HGF cells between control group and zoledronic acid groups was significant (P < 0.05) at three concentrations of zoledronic acid (1.0, 5.0, 10.0 µmol/L). The cell proliferation test showed an concentration-time dependent effect. With increase of concentration and time, the cell proliferation was significantly inhibited. There was significant difference in absorbance value among the different concentration groups (P < 0.05).

CONCLUSIONSZoledronic acid can inhibit HGF proliferation and promote its apoptosis.

Apoptosis ; drug effects ; Bisphosphonate-Associated Osteonecrosis of the Jaw ; etiology ; Bone Density Conservation Agents ; pharmacology ; Cell Proliferation ; drug effects ; Coloring Agents ; Diphosphonates ; pharmacology ; Dose-Response Relationship, Drug ; Fibroblasts ; drug effects ; physiology ; Flow Cytometry ; Gingiva ; cytology ; Humans ; Imidazoles ; pharmacology ; Tetrazolium Salts ; Thiazoles ; Time Factors

Objective To evaluate the clinical outcomes in patients with both column acetabular fractures involvement posterior wall using lag screw through single ilioinguinal approach.Methods Between August 2008 to August 2014,35 consecutive patients with both columns acetabular fractures and fracture also involved posterior wall and fixed by lag screws were retrospectively analyzed.There were 25 males and 10 females.The average age was 44.4+ 12.5 years (range,18-72 years).According to Letournel classifications,the acetabular fracture involved both columns and posterior wall in all cases,and 2 cases with additional seagull sign.The surgeon reduced posterior wall by pressing the fragment through a small tunnel in the soft tissue leaning against the external cortex of iliac bone and fixed the fragment using lag screws from the anterior side.Clinical and functional outcomes were assessed using the modified Merle d'Aubigné scoring system.Radiographic results were evaluated based on Matta scoring system.Results The patients were follow for an average of 44.7+ 18.9 months (range,24-96 months).The average operative time was 257.7±60.4 min (range,160-490 min).The average blood loss during the operating was 742.9±+614.5 ml (range,300-4 000 ml).Loss of reduction of the posterior wall was not found in any case.At the latest follow-up,radiographic results were excellent in 11,good in 19,and poor in 5.The average modified Merle d'Aubigné score was 16.4 (range,13-18).There were 8 cases of excellent,23 cases of good,and 4 cases of fair.Four patients developed deep venous thrombosis of the lower extremities after 3-4 days of operation.Warfarin was used for anticoagulation therapy and thrombi disappeared 6 months postoperatively.One case had superficial wound infection and was treat with vacuum sealed drainage (VSD) and anti-infection therapy.The incision healed without infection after one month.One case with poor quality of reduction and remaining seagull sign was treated with total hip arthroplasty after 35 months because of severe traumatic arthritis.The total reoperation rate was 5.7％ (2/35).latrogenic sciatic nerve injury as well as heterotopic ossification was not found in any case.Conclusion Satisfactory quality of reduction and good clinical outcomes can be achieved in patients with acetabular fractures involved both columns and posterior wall by single ilioinguinal approach and lag screw fixation for posterior wall.Iatrogenic sciatic nerve injury as well as heterotopic ossification was not found in any case.

Objective To evaluate the clinical outcomes of ilioinguinal approach with short infrapectineal buttress plate fixing posterior column for patients with complex acetabular fractures. Methods Data of 29 consecutive patients (male 22, fe?male 7, age range 25-72 years, average age 53 ± 6.3 years) with complex acetabular who had operated by single ilioinguinal ap?proach with infrapectineal buttress plate from September 2008 to August 2012 were retrospectively analyzed. According to Letour?nel?Judet classifications, there were 4 cases of anterior column and posterior hemi?transverse, 11 cases of associated both?column, 4 of T?shaped and 6 with seagull sign. The acetabular fractures model was printed preoperatively by 3D printing technique for sur?gical simulation, open reduction and internal fixation through single ilioinguinal approach with a short infrapectineal plate fixing posterior column, particularly an ox horn shape K?wire sleeve was developed for drilling and screw insertion using flexible screw?driver. Clinical, radiographic, and functional outcomes assessed by the modified Merle d’Aubigné score were collected. Results Two patients were lost to follow?up, including one patient who died at 15 days postoperative because of pulmonary embolism, and the other one who had moved abroad at 12 months postoperative. The remaining 27 (93%) had a mean follow?up of 40 months (range, 24-75 months). The operating time was 180±35.5 min;the time for the fracture union was 3.5±0.9 month;blood loss during the operating was 500±43.9 ml;no case had fracture re?displacement. At the latest follow?up, radiographic grades were excellent in 17, good in 8, poor in 4, including one poor patient who had a total hip arthroplasty (3%) at 35 months after the internal fixation. The average modified Merle d’Aubigné score was 16 (range, 10-18), and categorized as excellent in 12, good in 8, fair in 5, and poor in 2. Three patients had developed deep venous thrombosis of the lower extremities, and one of them died of pulmonary embo?lism 15 days after operation; two cases of deep venous thrombosis of the lower extremity were found 3-4 days after operation. Thrombosis disappeared 6 months after anticoagulant therapy by warfarin. One case who had superficial wound infection after oper?ation was treated by anti?infection and VSD negative pressure, and incision was healed after one month. One case with poor quality of fracture reduction and seagull sign was treated with total hip arthroplasty after 35 months because of traumatic arthritis. The to?tal complication rate was 24%(7/29). Conclusion In the patient with complex acetabular fractures combining posterior column medial displacement, single Ilioinguinal approach with infrapectineal buttress plates could achieve a stable concentric hip joint, and immediate postoperative ambulation can result in reasonable clinical, radiographic, and functional outcomes.

Objective To assess the effect of preoperative administration of tranexamic acid (TXA) on the hidden blood loss in the surgery of intertrochanteric fractures with proximal femoral nail anti-rotation (PFNA).Methods Eighty patients with intertrochanteric fracture were treated with PFNA in our hospital from November 2015 to July 2016.They were 15 men and 65 women,with a mean age of 72.6 years.Of them,39 were included into TXA group where TXA was administered preoperatively and 41 were assigned into the control group where no TXA was used preoperatively.Blood routine examinations were carried out on one day before operation,the first and third days after operation.The surgical blood loss,operative blood transfusion,24-hour drainage after operation,and postoperative 3-day blood transfusion were recorded.The total and hidden blood losses were calculated according to the formula.The 2 groups were compared in terms of blood loss and complications.Results In TXA group,the total blood loss (1,632.3 ± 849.2 mL),the hidden blood loss (1,270.9 ± 623.3 mL) and the transfusion rate (28.2％) were significantly lower than those in the control group (2,014.8 ± 924.7 mL,1,549.1 ± 624.9 mL and 56.1％) (P ＜ 0.05).There was no significant difference between the 2 groups in visible blood loss (361.4 ± 154.3 mL for TXA group versus 465.7 ± 191.3 mL for the control group) (P ＞ 0.05).Deep venous thrombosis occurred in 2 patients,limb swelling in one patient and wound dehiscence in one patient in TXA group while deep venous thrombosis occurred in one patient,limb swelling in 3 patients and wound dehiscence in none in the control group,showing no significant differences between the 2 groups (P ＞ 0.05).No infection was observed in either group.Conclusion Preoperative administration of TXA can reduce surgical hidden blood loss and transfusion rate as well but will not increase the risk of deep vein thrombosis in the surgery of intertrochanteric fractures with PFNA.

Neuromyelitis optica spectrum disorders (NMOSD) are a severe autoimmune inflammatory demyelinating disease of the central nervous system. NMOSD complicated with immune thrombocytopenia (ITP) is rare. This paper reports a case of NMOSD who was misdiagnosed as multiple sclerosis for many years, and then developed thrombocytopenia. ITP was diagnosed by perfect examination. After immunosuppression and thrombopoiesis therapy, the platelets returned to normal. The review of the case and literatures can help to improve the understanding of this kind of disease, timely diagnose and treat patients, and avoid serious complications.

Objective:To investigate the clinical efficacy of lenalidomide combined with bortezomib and dexamethasone (RVd) regimen in treatment of newly diagnosed multiple myeloma (NDMM) patients and its effect on the levels of regulatory T cells (Treg cells) and natural killer (NK) cells.Methods:Thirty-eight NDMM patients who were admitted to the Second Affiliated Hospital of Bengbu Medical College from September 2019 to May 2022 were selected for a prospective study, and were divided into control group (18 cases) and observation group (20 cases) according to random number table method. The control group was treated with bortezomib+epirubicin+dexamethasone (VAd) regimen, and the observation group was treated with RVd regimen. The efficacy and safety were compared between the two groups. The levels of Treg cells (CD4 + CD25 + FOXP3 +) and NK cells (CD3 - CD56 + CD16 +) before and after treatment in the two groups were detected by flow cytometry, and the results were compared. Results:After 4 courses of treatment, the objective response rate (ORR) of the observation group was 95.0% (19/20), which was higher than that of the control group [77.8% (14/18)], and the difference was statistically significant ( P = 0.016). Before treatment, there was no statistical difference in the levels of Treg cells and NK cells between the two groups ( P values were 0.381 and 0.650). After treatment, the level of Treg cells in the control group increased from (1.5±0.5)% before treatment to (4.7±1.3)% ( P = 0.008), while the level of Treg cells in the observation group increased from (1.4±0.5)% before treatment to (6.8±1.5)% ( P = 0.001), and the level in the observation group was higher than that in the control group ( P = 0.027); the level of NK cells in the control group increased from (16±6)% before treatment to (20±5)% ( P = 0.004), while the level of NK cells in the observation group increased from (16±6)% before treatment to (24±6)% ( P = 0.006), and the level in the observation group was higher than that in the control group ( P = 0.032). The incidence rates of thrombocytopenia and neutropenia in the observation group were higher than those in the control group, and the differences were statistically significant ( P values were 0.012 and 0.027), which was reversible after active treatment. There was no statistical difference in the incidence rates of other adverse reactions (all P>0.05). Conclusions:RVd regimen for NDMM is clinically effective, safe and reliable, and the patients' levels of Treg cells and NK cells elevate after treatment.

Objective To evaluate the clinical treatment of unstable pelvic fractures by posterior ring fixation using percutaneous minimally invasive pedicle screws following anterior ring fixation.Methods From January 2010 to January 2016,31 patients with unstable pelvic fracture were treated at our department.They were 20 males and 11 females,with an average age of 44.4 years (range,from 18 to 65 years).According to the Tile classification,6 cases were type B2,8 cases type B3,12 cases type C1,and 5 cases type C2.The anterior pelvic ring was fixated in supine position first,and the posterior pelvic ring was fixated next using percutaneous minimally invasive pedicle screws.The operation time,intraoperative bleeding,and frequency of fluoroscopy needed for the posterior ring fixation were recorded.Reduction quality,complications like loss of reduction and pelvic function at the final follow-up were also assessed.Results For the posterior ring fixation in the 31 patients,the operation time ranged from 40 to 60 minutes (average,50.7 minutes),blood loss from 30 to 80 mL (average,42.9 mL),and fluoroscopic frequency from 7 to 12 times (average,9.7 times).By the Tornetta evaluation,the reduction was rated postoperatively as excellent in 15 cases and as good in 16 ones,yielding an excellent and good rate of 100％.Twenty-nine patients obtained complete follow-up for 12 to 83 months (average,34.7 months),and they achieved bone union after an average of 14.5 weeks (range,from 12 to 16 weeks).According to the Majeed criteria for pelvic function at the final follow-up,10 cases were rated as excellent,16 cases as good,and 3 cases as fair,giving an excellent and good rate of 89.7％.No reduction loss,incision infection,vascular or nerve injury,screw loosening or breakage,or fracture displacement was observed during follow-up.Conclusion In the treatment of unstable pelvic fractures,posterior ring fixation using percutaneous minimally invasive pedicle screws following anterior ring fixation can lead to less operation time,intraoperative bleeding and fluoroscopic radiation,and satisfactory fracture reduction and functional recovery of the pelvis as well.