Objective To reveal the thermally induced denaturation of wild-type human γ D-crystallin(HGD)and congenital cataract-related mutant(HGD P23T),and compare the differences in the structural changes between wild-type and mutants during a heating process.Methods HGD and HGD P23T were expressed and purified.The temperature-dependent intrinsic fluorescence intensity and static light scattering intensity of the protein samples were measured to reveal the temperature-dependent folding and aggregation structural changes of HGD and HGD P23T.Results When the temperature was below 70℃,the barycentric mean of the intrinsic fluorescence of HGD and HGD P23T shifted towards a longer wavelength with increasing temperature and the fluorescence intensity de-creased indicating the unfolded protein conformations.The conformational stability of HGD P23T was weaker than that of HGD.When temperature was higher than 70℃,the static light scattering intensity increased significantly with temperature,indicating protein aggregation upon heating.Relative to the wild-type,HGD P23T showed a stronger aggregation potency.Conclusions Heating disrupts the folding conformation of Γd-crystallin,induces the unfolded protein to aggregate.The disease-associated P23T mutation significantly reduces the conformational stability of Γd-crystallin.

Objective To investigate the effect and mechanism of melatonin on the anoikis of melanoma cells.Methods The drug concentration of melatonin inhibiting melanoma cell line B16-F10 was optimized based on the effect on CCK-8 assay.An anti-anoikis of melanoma cell model was developed and divided it into four groups:The blank control group,the TrkB activator group,the melatonin group and the melatonin+TrkB activator group.Calce-in AM/EthD-1 fluorescence double staining was used to detect the anoikis of melanoma cells.Reactive oxygen spe-cies were detected using the fluorescent probe DCFH-DA.Western blot was used to detect the expression of Nrf2 protein and TrkB protein in each group.Results Melatonin significantly inhibited the proliferation of melanoma cells in a time-and dose-dependent manner with IC50 of 1×10-7 μmol/L.Its inhibitory effect was found to be related to in-duction of anoikis of melanoma cells.Melatonin could upregulate the generation of cellular reactive oxygen species(P<0.05),while addition of TrkB activator antagonized this effect.Melatonin could reduce the expression of Nrf2 protein and TrkB protein in melanoma cells(P<0.05),and the addition of TrkB activator could inhibite the effect of melatonin on the expression of Nrf2 protein and TrkB protein(P<0.05).Conclusions Melatonin can inhibit the pro-liferation of melanoma cell line B16-F10 through the mechanism of inducing anoikis.

Objective To explore the impact of change in oxygen concentration on the expression of angiopoietin-like protein 8(ANGPTL8)by endothelial cells(HPAECs)of human pulmonary artery,and the role and mecha-nism of ANGPTL8 in pulmonary hypertension(PH).Methods HPAECs were treated under hypoxic and hyperoxic conditions,and the expression level of ANGPTL8 was detected using Western blot and PCR.The changes in endo-thelial-mesenchymal transition(EndMT)and ERK signaling pathway activity were analyzed.Simultaneously,new-born rats were exposed to hyperoxia to develop a bronchopulmonary dysplasia(BPD)model.The expression of ANGPTL8 protein and changes in the ERK signaling pathway in lung tissue were observed.Results Under hypoxic condition,the protein expression of ANGPTL8 in HPAECs was significantly increased accompanied by inhibition of the ERK signaling pathway.ANGPTL8 promoted the EndMT process induced by hypoxia(P<0.05)and silencing the expression of ANGPTL8 resulted in a partial reversal of EndMT.The protein expression of ANGPTL8 was decreased in hyperoxia-exposed HPAECs and rat lung tissues accompanied by the activation of the ERK signaling pathway(P<0.05).Conclusions ANGPTL8 is highly sensitive to the change of oxygen concentration in HPAECs and closely correlated to its expression level and to the activity of ERK signaling pathway.This result suggests that ANGPTL8 may have potential regulatory effects on the development of PH.

Objective To investigate the effects of CD151 down-regulation combined with bevacizumab on colorectal cancer growth and microvessel density.Methods Human colorectal cancer cell line HT-29 and CD151--HT-29 cells strain(CD151 down-regulated HT-29 cells)were treated with bevacizumab.The cells were divided into four groups:control(HT-29)group,bevacizumab-treatment group,CD151--HT-29 group,and CD151--HT-29+bevacizumab-treatment group.Cell proliferation was observed in each group using the MTS assay.A subcutaneous xenograft model in nude mice was established,and the HT-29 control group and CD151--HT-29 group were treated with either 0.9％NaCl solution or bevacizumab.The growth of subcutaneous tumors in the four groups was ob-served,and the volume and weight of the tumors were recorded.Tumor tissues were collected for immunohistochem-ical staining of endothelial cells to assess microvessel density(MVD).Results Compared with the control group,cell proliferation was significantly reduced in the bevacizumab-treated group and CD151--HT-29 group(P<0.001).Cell proliferation in the CD151--HT-29+bevacizumab-treated group was slower than that in the single treatment groups(P<0.001).In the subcutaneous tumor model,the volume,weight,and MVD of tumors in the bevacizumab-treated and CD151--HT-29 groups were significantly reduced compared to the control group(P<0.01).In the CD151--HT-29+bevacizumab group,the tumor volume,weight,and CD34 expression were significantly lower than in the single treatment groups(P<0.01).Conclusions CD151 protein may play a role in the regulation of angiogenesis in colorectal cancer tissues and may have a synergistic effect with bevacizumab in in-hibiting microvessel formation in tumor tissues.

Objective To investigate the effect of total paeony glycoside(TPG)on cerebral ischemia-reperfusion injury(CI/RI)of rats.Methods The rats were randomly divided into sham surgery(sham)group,CI/RI model group(simple CI/RI group),positive control group(nimodipine group,5 mg/kg),low-dose TPG group(TPG-L group,27 mg/kg),a high-dose TPG group(TPG-H group,54 mg/kg)and a high-dose TPG+NOD-like receptor thermal protein domain associated protein 3(NLRP3)activator diethyl dithiocarbamate(DDC)group(TPG-H+DDC group,54 mg/kg TPG and 30 mg/kg DDC),with 18 rats in each,administered once a day for 7 consecutive days.After the administration,the neurological deficit score of the rats was evaluated.Nissl staining microscopy was applied to observe neuronal activity in brain tissue.2,3,5-triphenyltetrazolium chloride(TTC)staining microscopy was applied to detect the area of cerebral infarction in rats.The level of interleukin-1β and IL-18 in brain tissue was measured by ELISA method.Western blot was applied to detect the expression of purinergic receptor P2X ligand-gated ion channel 7(P2X7R)/NLRP3 signaling pathway related proteins and pyroptosis related proteins such as apoptosis associated speck like protein containing a CARD(ASC)and cysteine protease 1(caspase-1)proteins in brain tissue.Results Compared with the sham group,the neurological deficit score,infarct area,level of IL-1β and IL-18 in brain tissue and protein expression of P2X7R,NLRP3,ASC,and caspase-1 in brain tissue of rats in the simple CI/RI group were significantly increased(P<0.05),while the proportion of Nissl body positive cells in brain tissue was significantly reduced(P<0.05).The change in corresponding indicators of rats in the nimodipine group,TPG-L group,and TPG-H group was opposite to those in the simple CI/RI group(P<0.05).NLRP3 acti-vator DDC antagonized the inhibitory effect of TPG on cell pyroptosis in CI/RI rats.Conclusions TPG may inhibit brain injury in CI/RI rats by down-regulating the P2X7R/NLRP3 pathway.

Objective To investigate the impacts of usnea acid(UA)on the proliferation of human ovarian cancer(OC)cells.Methods Ovarian adenocarcinoma cells SKOV3 were randomly divided into control group,L-UA group,M-UA group,H-UA group(with 10,20,and 50 μmol/L UA added respectively),pcDNA3.1-NC group and pcDNA3.1-PD-1 group.RT-qPCR method was applied to detect the expression of programmed cell death 1(PD-1)and programmed cell death ligand 1(PD-L1)in SKOV3 cells.CCK-8 assay and plate method were applied to detect the effect of UA on SKOV3 cell proliferation.The effect of UA on SKOV3 cells apoptosis was examined by flow cytometry.Mouse ovarian epithelial cancer cell line ID8 cells were collected to construct an OC mouse model,and the mass and volume of OC tumors were recorded.Immuno-histochemical microscopy was applied to detect the infiltration of PD-1,PD-L1,and CD8+T cells.Results The PD-1 mRNA,PD-L1 mRNA,colony count,A450 val-ue,PCNA protein,PD-1 protein,and PD-L1 protein in the L-UA,M-UA,and H-UA groups were lower than those in the control group,the apoptosis rate and Bax protein were higher in the control group(P<0.05).Com-pared with the H-UA group and the pcDNA3.1-NC group,the PD-1 mRNA,PD-L1 mRNA,colony count,A450 value,PCNA protein,PD-1 protein,and PD-L1 protein in the pcDNA-3.1-PD-1 group increased and the apoptosis rate and Bax protein decreased(P<0.05).The quality,volume,positivity rate of PD-1and PD-L1 of OC tumors in the UA group was lower than that in the control group,the counting number of CD8+T cell infiltration was higher than control group(P<0.05).Conclusions UA may inhibit progression of OC cell line SKOV3 by regula-ting cell proliferation,apoptosis,and immune escape.

Objective To investigate the effects of curcumin on the proliferation,migration,invasion,and apoptosis of pheochromocytomacell line PC12.Methods PC12 cells were incubated with different concentrations of curcumin.Cell proliferation was assessed using the CCK-8 assay to determine the IC50.The scratch assay was used to evaluate cell migration and Transwell chambers were employed to assess cell invasiveness.Flow cytometry was used to analyze apoptosis.qPCR was conducted to measure the mRNA expression of pro-apoptotic(Bax)and anti-apoptotic(Bcl-2)genes,and Western blot was performed to detect Bax and Bcl-2 protein expressions.Results Curcumin(10-80 μmol/L)inhibited PC12 cell proliferation in a concentration-dependent manner,with an IC50 as 29 μmol/L.Curcumin also suppressed PC12 cell migration in a concentration-dependent mode;the migration rate decreased from 66％in the control group down to 51％,5％,and 0.5％in the 10,20,and 30 μmol/L curcumin groups,respectively.Curcumin at concentrations of 20-30 μmol/L significantly reduced PC12 cell invasiveness(P<0.000 1).Moreover,curcumin significantly promoted PC12 cell apoptosis;the percentage of apoptotic cells increased by 2.25％,18.53％,and 26.89％in the 10,20,and 30 μmol/L curcumin groups as compared to those of control group,respectively.Curcumin treatment resulted in a significant up-regulation of Bax mRNA and protein expression,and a significant down-regulation of Bcl-2 mRNA and protein expression(P<0.05).Conclusions Curcumin may significantly inhibit the proliferation,migration,and invasion of PC12 cells and arouse cell apopto-sis.Its pro-apoptotic effect may be associated with alterations in the expression of Bax and Bcl-2 genes.

Objective To construct an injectable hydrogel(IH)entrapment system of bone marrow mesenchymal stem cells(BM-MSCs)and to explore its effect of promoting articular cartilage repair and underlying mechanism.Methods The primary BM-MSCs of mice were cultured by whole bone marrow adherent method.The differentiation potential was identified by alizarin red(ARS),oil red O(ORO)and Alcian blue(AB)staining,and the expres-sion of CD44,CD90,CD105,CD34 and CD45 on the surface was detected by flow cytometry.BM-MSCs loaded with IH(BM-MSC-IH)was prepared,and the rheological properties of BM-MSC-IH were tested by rheological test.The model of full-thickness injury of knee cartilage in mice was established,and the mice were randomly divided into normal saline group,BM-MSC group and BM-MSC-IH group with 8 in each.The mice were injected with BM-MSC-IH 0.2 mL,cell suspension 0.2 mL and 0.9％NaCl solidion,independently.After 6 and 12 weeks of in-jection,the repair of knee cartilage in mice was observed by safranin-fast green staining,and the expression of typeⅡ collagen in knee joint was examined by immunohistochemical staining microscopy.Results The primary cultured cells showed positive expression of CD44,CD90 and CD105,but negative expression of CD34 and CD45,and had the potential of osteogenic,adipogenic and chondrogenic differentiation.The constructed BM-MSC-IH sys-tem was solid colloid,and the rheological properties were consistent with the basic characteristics of hydrogel.BM-MSC-IH effectively promoted the regeneration of mouse knee cartilage and increase the local expression of typeⅡ collagen.Conclusions Bone marrow-mesenchymal stem cells embedded with injectable hydrogel effectively pro-mote the repair of injured cartilage and the underlying mechanism is potentially promoting the differentiation of chondrocytes and the expression of type Ⅱ collagen.Its effect is better than application of BM-MSCs alone.

Objective To explore the effect of nuclear respiratory factor 1(NRF1)activation of ATP binding cas-sette transporter C1(ABCC1)transcription on cisplatin resistance in lung adenocarcinoma cells and its potential mechanisms.Methods The expression levels of ABCC1 in lung adenocarcinoma tumor tissues were analyzed by on-cogenomic datasets.Cells were grouped into:sh-NC,sh-ABCC1,sh-NC+oe-NC,sh-NRF1+oe-NC and sh-NRF1+oe-ABCC1.Real-time polymerase chain reaction(RT-qPCR)was used to detect the expression of ABCC1 in lung adenocarcinoma cells.Cisplatin-resistant lung adenocarcinoma cell strains were constructed to detect the expression level of ABCC1.Cell proliferation,migration and invasion were assessed by colony formation and Transwell assay.The IC50 values of drug-resistant lung adenocarcinoma cells treated with different doses(0,0.001,0.002,0.004,0.008,0.016 and 0.032 mg/mL)of cisplatin were detected by CCK-8 assay.Western blot was used to detect the protein expression of epithelial-mesenchymal transition markers(E-cadherin,N-cadherin).Dual luciferase and ChIP assays were performed to verify the binding relationship between NRF1 and ABCC1.Results ABCC1 was highly expressed in lung adenocarcinoma tumor tissues and cells.Compared with cisplatin-sensitive lung adenocarci-noma cells,ABCC1 was highly expressed in cisplatin-resistant lung adenocarcinoma cells,and knockdown of ABCC1 significantly inhibited cell proliferation,migration and invasion,and increased the expression of E-cadherin(P<0.05)and decreased the expression of N-cadherin(P<0.05).Knockdown of ABCC1 significantly increased the sensitivity of lung adenocarcinoma cells to cisplatin(P<0.05).In addition,dual luciferase and ChIP experi-ments confirmed the binding relationship between NRF1 and ABCC1 promoter de-binding,and NRF1 could activate the transcription of ABCC1.Knockdown of NRF1 attenuated the inhibitory effect of over-expressed ABCC1 on the proliferation,migration,invasion and cisplatin-resistance of lung adenocarcinoma cells(P<0.05).Conclusions This study revealed the effect of the NRF1/ABCC1 axis enhancement is a potential strategy to overcome the barrier of cisplatin-resistance in chemotherapy of lung adenocarcinoma.

Objective To analyze the impact of low-dose esketamine combined with sufentanil on peri-operative in-flammation and immune function of elderly patients undergoing total hip arthroplasty(THA).Methods From January 2019 to January 2022,120 elderly patients who underwent THA under combined spinal-epidural an-esthesia and received patient-controlled intravenous analgesia(PCIA)were included.The patients were divided into a control group(analgesic drugs:sufentanil+granisetron)and an observation group(analgesic drugs:esket-amine+sufentanil+granisetron).Pain intensity was assessed using the Visual Analog Scale(VAS).Serum level of interleukin(IL)-6,tumor necrosis factor(TNF)-α and C-reactive protein(CRP)was measured by ELISA.CD4+and CD8+cells were detected using a CytoFLEX flow cytometer,and the CD4+/CD8+ratio was calculated.The inci-dence of adverse reactions in both groups was recorded and compared.Results In resting state,the post-surgical VAS scores in the observation group patients were significantly lower than those in control group at 48 and 72 h.In the active state,the VAS scores recorded for the observation group following surgery remained notably lower than those of the control group at 24,48,72 h(P<0.05).Compared with 24 h before surgery in the same group,the level of IL-6,TNF-α,and CRP in the control group significantly increased at 24,48,and 72 h after surgery,while in the observation group,they increased at 24 and 48 h after surgery and returned to pre-operative level at 72 h(P<0.05).The observation group exhibited significantly lower level of IL-6,TNF-αand CRP as compared to the control group over the 24,48,72 h following surgery(P<0.05).Compared with 24 h before surgery in the same group,CD8+(％)increased,while CD4+(％)and CD4+/CD8+decreased in the observation group at 24,48,and 72 h after surgery.However,72 h after surgery,these values returned to the level before operation.Twenty four,48,and 72 h after sur-gery,the observation group displayed notably elevated level of CD8+(％),CD4+(％)and CD4+/CD8+as compared to the control group(P<0.05).No significant variation in the occurrence of adverse reactions was observed between the two groups.Conclusions Low-dose esketamine combined with sufentanil can reduce peri-operative inflammation in elderly patients undergoing THA with minimal impact on immune function.