OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

AIM:To observe the role of enoyl-coenzyme A hydratase/L-3-hydroxyacyl-coenzyme A dehydroge-nase(Ehhadh)in renal tubulointerstitial inflammation in high-fat diet(HFD)fed mice,and to explore its molecular mecha-nism.METHODS:Twenty-four C57BL/6N mice were randomly divided into 4 groups:standard diet(SD)group,HFD group,HFD with Ehhadh overexpression(HFD+Ehh)group and HFD with blank vector(HFD+Vec)group.Each group consisted of 6 mice.The HFD mice were fed with a diet containing 60%fat,20%protein,and 20%carbohydrates for 16 weeks.Briefly,at the end of 8 weeks,the mice in HFD+Ehh or HFD+Vec group were injected with adeno-associated virus 9(AAV9)-Ehhadh or AAV9-vector via the tail vein,and then continued another 8-week HFD feeding.At the end of the experiments,the renal function and morphological changes were observed.The protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),caspase-1 p10,interleukin-1β(IL-1β)and IL-18 in the kidney were detected by Western blot and immunohistochemistry.Immunofluorescence staining was used to detect the colocalization of Ehhadh and peroxisomal biogenesis factor 14(Pex14).ELISA was used to detect the content of IL-1β and IL-18 in the urine.Lipid droplet formation in renal tissues was detected by Nile red staining.Absolute quantitative lip-idomic analysis were used to detect the differential lipid species in renal cortices of the mice in SD,HFD and HFD+Ehh groups.RESULTS:Compared with SD group,the expression of Ehhadh protein was significantly decreased in the peroxi-some of renal tubular epithelium cells in HFD-fed mice(P<0.01).Overexpression of Ehhadh significantly improved renal function(P<0.01)and alleviated the morphological changes of renal tubular epithelial cells in HFD group.Moreover,it significantly inhibited the expression of inflammatory cytokines IL-1β and IL-18 and macrophage infiltration in renal tu-bule interstitium of HFD-fed mice(P<0.01).At the same time,Ehhadh overexpression inhibited HFD-induced NLRP3 inflammasome activation(P<0.01).It also attenuated lipid deposition in renal tubular epithelium cells(P<0.01)and promoted the β-oxidation of long-chain fatty acid such as cholesterol and phospholipids in peroxisomes.CONCLUSION:The Ehhadh inhibits tubulointerstitial inflammation by promoting long-chain fatty acid β-oxidation in peroxisomes and in-hibiting the activation of NLRP3 inflammasome in HFD-fed mice.

OBJECTIVE To screen and obtain nanobodies with neutralizing activity against granulo-cyte-macrophage colony stimulating factor(GM-CSF)to contribute to investigations into the mecha-nism of inflammation interventions and the development new drugs.METHODS Recombinant human GM-CSF was subcutaneously injected to immunize camels.Peripheral blood was collected after five immunizations,and mononuclear cells were isolated.Total mRNA was extracted,and the variable domains of the heavy chain of heavy-chain antibody(VHH,also called nanobody)genes were obtained by PCR amplification after reverse transcription.The genes were cloned into the pADSCFV-S phage display vector and electrotransformed into TG1 competent cells to construct a nanobody immune library that was screened with recombinant human GM-CSF immobilized on a solid phase.The VHH genes specifi-cally binding to human GM-CSF were cloned into the pABG eukaryotic expression vector before VHH-Fc samples were prepared by using the human embryonic kidney 293 fibroblast expression system.The binding activity of candidate VHH-Fc molecules to GM-CSF was investigated through ELISA response curves,and binding colorimetric values with different antigens were detected to determine their specificity.The binding affinity between VHH-Fc candidates and GM-CSF was measured using biolayer interferom-etry(BLI).The inhibition rate curve of VHH-Fc candidates on GM-CSF was detected through cell prolif-eration assays to determine its neutralization activity.The Uncle system was used to investigate its thermal stability.100 μg of VHH-Fc was injected into mice via the tail vein,and the serum concentration of VHH-Fc was quantitatively detected by ELISA to examine its pharmacokinetic curve in mice.RESULTS The camel serum titer of anti-human GM-CSF antibodies was higher than 1:800 000 after the fifth immuni-zation,and the capacity of the constructed nanobody library was about 5.55×107.Following the screening process,five candidate VHH-Fc molecules specifically binding to human GM-CSF were obtained.Among these,22N10 effectively neutralized the cell proliferation-promoting activity of GM-CSF(the IC50 value was 17.23 nmol·L-1).Subsequent studies revealed that 22N10 interacted with human GM-CSF with an affinity of 1.97×10-8 mol·L-1,blocked the binding of GM-CSF to its receptor CSF2Rα,exhibited good thermal stability(Tm1=59.2℃),and showed favorable metabolic characteristics in mice.CONCLU-SION A new candidate nanobody molecule 22N10 targeting human GM-CSF is obtained which is expected to facilitate the drug development and mechanism investigations.

AIM:To observe the role of enoyl-coenzyme A hydratase/L-3-hydroxyacyl-coenzyme A dehydroge-nase(Ehhadh)in renal tubulointerstitial inflammation in high-fat diet(HFD)fed mice,and to explore its molecular mecha-nism.METHODS:Twenty-four C57BL/6N mice were randomly divided into 4 groups:standard diet(SD)group,HFD group,HFD with Ehhadh overexpression(HFD+Ehh)group and HFD with blank vector(HFD+Vec)group.Each group consisted of 6 mice.The HFD mice were fed with a diet containing 60%fat,20%protein,and 20%carbohydrates for 16 weeks.Briefly,at the end of 8 weeks,the mice in HFD+Ehh or HFD+Vec group were injected with adeno-associated virus 9(AAV9)-Ehhadh or AAV9-vector via the tail vein,and then continued another 8-week HFD feeding.At the end of the experiments,the renal function and morphological changes were observed.The protein expression levels of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),caspase-1 p10,interleukin-1β(IL-1β)and IL-18 in the kidney were detected by Western blot and immunohistochemistry.Immunofluorescence staining was used to detect the colocalization of Ehhadh and peroxisomal biogenesis factor 14(Pex14).ELISA was used to detect the content of IL-1β and IL-18 in the urine.Lipid droplet formation in renal tissues was detected by Nile red staining.Absolute quantitative lip-idomic analysis were used to detect the differential lipid species in renal cortices of the mice in SD,HFD and HFD+Ehh groups.RESULTS:Compared with SD group,the expression of Ehhadh protein was significantly decreased in the peroxi-some of renal tubular epithelium cells in HFD-fed mice(P<0.01).Overexpression of Ehhadh significantly improved renal function(P<0.01)and alleviated the morphological changes of renal tubular epithelial cells in HFD group.Moreover,it significantly inhibited the expression of inflammatory cytokines IL-1β and IL-18 and macrophage infiltration in renal tu-bule interstitium of HFD-fed mice(P<0.01).At the same time,Ehhadh overexpression inhibited HFD-induced NLRP3 inflammasome activation(P<0.01).It also attenuated lipid deposition in renal tubular epithelium cells(P<0.01)and promoted the β-oxidation of long-chain fatty acid such as cholesterol and phospholipids in peroxisomes.CONCLUSION:The Ehhadh inhibits tubulointerstitial inflammation by promoting long-chain fatty acid β-oxidation in peroxisomes and in-hibiting the activation of NLRP3 inflammasome in HFD-fed mice.

Purpose To investigate the protective effect and mechanism of A-485 on renal tubular injury in diabetic mice.Methods Eighteen male C57BL/6J mice were randomly divided into three groups:Control group,diabetic kidney dis-ease(DKD)group and A-485 treatment group.The DKD mice model was established by feeding high-fat diet for 8 weeks and intraperitoneal injection of streptozotocin for 5 days.Subsequent-ly,the A-485 treatment group was given A-485(10 mg/kg/day)by intraperitoneal injection every other day for 4 weeks.After treatment,the renal function,P300 enzyme activity and lipid deposition in renal tissue were measured.Western blot a-nalysis was performed to detect SREBP-1,FASN,ACC,ChREBP,P300,CBP,H3K18ac and H3K27ac protein levels.Results Compared with control mice,the levels of FBG,BUN,Scr and UAE were significantly increased in diabetic mice(FBG:2.52 times,BUN:2.89 times,Scr:2.13 times,UAE:4.21 times),while diabetic mice treatment with A-485 exhibi-ted a remarkable decrease on BUN,Scr and UAE(BUN:0.511 times,Scr:0.636 times,UAE:0.574 times,P＜0.01).The results of the transmission electron microscopy and oil red O stai-ning showed that A-485 treatment prevents lipid droplets forma-tion and up-regulation of SREBP-1,FASN,ACC and ChREBP in renal tubular cells of diabetic mice(SREBP-1:0.544 times,FASN:0.449 times,ACC:0.306 times,ChREBP:0.317 times,P＜0.01).Furthermore,A-485 intervention downregu-lated the enzyme activity of P300(0.546 times)and suppressed the expression of H3K18ac(0.337 times)and H3K27ac(0.308 times,P＜0.01).Conclusion A-485 can significant-ly improve renal lipid metabolic disorder in diabetic mice,which may be achieved by inhibiting p300-induced H3K18ac and H3K27ac.

Objective:To observe the corneal morphology and visual quality after transepithelial photorefractive keratectomy (Trans-PRK) with smart pulse technique (SPT) and 1 050 Hz cutting frequency in the correction of myopia and astigmatism.Methods:A self-controlled case series study was conducted.Sixty five eyes of 33 patients who underwent Trans-PRK surgery in Ineye Hospital of Chengdu University of TCM from July 2017 to June 2018 were followed up for 6 months.The uncorrected visual acuity (UCVA) converted to logarithm of the minimum angle of resolution (LogMAR) unit, best corrected visual acuity (BCVA) (LogMAR), and spherical equivalent (SE) of the subjects were recorded.The anterior corneal surface symmetry index (SI), the anterior corneal surface Q value in the range of 6, 7, 8, and 9 mm diameter, the spherical aberration, coma, trefoil and total higher-order aberration of the anterior corneal surface, the strehl ratio (SR), and the modulation transfer function (MTF) of 10, 20, 30, and 40 c/d in the horizontal and vertical meridian directions before and after surgery were measured with Sirius corneal topography analyzer.The differences of each index among different time points were compared, and the correlation between indexes was analyzed by Pearson correlation analysis.This study followed the Declaration of Helsinki.The study protocol was approved by the Medical Ethics Committee of Ineye Hospital of Chengdu University of TCM (No.2020yh-004). All patients signed the informed consent form before surgery.Results:The average preoperative BCVA and SE were -0.09±0.06 and (-4.24±1.24)D.The mean UCVA and SE at 7 days, 1, 3 and 6 months postoperatively were -0.10±0.08 and (0.03±0.63)D, -0.12±0.06 and (0.08±0.53)D, 1.16±0.06 and (0.02±0.79)D, -0.18±0.05 and (0.08±0.37)D, respectively.The SI at different time points after the surgery were significantly higher than that before operation (all at P<0.05). At 1, 3 and 6 months after surgery, the Q value of anterior corneal surface in different diameter ranges increased from negative to positive, showing statistically significant differences (all at P<0.05). At each time point after surgery, the trefoil and total higher-order aberrations of the anterior corneal surface increased to varying degrees.Coma at 7 days and 6 months after surgery were significantly higher than that before surgery, and spherical aberration at 3 and 6 months after surgery were significantly higher than that before surgery (all at P<0.05). The SR values at 3 and 6 months after operation were significantly higher than that before operation (all at P<0.05). At 6 months after operation, the MTF values at different spatial frequencies of the horizontal meridian and the MTF values at 30 and 40c/d spatial frequencies of the vertical meridian were lower than those before operation, and the differences were statistically significant (all at P<0.05). The correlation analysis showed that the Q value of different diameter ranges was positively correlated with spherical aberration ( r=0.798-0.925, P<0.05), total higher-order aberration ( r=0.596-0.630, P<0.05), SI ( r=0.235-0.303, P<0.05) and corneal ablation depth ( r=0.583-0.659, P<0.05) at 6 months after surgery.SI was positively correlated with spherical aberration ( r=0.307, P<0.05), coma ( r=0.424, P<0.05), total higher-order aberration ( r=0.300, P<0.05), corneal ablation depth ( r=0.227, P<0.05), and eccentric cutting amount ( r=0.281, P<0.05). There was no correlation between SR and aberration, corneal ablation depth, eccentric cutting amount, etc.(all at P≥0.05). Conclusions:Trans-PRK using SPT to correct myopic astigmatism can improve vision, stabilize diopter, enhance retinal imaging quality, increase the asymmetry of the anterior corneal surface, and introduce different degrees of higher-order aberrations.

ObjectiveTo investigate the effects of the inoculation of potassium-solubilizing bacteria on the rhizosphere soil microenvironment of Paris polyphylla var. yunnanensis. MethodThe effects of different potassium-solubilizing bacteria on the physical and chemical properties， microbial community structure， and soil enzyme activity in the rhizosphere soil of P. polyphylla var. yunnanensis were investigated by pot planting at room temperature. The correlation of various indexes was analyzed. ResultThe inoculation with different potassium-solubilizing bacteria could significantly affect the physical and chemical properties of rhizosphere soil of P. polyphylla var. yunnanensis. The mass fractions of available nitrogen， available phosphorus， and available potassium were 24.5-90.5 mg·kg^-1， 2.53-25.9 mg·kg^-1， and 132-312 mg·kg^-1， respectively， and the soil pH was 7.08-7.75， which were in line with the optimal ranges of P. polyphylla var. yunnanensis planting. The inoculation of different potassium-solubilizing bacteria could affect the number of bacteria， actinomycetes， and fungi in rhizosphere soil to varying degrees. The transformation of soil from "fungal type" to "bacterial type" marks the improvement of soil fertility. It also affected the enzyme activity of rhizosphere soil， and the activities of neutral phosphatase， protease， and polyphenol oxidase showed an increasing trend. The correlation analysis showed that the number of bacteria was negatively correlated with the number of fungi （r=-0.856， P<0.01）， positively correlated with the number of actinomycetes， the content of available nitrogen and available potassium， and negatively correlated with soil pH. ConclusionThe inoculation of potassium-solubilizing bacteria can effectively improve the content of available potassium， available nitrogen， available phosphorus， and other nutrients in the rhizosphere soil of P. polyphylla var. yunnanensis， improve soil fertility， alleviate the continuous cropping obstacles of P. polyphylla var. yunnanensis， and lay a theoretical foundation for the green and sustainable development of P. polyphylla var. yunnanensis.

Bone defects have always been an important cause of threat to human health, and artificial biomimetic bone repair replacement materials are currently one of the most effective and feasible solution approaches to treat bone damage. To develop artificial bone biomimetic materials, an in vitro biomimetic mineralization system must be constructed first to study in vitro biomimetic mineralization mechanism of natural bone matrix. Collagen is a template for mineralization, and its properties such as crosslinking degree, diameter, osmotic pressure, and surface charge can all directly affect mineralization progress. The biochemical and mechanical environments in which mineralization occurs are also quite distinct in their effects on mineralization process, particularly noncollagenous proteins and fluid shear stress (FSS). FSS is considered to be the main mechanical stimulation of bone tissues in micro-environment, which is of great significance to bone growth, repair and health maintenance. FSS at different levels and loading regimes has significant effects on transformation of amorphous calcium phosphate to bone apatite, self-assembly and directional alignment of collagen fibrils, and formation of hierarchical intrafibrillar mineralization. In this paper, the factors affecting collagen mineralization and their mechanism were summarized, with focus on regulation of FSS on collagen mineralization, and development direction in future was also prospected.

Objective:To research the metabolomic alterations of human lung bronchial epithelial cells infected with human rhinovirus 1B (HRV1B).Methods:Untargeted metabolomics was used to determine the metabolomic alterations in human lung bronchial epithelial cells (BEAS-2B) 6 h, 12 h and during the dynamic process (6 h∶12 h) after HRV1B infection.Results:A total of 93 differentially significant metabolites (DSMs) (47 DSMs were up-regulated and 46 DSMs were down-regulated) and 88 DSMs (37 DSMs were up-regulated and 51 DSMs were down-regulated) at post infection of HRV1B in BEAS-2B at 6 h or 12 h, respectively. A total of 30 DSMs (12 DSMs were up-regulated and 18 DSMs were down-regulated) in a dynamic process (6 h∶12 h) after HRV1B infection. Unknown metabolites took up most proportions. The trends of fatty acid, lipid, amino acid, nucleotide and carbohydrate were increased along with the prolonging of HRV1B infection. DSMs such as Diisononyl phthalate was co-detected DSMs among three groups.Conclusions:Metabolites such as fatty acid, lipid, amino acid, nucleotide and carbohydrate of BEAS-2B cells are changed induced by HRV1B infection.

Objective:To compare the clinical outcome of transepithelial photorefractive keratectomy (TransPRK) using 1 050 Hz ablation frequency and intelligent pulse technique (SPT) and small incision lenticule extraction (SMILE) for myopia and astigmatism.Methods:A cohort study was performed.Eighty-five eyes of 43 patients who received TransPRK for myopia and 85 eyes of 46 patients who received SMILE for myopia in the Ineye Hospital of Chengdu University of TCM were enrolled from August 2017 to April 2018.The follow-up duration was 6 months.The changes of visual acuity and diopter were observed and compared before and after operation, and the predictability, stability, safety, effectiveness and long-term vision were compared between the different surgeries.This study complied with the Declaration of Helsinki and the study protocol was approved by the Ethics Committee of Ineye Hospital of Chengdu University of TCM.Results:The refractive power tended to be emmetropic and relatively stable in the TransPRK group, and the refraction varied from mild hyperopia to emmetropic gradually during 6 months after SMILE.There was no significant difference in the spherical equivalent (SE) between the two groups before and after operation (all at P>0.05). No significant difference was found in mean validity index between the two groups at 6 months after surgery (1.189±0.248 vs.1.120±0.205; t=1.862, P=0.065). The uncorrected visual acuity (UCVA) in the SMILE group was significantly higher than that in the TransPRK group at 7 days and 1 month after surgery ( P<0.05), and there was no significant difference in UCVA between the two groups at 3 months and 6 months after surgery ( P>0.05). The safety index at 6 months after surgery in the TransPRK group was 1.209±0.222, which was significantly higher than 1.143±0.178 in the SMILE group, with a significant difference between the two groups ( t=2.024, P=0.045). Conclusions:The predictability, stability, safety, effectiveness and long-term vision are good after TransPRK with SPT and SMILE for myopia and astigmatism.The safety index is better in TransPRK compared with SMILE, and the restoration of vision is faster after SMILE than that after TransPRK.