Objectives To investigate the impact of ischemic preconditioning (IPC) on dynamics of homing of endothelial progenitor cells (EPCs) after renal ischemia reperfusion injury (IR).Methods Sixty male Sprague-Dawley rats were randomly divided into three groups after right-side kidney nephrectomy:for sham-operated rats,lumbotomy without vascular clamping was performed; IR rats were clamped renal blood vessels for 40 minutes while IPC rats were pre-treated with 15 min ischemia and 10 min reperfusion.At 3,12,24 h,and 3 days after reperfusion,the pool of circulating,kidneys,lungs and spleens were harvested.The extent of renal injury was assessed by biochemical and histological examination.The dynamics of homing of EPCs was observed by flow cytometry.Results The rats in IPC group exhibited significant improvements in renal function and morphology.Compared with IR group and sham group,the number of EPCs in blood was increased in the IPC group at 12 h and 24 h after reperfusion (P ＜ 0.05).The number of EPCs in kidney was increased at all times pointin the IPC group and IR group as compared to the sham group (P ＜ 0.05.In addition,EPCs number was increased in IPC group compared with the IR group at 12 h and 24 h [(11.36±0.66)％ vs (6.37±0.69)％,(6.31±0.70)％ vs (4.40±0.60)％,all P＜ 0.05].Compared with IR group and sham group,the number of EPCs in the lung was increased in the IPC groups at 12 h after reperfusion [(2.95±0.66)％ vs (1.78±0.59)％,(1.66±0.61)％,all P ＜ 0.05].The number of EPCs in spleen was increased in the IPC group at 72 h as compared with the IR group and sham group [(0.55±0.06)％ vs (0.34±0.07)％,(0.31±0.06)％,all P ＜ 0.05].Conclusions Endogenous EPCs may home to injured kidney after IPC.EPCs can also gather in the lungs and spleen.

Objective To study the relationship of soluble LAIR (sCD305 and CD3060) expression in recipient serum with cytomegalovirus (CMV) pneumonitis after renal transplantation. Methods Nineteen serum specimens from recipients were divided into CMV pneumonitis group (n=10) and control group (n=9). Then the concentrations of sCD305 and CD3060 were quantitated with sandwich ELISA. The data were analyzed by using student t test. Results sCD305 was skewness distributed in both 2 groups, was 0.000-3.039 μg/L in CMV pneumonitis group and 0.000-8.375 μg/L in con-trol group. CD3060 was skewness distributed in CMV pneumonitis group and the concentration was 0.000-0.017μg/L. CD3060 was mormally distributed in control group and the concentration was 0.046±0.035 μg/L. There was significant difference of CD3060 (P=0.000) concentrations and no sig-nificant difference of sCD305(P=0.316) concentrations in 2 groups, respectively. Conclusions The concentration of CD3060 is low in CMV pneumonitis patients. The combination of CMV PP65 antigen detection and CD3060 detection is helpful for the early and precise diagnosis of CMV pneumonitis in renal transplantation patients.

Objective To investigate the effect of POSTN protein on the proliferation of chondrocytes of tibial plateau in old rats.Methods Cartilage cells collected from the tibial plateau of old rats were cultured in vitro to the third generation.Then the cells were divided into 3 groups:POSTN group,PBS group and POSTN antibody group.The proliferations of the three groups at 24 h,48 h and 72 h were determined by EDU method.The expression of Notch1 protein was detected by immunohistochemistry in all groups at the same time.Female 20-month-old Sprague-Dawley rats were randomly divided into 3 groups:POSTN protein injection group,Phosphate Buffered Saline (PBS) injection group and POSTN antibody injection group.Twelve weeks after the operation,related reagents were injected 3 times consecutively at day 1,day 3,day 5 and EDU was injected into joints at day 1.At 2 weeks after injection,the rats were killed and the knee tibial plateau was taken to observe the proliferation of the cartilage cells.Results At 24 h,there were differences between the three groups O(F=27.32,P=0.017).The proliferation rates of POSTN group [(23±8)％] and PBS group [(21±10)％] were higher than that of POSTN antibody group (16±5)(P=0.003,P=0.011).At 48 h,there were differences between the three groups (F=35.34,P＜0.01).The proliferation rate of POSTN group [(36±11)％] was higher than that of the other groups [(22±6)％],(18±6)％(P=0.021,P＜0.01).At 72h,there were differences between the three groups (F=52.62,P=0.000).The proliferation rate of POSTN group [(56±17)％] was the highest one,the proliferation rate of PBS group [(31±8)％] was the medium,and the POSTN antibody group [(26±7)％] was the lowest one (all P＜0.05).As for Notch1 protein expression in chondrocytes,there were differences between the three groups (F=26.72,P＜0.01).The Notch1 protein was the most frequently expressed in POSTN protein-injection group and the least in the anti-POSTN group.In rats,the proliferation rate of the chondrocytes in the medial tibia plateau of the knee of POSTN protein injection group [(36±14)％],which was the highest,and that of the POSTN antibody injection group [(10 ±4)％] was the lowest (all P＜0.05).Conclusion POSTN protein can promote the proliferation of chondrocytes knee OA rats.POSTN antibody injection has been shown to induce the proliferation of chondrocytes.The POSTN protein may promote the proliferation of chondrocytes by activating the Notch signaling pathway.

Objective:By studying the gait changes of patients with knee osteoarthritis (KOA), the study provided a theoretical basis for the quantitative indicators obtained from gait analysis to the diagnosis of KOA. And it provided a gait reference for the clinical diagnosis, treatment, rehabilitation, prevention and efficacy evaluation of KOA.Methods:A total of 30 patients (KOA group) with KOA hospitalized in our hospital from May 2021 to October 2021 and 30 healthy people (control group) were compared for gait changes. The t test, Mann-Whitney U test and Fisher′s exact test were used to compare differences between groups. Results:The KOA group were greater than the control group in terms of step time [(642±81) ms and (548±62) ms, t=-5.01, P<0.001], gait cycle [(1 284±168) ms and (1 076±114) ms, t=-5.61, P<0.001], double support time [(531±125) ms and (331±51) ms, t=-8.10, P<0.001], double support time period proportion (0.417±0.063 and 0.309±0.023, t=-8.50, P<0.001), total support time [(914±135) ms and (678±107) ms, t=-7.52, P<0.001], total support time period proportion (0.711±0.027 and 0.627±0.044, t=-8.87, P<0.001), and left static standing time (55.7±8.4 and 51.5±2.2, t=-2.65, P=0.012), for which the differences were statistically significant. The KOA group were lower than the control group in terms of single support time period proportion (0.287±0.030 and 0.334±0.013, t=7.80, P<0.001), right static standing time (44.3±8.4 and 48.5±2.3, t=2.65, P=0.012), step length [(36±8) cm and (52±5) cm, t=9.97, P<0.001], stride length [(70±16) cm and (103±8) cm, t=10.00, P<0.001], velocity [(0.60±0.18) m/s and (1.05±0.19) m/s, t=9.54, P<0.001], left knee range of motion [(42±17)° and (63±4) °, t=6.49, P<0.001], and right knee range of motion [(37±18) ° and (62±3)°, t=7.54, P<0.001], for which the differences were statistically significant. Conclusion:Gait analysis can quantitatively evaluate the condition of patients with KOA, making it possible to transform the diagnostic criteria of KOA from qualitative to quantitative.

Objective:To explore the effects of Alpha-2-macroglobulin-rich serum (A2MRS) on knee post-traumatic osteoarthritis (PTOA).Methods:The knee PTOA models were constructed by transection of the anterior cruciate ligament (ACL) in 80 SD male rats, aged 2 months and weighing from 250 to 300 g, which were randomized into 4 groups ( n=20): a high dose group (A2MRS containing 20 μg/μL A2M administered), a low dose group (A2MRS containing 10 μg/μL A2M administered), a positive control group (normal saline administered), and a blank control group (the knee joint cut pseudooperatively and normal saline administered). HE, toluidine blue staining, safranine O staining, modified Mankin scoring and Osteoarthritis Research Society International (OARSI) scoring were conducted to evaluate and compare the therapeutic effects of A2MRS on the knee PTOA among the 4 groups. Results:The rat cartilage was thinner with patchy and cracked surface, and the chondrocytes were reduced and distributed unevenly in the positive control group, compared with the blank control group. The modified Mankin score (3.89±0.93) and OARSI score (10.05±0.72) in the positive control group were significantly higher than those in the blank control group (0.67±0.07 and 3.10±0.29) ( P<0.05). The rat cartilage was thicker with basically complete and crack-free surface, and the chondrocytes were increased and distributed more evenly in the high dose group and the low dose group, compared with the positive control group. The modified Mankin scores (1.33±0.50 and 1.56±0.53) and OARSI scores (6.30±0.64 and 4.75±0.66) in the high dose group and the low dose group were significantly lower than those in the positive control group ( P<0.05). However, there were no such differences between the high dose group and the low dose group ( P>0.05). Conclusion:A2MRS effectively delays the pathological process of knee PTOA.

Objective:To investigate the differences in gene expression levels in knee chondrocytes and chondrons in vitro.Methods:The chondrocytes and chondrons were isolated from full thickness of the 8-months ( n=5) rabbit knees cartilage. Chondrons from right knee were enzymatically isolated using 0.3% dispase and 0.2% collagenase-2 with shaking for 3 hours. Chondrocytes were isolated by 0.4% Pronase and 0.025% collagenase-2 from left knee. The mRNA levels in chondrocytes and chondrons were analyzed by quantitative real-time PCR, including matrix proteins [aggrecan(Agg), collagen(Col-2), Col-6A6, Col-10, Col-11], MMPs and inhibitors (MMP-1, MMP-3, MMP-9, MMP-13, TIMP-1, TIMP-2, TIMP-3), cytoskeletal proteins (Sox-9, vinculin, tubulin, actin), cytokines (IL-β, TNF-α). Analysis was performed using SPSS 16.0 statistical software, and the two-group comparisons were considered as significant by t-test at P<0.05. Results:Compared to the chondrocytes, the Agg [(5.78±0.90) vs (1.89±0.27), t=9.26, P<0.001], Col-2 [(6.29±0.76) vs (3.06±0.60), t=7.46, P<0.001], Col-6A6 [(0.89±0.18) vs (0.22±0.06), t=7.90, P<0.001], Col-10 [(3.83±0.76) vs (1.00±0.26), t=7.88, P<0.001] and TIMP-1 [(1.98±0.85) vs (1.03±0.34), t=2.32, P=0.049], TIMP-2[(3.46±1.50) vs (1.52±1.06), t=2.36, P=0.046], TIMP-3 [(3.96±0.50) vs (1.36±0.18), t=10.94, P<0.001], Sox-9 [(7.09±2.93) vs (3.24±0.77), t=2.84, P=0.022], vinculin [(3.42±1.69) vs (1.46±0.68), t=2.41, P=0.043], tubulin[(9.34±0.71) vs (2.35±0.80), t=14.61, P<0.001] showed higher expression in the chondrons. Compared to the chondrocytes, the MMP-1 [(1.02±0.30) vs (2.67±0.45), t=6.91, P<0.001], MMP-3 [(1.21±0.32) vs (2.52±0.79), t=3.44, P=0.009], MMP-13 [(1.23±0.34) vs (3.42±0.86), t=5.30, P=0.007], IL-1β [(1.02±0.14) vs (2.70±0.49), t=7.37, P<0.001], TNF-α [(0.99±0.08) vs (3.15±0.54), t=8.85, P<0.001] showed lower expression in the chondrons. There were no difference between chondrons and chondrocytes for Col-11, MMP-9, actin ( P>0.05). Conclusion:The gene expression of extracellular matrix components are higher and the gene expression levels of inflammatory factors and MMPs are decreased in chondrons compared with the chondrocytes, suggesting the chondrons have more multiplication potential as seeding cells for tissue-engineered cartilage.

ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） of Qingxin Lianziyin（QXLZY） benchmark samples， in order to clarify the key quality attributes and provide a reference for the quality evaluation of QXLZY. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of QXLZY benchmark samples was developed by using a YMC Hydrosphere C₁₈ column（4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-10 min， 5%-20%A； 10-20 min， 20%A； 20-25 min， 20%-24%A； 25-40 min， 24%-30%A； 40-55 min， 30%-50%A； 55-65 min， 50%-100%A； 65-75 min， 100%A； 75-75.1 min， 100%-5%A； 75.1-90 min， 5%A）， and the detection wavelength was 360 nm. Ultra-high performance liquid chromatography-linear ion trap/orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） with electrospray ionization（ESI） was used to identify the components of QXLZY benchmark samples by accurate relative molecular weight and multilevel MS fragment ion information， the detection conditions were positive and negative ion modes and data dependency scanning mode. TLC identification methods for Ophiopogonis Radix， Lycii Cortex， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma in QXLZY were established. ResultA total of 15 characteristic peaks were identified from Glycyrrhizae Radix et Rhizoma， Plantaginis Semen and Scutellariae Radix， and the relative standard deviations of the retention times of 15 characteristic peaks in 15 batches of QXLZY benchmark samples were≤3% with peak 8（baicalin） as the reference peak. A total of 100 compounds， including flavonoids， organic acids， saponins， amino acids and others， were identified in the benchmark samples by UHPLC-LTQ-Orbitrap MS. The established TLC had good separation and was suitable for the identification of Ophiopogonis Radix， Lycii Cortex， Nelumbinis Semen， Poria， Astragali Radix and Ginseng Radix et Rhizoma in QXLZY. ConclusionThe material basis of QXLZY benchmark samples is basically determined by MS designation and source attribution. The established specific chromatogram and TLC of QXLZY are simple， stable and reproducible， which can provide a reference for the development and quality control of QXLZY.

ObjectiveTo establish the specific chromatogram and thin layer chromatography（TLC） identification method of Kaixinsan（KXS） samples， in order to clarify the key quality attributes and provide reference for the quality evaluation of KXS. MethodHigh performance liquid chromatography（HPLC） specific chromatogram of KXS was developed with YMC Hydrosphere C₁₈ column（4.6 mm×250 mm， 5 μm）， the mobile phase was acetonitrile（A）-0.2% formic acid aqueous solution（B） for gradient elution（0-15 min， 2%-20%A； 15-25 min， 20%-25%A； 25-30 min， 25%-30%A； 30-45 min， 30%-31%A； 45-50 min， 31%-44%A； 50-65 min， 44%-45%A； 65-73 min， 45%-75%A； 73-95 min， 75%-100%A； 95-105 min， 100%A； 105-105.1 min， 100%-2%A； 105.1-120 min， 2%A）， the detection wavelength was 320 nm. Ultra high performance liquid chromatography-linear ion trap-electrostatic field orbitrap mass spectrometry（UHPLC-LTQ-Orbitrap MS） was used to identify the chemical components of KXS with electrospray ionization（ESI）， negative ion mode and scanning range of m/z 50-2 000. TLC identification methods for Poria and Ginseng Radix et Rhizoma in KXS were established. ResultThere were 11 common peaks in the specific chromatogram of KXS， attributed to Polygalae Radix， Poria and Acori Tatarinowii Rhizoma. Taking peak 9（α-asarone） as the reference peak， the relative standard deviations of the retention times of 15 batches of KXS samples were<0.2%. A total of 34 compounds were identified by UHPLC-LTQ-Orbitrap MS， including terpenoids， phenylpropanoids， oligosaccharides and ketones. The established TLC had good separation and was rapid， reliable， simple， feasible， suitable for the identification of Poria and Ginseng Radix et Rhizoma in KXS. ConclusionThe specific chromatogram and TLC of KXS are stable and reproducible. The material basis of KXS is basically clarified by MS， which can provide a reference for the development and quality control of KXS.