Objective: To establish a method for determining the molecular weight and molecular weight distribution of Poly(Lactide-co-Glycolide Acid) (PLGA) using Size Exclusion Chromatography-Refractive Index-Multiangle Laser Light Scattering (SEC-RI-MALLS) and Size Exclusion Chromatography-Refractive Index (SEC-RID), and to compare the results obtained from these two methods.
Methods: For SEC-RI-MALLS, tetrahydrofuran was used as the mobile phase, Shodex GPC KF-803L was employed as the chromatographic column with a flow rate of 1 mL·min^-1, column temperature at 30 ℃, and an injection volume of 100 μL. For SEC-RID, tetrahydrofuran was also used as the mobile phase, Agilent PLgel 5 μm MIXD-D was used as the chromatographic column with a flow rate of 1 mL·min^-1, column temperature at 30 ℃, differential detector temperature at 35 ℃, and an injection volume of 20 μL. The molecular weight and molecular weight distribution were calculated using Agilent’s GPC software. The newly established methods were validated methodologically, and the molecular weight and molecular weight distribution of 13 batches of samples were determined.
Results: The precision, accuracy, stability, and repeatability tests for SEC-RI-MALLS showed RSD values of 1.35%, 1.58%, 1.53%, and 1.26%, respectively. The SEC-RID method exhibited good linearity (r=0.999 9), with RSD values for precision, accuracy, stability, and repeatability tests (n=6) of 2.05%, 1.62%, 1.30%, and 2.97%, respectively. The results obtained from SEC-RI-MALLS were lower than those from SEC-RID, and the molecular weight distribution coefficient was smaller, but the results from the paired T-test performed with the value measured by SEC-RID method and the value measured by SEC-RI-MALLS method multiplied a conversion coefficient of 1.5 showed no significant difference between the two methods.
Conclusion: Both methods are stable and reliable, and can be used for the determination of PLGA molecular weight and molecular weight distribution based on the specific situations.

OBJECTIVE: Based on the PTEN-induced hypothetical kinase 1 (PINK1)/Parkin pathway, the effect of acupuncture pretreatment on the expression of mitochondrial autophagy-related proteins in gastrocnemius muscle tissue of rats with exercise-induced muscle damage (EIMD) was observed, and the underlying mechanism of acupuncture pretreatment for the prevention and treatment of EIMD was explored. METHODS: Of 88 SD male rats, aged 6 weeks, 8 rats were randomly selected as a blank group, and the remaining 80 rats were randomized into a model group and an acupuncture pretreatment group, with 40 rats in each group. Either the model group or the acupuncture pretreatment group was subdivided randomly into 5 subgroups with 8 rats in each one according to the time points of sample collection, 0 h, 12 h, 24 h, 48 h and 72 h after modeling. An intermittent downhill running centrifugal exercise was carried out on an animal experimental treadmill to establish the EIMD model in the model group and the acupuncture pretreatment group. The rats in the acupuncture pretreatment group received acupuncture at "Guanyuan" (CV6) and bilateral "Zusanli" (ST36), once a day for 20 min each time, for 7 consecutive days before EIMD model preparation. Transmission electron microscopy was used to observe the ultrastructure of gastrocnemius muscle tissue in each group. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) and catalase (CAT) in serum were detected by ELISA. Western blot was used to detect the protein expression of PINK1, Parkin, sequestosome 1 (p62) and microtubule-associated protein light chain 3B (LC3B) in rat gastrocnemius muscle tissue. Real-time PCR was adopted to detect the mRNA expression of PINK1, Parkin, p62 and LC3B in rat gastrocnemius muscle tissue. RESULTS: Compared with the blank group, the mitochondria of gastrocnemius muscles showed obvious swelling in the 0 h, 12 h, 24 h, and 48 h model subgroups , autophagosomes were formed in the 12 h and 24 h model subgroups, and the mitochondrial morphology returned to normal gradually in the 72 h model subgroup. The serum MDA contents of rats in 5 model subgroups increased (P<0.01, P<0.05). The contents of SOD and CAT in the subgroups of 0 h, 12 h, 24 h and 48 h decreased (P<0.05, P<0.01). The protein and mRNA expression levels of PINK1, Parkin and LC3B in gastrocnemius muscle tissue of rats in 0 h, 12 h and 24 h subgroups were elevated (P<0.01); and the protein and mRNA expression levels of p62 in the 0 h, 12 h, 24 h and 48 h subgroups were reduced (P<0.01, P<0.05). Compared with the model subgroup at the same time point, the myofibril damage and the degree of mitochondrial swelling were mild in each acupuncture pretreatment subgroup, and the numbers of autophagosomes were fewer. The contents of MDA in the acupuncture pretreatment subgroups decreased at 0 h, 12 h, 24 h, and 48 h (P<0.05, P<0.01). The contents of SOD and CAT in the 12 h acupuncture pretreatment subgroup increased (P<0.05, P<0.01). The protein and mRNA expression levels of PINK1 and Parkin in the 0 h, 12 h, and 24 h acupuncture pretreatment subgroups decreased (P<0.01, P<0.05). The protein and mRNA expression levels of LC3B in the 12 h acupuncture pretreatment subgroup decreased (P<0.01), and that of p62 in the 0 h and 24 h acupuncture pretreatment subgroups increased (P<0.01, P<0.05). CONCLUSION The intermittent downhill running centrifugal exercise induces the excessive mitochondrial autophagy. Acupuncture pretreatment may attenuate EIMD, and the underlying mechanism is related to the regulation of PINK1/Parkin signaling pathway expression, reducing oxidative stress damage in skeletal muscle cells, and inhibiting mitochondrial autophagy overactivation.
Animals ; Ubiquitin-Protein Ligases/genetics* ; Male ; Rats ; Acupuncture Therapy ; Protein Kinases/genetics* ; Rats, Sprague-Dawley ; Mitophagy ; Humans ; Muscle, Skeletal/metabolism* ; Physical Conditioning, Animal ; Muscular Diseases/physiopathology* ; Signal Transduction

Triple-negative breast cancer (TNBC) is a highly aggressive malignancy predominantly managed via chemotherapy. Our clinical sample analysis revealed a significant correlation between elevated CD24 expression in TNBC tumor cells and patient survival rates. We developed a novel antibody-drug conjugate (ADC), named HN03, consisting of an antibody with engineered cysteines for site-specific conjugation with a low toxic nitric oxide (NO) precursor as its payload through a novel Pt(IV)-mediated bioorthogonal self-cleavable linker. HN03 specifically targets tumor cells expressing high levels of CD24, concurrently generating cisplatin and releasing NO upon activation. HN03 also exhibited potent in vitro and in vivo antitumor activity. It significantly reduced tumor growth at various doses, prevented tumor metastasis, with markedly lower toxicity than traditional chemotherapy agents. We found that a key mechanism of its action involved inducing apoptosis and endoplasmic reticulum stress, substantially decreasing the number of M2-type macrophages. Overall, HN03 stands out as a promising therapeutic option for TNBC, offering a targeted treatment with reduced side effects and the potential for improved outcomes. Furthermore, using Pt(IV) in the linker and an NO precursor as the payload enhances the versatility of the Antibody-NO donor Conjugate (ANC), offering new avenues for the design of the next generation of ADCs.

In this study, we constructed a series of recombinant Fc variants of immunoglobulin G4 (IgG4), screened the fragment crystallizable (Fc) variants with significantly prolonged serum half-lives, and analyzed the relationship between mutation site and half-life, aiming to provide a theoretical basis for the development of IgG4 antibodies and Fc fusion protein-based drugs. Nine gene sites were selected for mutation, and different mutation sites were combined. The variant expression plasmids pET24b-Fc were constructed by molecular cloning and point mutation. The plasmids were transformed into Escherichia coli BL21(DE3) for the expression of different recombinant proteins of Fc. Fc2 and Fc3 variants had slightly lower recombinant protein yields, and the expression of other variants was not affected. The toxicity of different Fc variants was determined by cell counting kit-8 (CCK-8) and calcein acetoxymethyl ester/ propidium iodide (calcein AM/PI) in vitro and enzyme-linked immuno sorbent assay (ELISA) in vivo. The results showed that the recombinant Fc variants had good biocompatibility and safety. Finally, the Fc variants were labeled with fluorescent markers, and the effects of different mutations on their serum half-lives were investigated by in vivo experiments. The Fc5 variant with prolonged serum half-life was successfully screened out, which provided a theoretical and practical basis for the optimal design of IgG4 subtype antibody and Fc fusion protein drugs.
Immunoglobulin G/blood* ; Immunoglobulin Fc Fragments/biosynthesis* ; Half-Life ; Animals ; Escherichia coli/metabolism* ; Humans ; Recombinant Fusion Proteins/biosynthesis* ; Recombinant Proteins/biosynthesis* ; Mice

Poly(lactic-co-glycolide)(PLGA)is a key excipient in long-acting sustained-release preparations,and its degradation properties directly affect the drug release behavior.In this study,PLGA microspheres were prepared by microfluidic techniques,and the morphology changes of the microspheres were observed by scanning electron microscopy(SEM).In alkaline environment,due to the accelerated hydrolysis of ester bonds,the surface of the microspheres was rapidly dissolved and eroded,and the degradation rate was significantly higher than that in acidic environment.High temperature accelerated the degradation of PLGA microspheres.Under neutral and alkaline conditions,the microspheres showed aggregation and adhesion.Under acidic conditions,the microspheres gradually decomposed into irregular fragments.The high ionic strength further promoted the surface corrosion of the microspheres,especially under extreme pH conditions.Simultaneously,PLGA microspheres encapsulating coumarin were prepared to simulate the microsphere formulation.The release rate of coumarin after degradation of the microspheres under different conditions was observed by measuring the absorbance with ultraviolet-visible spectrophotometry.The results were consistent with those of the blank microspheres.This study revealed that the degradation of PLGA microspheres was significantly pH-dependent,temperature sensitive and ion strength responsive.These findings not only helped to understand and optimize the long-term stability and controlled release performance of drug-carrying microspheres,but also provided a theoretical basis for further improvement of PLGA-based drug carrier design.

Objective To explore the relationship between serum chitosinase 3-like protein 1(CHI3L1)and Syndecan-1(SDC1)levels and bone metabolism in elderly patients with type 2 diabetes mellitus and their predictive efficacy on osteoporosis.Methods A total of 412 elderly patients with type 2 diabetes admitted to this hospital from May 2019 to May 2023 were included in this study,and were divided into normal bone mass group(n=151),reduced bone mass group(n=138)and osteoporosis group(n=123)according to the iffer-ences in bone mineral density.Serum CHI3L1 and SDC1 levels were detected by enzyme-linked immunosor-bent assay,and serum levels of type 1 collagen cross-linked carboxyl terminal peptide(CTX),25-hydroxyvita-min D[25-(OH)D],osteocalcin(OC),and type 1 procollagen N-terminal propeptide(P1NP)were deter-mined by automatic chemiluminescence immunoassay.Pearson correlation analysis was used to investigate the relationship between serum CHI3L1,SDC1 and bone metabolism in elderly patients with type 2 diabetes.Re-ceiver operating characteristic(ROC)curve was drawn to evaluate the predictive value of serum CHI3L1 and SDC1 on osteoporosis in elderly patients with type 2 diabetes.Multivariate Logistic regression analysis was used to investigate the influencing factors of osteoporosis in elderly patients with type 2 diabetes.Results There were significant differences in diabetes course,fasting blood glucose,HbA1c and HDL-C a-mong normal bone mass group,decreased bone mass group and osteoporosis group(P＜0.05).The levels of serum CHI3L1,25-(OH)D,P1NP and osteocalcin in osteoporosis group were lower than those in osteopenia group,and those in osteopenia group were lower than those in normal bone mass group,the differences were statistically significant(P＜0.05).Serum SDC1 and CTX levels in osteoporosis group were higher than those in osteopenia group,and those in osteopenia group were higher than those in normal bone mass group,the differences were statistically significant(P＜0.05).Serum CHI3L1 was positively correlated with 25-(OH)D,P1NP and OC(P＜0.05),and negatively correlated with CTX(P＜0.05).Serum SDC1 was negatively correlated with 25-(OH)D,P1NP,OC(P＜0.05),and positively correlated with CTX(P＜0.05).The area under the curve(AUC)of serum CHI3L1,SDC1 and their combination predicted osteoporosis in elderly pa-tients with type 2 diabetes were 0.851,0.772 and 0.904,respectively.Multivariate Logistic regression analysis showed that long duration of diabetes,increased HbA1c,high expression of OC,CHI3L1＞4.16 ng/mL,SDC1≥50.94 ng/mL were all influential factors for osteoporosis in elderly patients with type 2 diabetes(P＜0.05).Conclusion Low expression of CHI3L1 and high expression of SDC1 in serum are associated with ab-normal bone metabolism in elderly patients with type 2 diabetes.These two indexes are expected to be used as biological markers to predict osteoporosis in elderly patients with type 2 diabetes.

Objective:To investigate the mechanism of Juanbi Capsules in the intervention of knee osteoarthritis by inhibiting lipid peroxidation and chondrocyte ferroptosis through activating nuclear factor E2 related factor (Nrf2)/glutathione peroxidase 4 (GPx4) signaling pathway.Methods:Totally 45 rats were randomly divided into blank group, model group and Juanbi Capsules low-, medium- and high-dosage groups, with 9 rats in each group. Except the blank group, the other groups were injected with sodium monoiodoacetate (MIA) to establish knee osteoarthritis model. From the third week of modeling, rats in Juanbi Capsules low-, medium- and high-dosage groups were gavaged with 108.05, 216.09, and 432.18 mg/kg of Juanbi Capsules suspension, and rats in the blank group and model group were gavaged with equal volume of normal saline, once a day, for 28 consecutive days. HE and safranine fast green staining were used to observe the pathological changes of cartilage tissue, and Mankin's score was performed; the levels of MDA, Fe 2+, glutathione (GSH) in articular cartilage were detected by biochemical kit; Western blot was used to detect the protein expressions of SLC7A11, GPx4, acsll4 and Nrf2 in rat articular cartilage. Results:Compared with the model group, the Mankin's score was significantly lower in the Juanbi Capsules middle- and high-dosage groups ( P<0.01); the MDA level decreased in Juanbi Capsules low-, medium- and high-dosage groups ( P<0.01), GSH level increased ( P<0.01), and and Fe 2+ level decreased Juanbi Capsules middle- and high-dosage groups ( P<0.01, P<0.05); the protein expressions of Nrf2, SLC7A11 and GPx4 in cartilage tissue of Juanbi Capsules middle- and high-dosage groups increased ( P<0.01 or P<0.05), the expression of ACSl4 protein decreased ( P<0.01 or P<0.05), and SLC7A11 protein expression increased in Juanbi Capsules low-dosage group ( P<0.05). Conclusion:Juanbi Capsule may inhibit the ferroptosis of rat articular cartilage by activating the Nrf2/GPX4 signaling pathway.

Hypereosinophilia is the term given to a group of highly heterogeneous diseases that are characterized by an increase in the absolute count of peripheral eosinophils,which can accumulate in one or more organs.Although parasitic infection is one of the common causes of eosinophilia,it is often ignored.So far,no clear diagnostic guideline for hepatic sparganosis exists worldwide,and only scattered cases have been reported.We reported a case of a patient with hypereosinophilia induced by hepatic sparganosis,with multiple organ involvement,and discussed the clinical characteristics and treatment plan.This report will help clinicians better understand the differential diagnosis of hepatic sparganosis and the diagnosis and treatment of hypereosinophilia.

This study aims to investigate the effect of salvianolic acid B (Sal B), the active ingredient of Salvia miltiorrhiza, on H9C2 cardiomyocytes injured by oxygen and glucose deprivation/reperfusion (OGD/R) through regulating mitochondrial fission and fusion. The process of myocardial ischemia-reperfusion injury was simulated by establishing OGD/R model. The cell proliferation and cytotoxicity detection kit (cell counting kit-8, CCK-8) was used to detect cell viability; the kit method was used to detect intracellular reactive oxygen species (ROS), total glutathione (t-GSH), nitric oxide (NO) content, protein expression levels of mitochondrial fission and fusion, apoptosis-related detection by Western blot. Mitochondrial permeability transition pore (MPTP) detection kit and Hoechst 33342 fluorescence was used to observe the opening level of MPTP, and molecular docking technology was used to determine the molecular target of Sal B. The results showed that relative to control group, OGD/R injury reduced cell viability, increased the content of ROS, decreased the content of t-GSH and NO. Furthermore, OGD/R injury increased the protein expression levels of dynamin-related protein 1 (Drp1), mitofusions 2 (Mfn2), Bcl-2 associated X protein (Bax) and cysteinyl aspartate specific proteinase 3 (caspase 3), and decreased the protein expression levels of Mfn1, increased MPTP opening level. Compared with the OGD/R group, it was observed that Sal B had a protective effect at concentrations ranging from 6.25 to 100 μmol·L^-1. Sal B decreased the content of ROS, increased the content of t-GSH and NO, and Western blot showed that Sal B decreased the protein expression levels of Drp1, Mfn2, Bax and caspase 3, increased the protein expression level of Mfn1, and decreased the opening level of MPTP. In summary, Sal B may inhibit the opening of MPTP, reduce cell apoptosis and reduce OGD/R damage in H9C2 cells by regulating the balance of oxidation and anti-oxidation, mitochondrial fission and fusion, thereby providing a scientific basis for the use of Sal B in the treatment of myocardial ischemia reperfusion injury.

BACKGROUND:As tissue engineering brings new hope to the worldwide problem of articular cartilage repair,the construction of light-curing 3D printed hydrogel scaffolds with biomimetic composition is of great significance for cartilage tissue engineering. OBJECTIVE:To construct a biomimetic methacryloylated hyaluronic acid/acellular Wharton's jelly composite hydrogel scaffold by digital light processing 3D printing technology,and to evaluate its biocompatibility. METHODS:Wharton's jelly was isolated and extracted from human umbilical cord,then decellulated,freeze-dried,ground into powder,and dissolved in PBS to prepare 50 g/L acellular Wharton's jelly solution.Methylallylated hyaluronic acid was prepared,lyophilized and dissolved in PBS to prepare 50 g/L methylallylated hyaluronic acid solution.Acellular Wharton's jelly solution was mixed with methacrylyacylated hyaluronic acid solution at a volume ratio of 1:1,and was used as bio-ink after adding photoinitiator.Methylacrylylated hyaluronic acid hydrogel scaffolds(labeled as HAMA hydrogel scaffolds)and methylacrylylated hyaluronic acid/acellular Wharton's jelly gel scaffolds(labeled as HAMA/WJ hydrogel scaffolds)were prepared by digital light processing 3D printing technology,and the microstructure,swelling performance,biocompatibility,and cartilage differentiation performance of the scaffolds were characterized. RESULTS AND CONCLUSION:(1)Under scanning electron microscope,the two groups of scaffolds showed a three-dimensional network structure,and the fiber connection of HAMA/WJ hydrogel scaffold was more uniform.Both groups achieved swelling equilibrium within 10 hours,and the equilibrium swelling ratio of HAMA/WJ hydrogel scaffold was lower than that of HAMA hydrogel scaffold(P＜0.05).(2)CCK-8 assay showed that HAMA/WJ hydrogel scaffold could promote the proliferation of bone marrow mesenchymal stem cells compared with HAMA hydrogel scaffold.Dead/live staining showed that bone marrow mesenchymal stem cells grew well on the two groups of scaffolds,and the cells on the HAMA/WJ hydrogel scaffolds were evenly distributed and more cells were found.Phalloidine staining showed better adhesion and spread of bone marrow mesenchymal stem cells in HAMA/WJ hydrogel scaffold than in HAMA.(3)Bone marrow mesenchymal stem cells were inoculated into the two groups for chondrogenic induction culture.The results of qRT-PCR showed that the mRNA expressions of agglutinoglycan,SOX9 and type Ⅱ collagen in the HAMA/WJ hydrogel scaffold group were higher than those in the HAMA hydrogel scaffold group(P＜0.05,P＜0.01).(4)These findings indicate that the digital light processing 3D bioprinting HAMA/WJ hydrogel scaffold can promote the proliferation,adhesion,and chondrogenic differentiation of bone marrow mesenchymal stem cells.