To summarize the clinical characteristics, antibody spectrum and neuroelectrophysiological features of autoimmune nodopathy(AN), and to explore the phenotypic differences among different antibody-positive subgroups.

OBJECTIVE To analyze the selection and rationales of comparators for pharmaceutical enterprises in their medical insurance access application， so as to provide a reference for promoting communication and consensus between enterprises and medical insurance authorities in this process. METHODS The application materials for drugs outside the catalogue that passed formal review published by the National Healthcare Security Administration from 2021 to 2025 were extracted， and then content analysis was used to systematically sort out relevant information of the declared drugs and comparators； the specific situations and rationales of pharmaceutical enterprises’ selection of comparators were analyzed. RESULTS A total of 1 341 declared drug documents were collected. Data analysis showed that 1 035 （77.18%） were submitted with positive comparators and 306 （22.82%） used blank comparators； 58 drugs （4.33%） took combination therapy as the reference， and 5 drugs （0.37%） referred to non-pharmacological （or non-single pharmacological） treatment regimens. Among competitive drugs declared by multiple enterprises， 50.00% of the enterprises submitted different comparators. A total of 4 basic conditions and 39 additional conditions were extracted as the rationales for selecting positive comparators. For blank comparators， 12 drug-related factors， 2 administrative factors， and 1 other factor were identified. More than 10% of the drugs did not state the rationale for comparator selection， and over 44% of drugs using blank comparators provided only one justification. CONCLUSIONS Pharmaceutical enterprises mainly select comparators based on their own interests in the medical insurance access application， and there are deficiencies in the adequacy and standardization of their selection basis and reasoning. It is recommended that enterprises follow the principled requirements of medical insurance authorities， and fully and normatively explain the reasons for selecting comparators in combination with the characteristics of their own products. Meanwhile， it is advisable to change the current open-ended statement form of selection reasons into a closed-ended answering mode， so as to highlight the priority of selection， standardize the declaration behavior of enterprises， and reduce communication divergences between the two parties.

ObjectiveTo investigate the potential mechanisms underlying the therapeutic effects of Bushen Huoxue Formulation （补肾活血方， BSHXF） in Parkinson's disease （PD） from the perspectives of synaptic vesicle cycling and intestinal barrier function. MethodsForty-eight male SD rats were randomly divided into four groups， control group， model group， Madopar group， and BSHXF group， with 12 rats in each group. Except for the control group， rats were administered rotenone by gavage once daily for 4 consecutive weeks to establish a PD rat model. After successful modeling， rats in the BSHXF group received concentrated BSHXF solution by gavage at a dose of 13.5 g/（kg·d）， while rats in the Madopar group received Madopar tablet suspension at 2 g/（kg·d）. Rats in the control group and the model group were administered distilled water by gavage at 10 ml/（kg·d）. All groups received continuous gavage treatment for 2 weeks. Motor function was evaluated using the open field test （total distance traveled， average speed， and total distance traveled in the central zone）， rotarod test （latency to fall）， and balance beam test （traversal time）. Histopathological changes in colon tissues were observed by HE staining. Western Blotting was used to detect the protein levels of α-synuclein （α-syn）， phosphorylated serine 129 α-synuclein （pSer129-α-syn）， vesicle-associated membrane protein 2 （VAMP2）， and synaptophysin （SYP） in the substantia nigra and colon tissues. The mRNA expression levels of α-syn， VAMP2， and SYP were detected by RT-PCR， and α-syn expression was examined by immunohistochemistry. Immunofluorescence staining was performed to assess the levels of tight junction protein 1 （ZO-1） and occludin in colon tissues， as well as tyrosine hydroxylase （TH） levels in substantia nigra tissues. ResultsCompared with the control group， rats in the model group showed reduced total distance traveled， average speed， and central-zone distance in the open field test， decreased latency to fall in the rotarod test， and prolonged traversal time in the balance beam test. The protein levels of pSer129-α-syn and α-syn， as well as α-syn mRNA expression were increased in the substantia nigra and colon tissues， whereas the protein and mRNA expression levels of VAMP2 and SYP were decreased. The relative integrated optical density of α-syn in the substantia nigra and colon tissues was increased， while the relative fluorescence intensities of ZO-1 and occludin in colon tissues and TH in the substantia nigra were decreased （P＜0.05）. HE staining showed focal necrosis in the mucosal layer of colon tissues， disappearance of some intestinal glands， extensive inflammatory cell infiltration， and proliferation of fibrous connective tissue in the submucosa. Compared with the model group， both the Madopar group and BSHXF group showed varying degrees of improvement in the above indicators （P＜0.05）. Pathological damage in colon tissues was also alleviated， with only mild fibrous tissue proliferation and limited diffuse inflammatory cell infiltration observed in the mucosal layer. Compared with the Madopar group， rats in the BSHXF group exhibited increased average speed， central-zone distance， and latency to fall in the rotarod test. The levels of pSer129-α-syn and α-syn proteins in the substantia nigra were decreased， while VAMP2 protein expression was increased. In colon tissues， SYP protein levels and mRNA expression were elevated. The relative fluorescence intensity of ZO-1 in colon tissues and TH in substantia nigra tissues was also increased （P＜0.05）. ConclusionBSHXF may improve motor dysfunction in PD by regulating synaptic vesicle cycling， restoring intestinal barrier integrity， and improving neuronal synaptic function through modulation of synaptic vesicle-associated protein expression in the substantia nigra and colon tissues.

ObjectiveTo investigate the potential mechanisms underlying the therapeutic effects of Bushen Huoxue Formulation （补肾活血方， BSHXF） in Parkinson's disease （PD） from the perspectives of synaptic vesicle cycling and intestinal barrier function. MethodsForty-eight male SD rats were randomly divided into four groups， control group， model group， Madopar group， and BSHXF group， with 12 rats in each group. Except for the control group， rats were administered rotenone by gavage once daily for 4 consecutive weeks to establish a PD rat model. After successful modeling， rats in the BSHXF group received concentrated BSHXF solution by gavage at a dose of 13.5 g/（kg·d）， while rats in the Madopar group received Madopar tablet suspension at 2 g/（kg·d）. Rats in the control group and the model group were administered distilled water by gavage at 10 ml/（kg·d）. All groups received continuous gavage treatment for 2 weeks. Motor function was evaluated using the open field test （total distance traveled， average speed， and total distance traveled in the central zone）， rotarod test （latency to fall）， and balance beam test （traversal time）. Histopathological changes in colon tissues were observed by HE staining. Western Blotting was used to detect the protein levels of α-synuclein （α-syn）， phosphorylated serine 129 α-synuclein （pSer129-α-syn）， vesicle-associated membrane protein 2 （VAMP2）， and synaptophysin （SYP） in the substantia nigra and colon tissues. The mRNA expression levels of α-syn， VAMP2， and SYP were detected by RT-PCR， and α-syn expression was examined by immunohistochemistry. Immunofluorescence staining was performed to assess the levels of tight junction protein 1 （ZO-1） and occludin in colon tissues， as well as tyrosine hydroxylase （TH） levels in substantia nigra tissues. ResultsCompared with the control group， rats in the model group showed reduced total distance traveled， average speed， and central-zone distance in the open field test， decreased latency to fall in the rotarod test， and prolonged traversal time in the balance beam test. The protein levels of pSer129-α-syn and α-syn， as well as α-syn mRNA expression were increased in the substantia nigra and colon tissues， whereas the protein and mRNA expression levels of VAMP2 and SYP were decreased. The relative integrated optical density of α-syn in the substantia nigra and colon tissues was increased， while the relative fluorescence intensities of ZO-1 and occludin in colon tissues and TH in the substantia nigra were decreased （P＜0.05）. HE staining showed focal necrosis in the mucosal layer of colon tissues， disappearance of some intestinal glands， extensive inflammatory cell infiltration， and proliferation of fibrous connective tissue in the submucosa. Compared with the model group， both the Madopar group and BSHXF group showed varying degrees of improvement in the above indicators （P＜0.05）. Pathological damage in colon tissues was also alleviated， with only mild fibrous tissue proliferation and limited diffuse inflammatory cell infiltration observed in the mucosal layer. Compared with the Madopar group， rats in the BSHXF group exhibited increased average speed， central-zone distance， and latency to fall in the rotarod test. The levels of pSer129-α-syn and α-syn proteins in the substantia nigra were decreased， while VAMP2 protein expression was increased. In colon tissues， SYP protein levels and mRNA expression were elevated. The relative fluorescence intensity of ZO-1 in colon tissues and TH in substantia nigra tissues was also increased （P＜0.05）. ConclusionBSHXF may improve motor dysfunction in PD by regulating synaptic vesicle cycling， restoring intestinal barrier integrity， and improving neuronal synaptic function through modulation of synaptic vesicle-associated protein expression in the substantia nigra and colon tissues.

Objective:To evaluate the value of a combined model based on primary lesion 18F-fluorodeoxyglucose ( 18F-FDG) PET/CT radiomics for predicting lymph node metastasis in non-small cell lung cancer (NSCLC) . Methods:A retrospective analysis was conducted on the clinical data of 203 NSCLC patients who underwent pre-treatment PET/CT imaging at Nanjing Drum Tower Hospital from June 2013 to July 2023. Patients were randomly assigned to the training set ( n=142) and the validation set ( n=61) at a ratio of 7∶3. A predictive model was developed in the training set, and its predictive performance and clinical application value were assessed in both the training and validation sets. Traditional PET/CT parameters and PET/CT radiomics features of the primary lesion were obtained by 3D-slicer software. Least absolute shrinkage and selection operator (LASSO), random forest, and extreme gradient boosting were performed to extract features. Support vector machine was used to construct a radiomics score (Radscore). Univariate and multivariate logistic regression analysis was used to predict the influencing factors of lymph node metastasis in NSCLC patients and to establish models. Predictive performance of the models was evaluated by receiver operator characteristic (ROC) curves and clinical application value was assessed by calibration curves and decision curve analysis (DCA) . Results:Among 203 NSCLC patients, 116 had lymph node metastasis, with 64 cases in the training set and 52 cases in the validation set. Three complementary classical machine learning methods were used for feature screening, and finally 10 radiomics features were obtained. The optimal threshold for Radscore-PET was 0.43 and the optimal threshold for Radscore-CT was 0.39. Univariate analysis showed that, sex ( OR=0.48, 95% CI: 0.24-0.95, P=0.036), tumor marker levels ( OR=3.81, 95% CI: 1.84-7.91, P<0.001), long diameter of tumor ( OR=2.56, 95% CI: 1.27-5.16, P=0.009), short diameter of tumor ( OR=3.73, 95% CI: 1.75-7.92, P=0.001), vacuolar sign ( OR=0.32, 95% CI: 0.12-0.86, P=0.024), ring-like metabolism ( OR=3.67, 95% CI: 1.33-10.13, P=0.012), maximum standardized uptake value (SUV max) ( OR=6.57, 95% CI: 3.03-14.25, P<0.001), metabolic tumor volume (MTV) ( OR=2.91, 95% CI: 1.43-5.92, P=0.003), total lesion glycolysis (TLG) ( OR=4.23, 95% CI: 2.08-8.59, P<0.001), Radscore-PET ( OR=21.93, 95% CI: 9.04-53.20, P<0.001) and Radscore-CT ( OR=13.72, 95% CI: 6.12-30.76, P<0.001) were all influencing factors for predicting lymph node metastasis in NSCLC patients. Multivariate analysis showed that, tumor marker levels ( OR=2.55, 95% CI: 1.11-5.90, P=0.028), vacuolar sign ( OR=0.26, 95% CI: 0.08-0.83, P=0.023), SUV max ( OR=5.94, 95% CI: 1.99-17.75, P=0.001), Radscore-PET ( OR=25.51, 95% CI: 5.92-110.22, P<0.001), and Radscore-CT ( OR=8.68, 95% CI: 2.73-27.61, P<0.001) were independent influencing factors for predicting lymph node metastasis in patients with NSCLC. Based on the above independent influencing factors, models were constructed: the traditional model (tumor marker levels, vacuolar sign, SUV max), the PET model (SUV max, Radscore-PET), the CT model (vacuolar sign, Radscore-CT), and the combined model (tumor marker levels, vacuolar sign, SUV max, Radscore-PET, Radscore-CT). ROC curve analysis showed that, the area under curve (AUC) of the traditional, PET, CT, and combined models in the training set were 0.75 (95% CI: 0.67-0.82), 0.90 (95% CI: 0.84-0.95), 0.85 (95% CI: 0.78-0.90), and 0.94 (95% CI: 0.88-0.97), respectively. The predictive value of the combined model was higher than that of the traditional model ( Z=5.01, P<0.001), the PET model ( Z=1.99, P=0.047), and the CT model ( Z=3.25, P=0.001). In the validation set, the AUCs for the traditional model, PET model, CT model, and combined model were 0.65 (95% CI: 0.52-0.77), 0.86 (95% CI: 0.74-0.93), 0.85 (95% CI: 0.73-0.93), and 0.90 (95% CI: 0.80-0.96), respectively. The predictive value of the combined model was superior to that of the traditional model ( Z=3.23, P=0.001). The sensitivity and specificity of the combined model in the training set were 84.37% and 91.03%, while in the validation set, the sensitivity and specificity were 82.61% and 94.74%, respectively. Calibration curves showed a good agreement between the predicted and actual probabilities in both the training and validation sets. DCA showed that the combined models had good discriminative ability in both the training and validation sets. Conclusions:Tumor marker levels, vacuolar sign, SUV max, Radscore-PET, and Radscore-CT are all independent influencing factors for predicting lymph node metastasis in patients with NSCLC. The combined model based on these factors demonstrates excellent predictive performance and clinical application value for predicting lymph node metastasis in NSCLC.

The rupture of carotid vulnerable plaques is the core pathological basis for major cardiovascular and cerebrovascular events. However， the insufficient alignment between existing animal models and the clinical disease and syndrome characteristics of traditional Chinese and western medicine has limited research progress. In this study， biomedical databases in China and abroad were systematically searched， and the modeling mechanisms and evaluation systems of carotid vulnerable plaque animal models were systematically assessed based on diagnostic criteria of both traditional Chinese and western medicine. Analysis of the clinical correspondence indicated that existing animal models can be categorized into four types： simple high-fat diet， surgical induction combined with high-fat feeding， genetic engineering combined with high-fat feeding， and drug induction combined with high-fat feeding. Among these， the compound strategy of surgical induction combined with high-fat feeding has become the current mainstream approach， showing good concordance with western medicine. The study found that the double balloon injury rabbit model and the ApoE^-/- mouse carotid artery tandem constriction combined with high-fat feeding model demonstrated a high degree of clinical correspondence with both traditional Chinese and western medicine in terms of vulnerable plaque imaging and pathological features. Nevertheless， existing models still face significant technical limitations in faithfully simulating plaque pathology and in translating findings to clinical applications. To address these challenges， integrating complex comorbidity mechanism construction， multimodal dynamic mechanism monitoring， and collaborative evaluation systems of traditional Chinese and western medicine could enable the development of highly concordant carotid vulnerable plaque disease-syndrome combination animal models. Such models would provide a reproducible experimental platform for targeted drug development to regulate plaque stability and for individualized precision treatment， as well as a theoretical basis for innovation in clinical diagnostic and therapeutic strategies.

ObjectiveTo investigate the possible mechanism of Bushen Huoxue Granule （补肾活血颗粒， BHG） in treating Parkinson's disease （PD） from the perspecitve of dopamine （DA） homeostasis. MethodsSeventy-two mice were randomly divided into blank group， model group， madopar group and BHG low-， medium- and high-dose groups， with 12 mice in each group. Except for the blank group， all mice were administered intraperitoneal injections of 1-methyl-4-phenyl-1，2，3，6-tetrahydropyridine （MPTP） for 7 consecutive days to induce a PD model. On the day following the injection， BHG low-， medium- and high-dose groups were administered BHG at doses of 1.25， 2.5， and 5.0 mg/（g·d） by oral gavage， respectively， while the madopar group received madopar tablets at dose of 0.093 8 mg/（g·d） by oral gavage. The blank group and the model group were given 0.2 ml/10 g of distilled water by gavage. All treatments were given once daily for 14 days. Open field test， pole climbing test and grip test were used to evaluate the behavior of mice in each group. Immunohistochemistry was used to detect the expression of tyrosine hydroxylase （TH） in striatum. Nissl staining was used to detect the activity of striatal neurons. The contents of DA and 3,4-dihydroxyphenylacetic acid （DOPAC） in striatum were detected by ultra performance liquid chromatography tandem mass spectrometry. The number and volume of synaptic vesicles were observed by transmission electron microscope. The expression of vesicular monoamine transporter 2 （VMAT2） in striatum was detected by immunofluorescence. Western Blot was used to detect the expression of extracellular signal-regulated kinase （ERK）， phosphorylated ERK （p-ERK）， cAMP response element-binding protein （CREB）， phosphorylated CREB （p-CREB） and VMAT2 in striatum. ResultsCompared to the blank group， mice in the model group showed a significant decline in total distance and average speed in the open field test， along with an increase in total resting time； in the pole test， both the time required for the mice to turn completely downward （T-turn） and the total time taken to reach the bottom of the pole （T-total） were prolonged； forelimb grip strength was reduced； in the striatum， the mean optical density of TH， the average fluorescence intensity of VMAT2 protein， and DA content all decreased， while the number of striatal neurons was reduced， and the DOPAC/DA ratio was elevated； the levels of p-ERK/ERK， p-CREB/CREB， and VMAT2 in the striatum significantly decreased （P＜0.01）； transmission electron microscopy revealed that both the number and volume of synaptic vesicles in striatal neurons were markedly reduced. Compared to the model group， mice in the madopar group and BHG low-， medium- and high-dose groups showed significant improvements in all the above indicators （P＜0.05 or P＜0.01）. Compared to madopar group， the BHG high-dose group exhibited increased DA content and elevated p-CREB/CREB ratio in the striatum （P＜0.05）. Compared to the BHG low-dose group， the BHG high-dose group showed increased total distance and mean velocity， decreased total resting time， T-turn， and T-total， as well as enhanced forelimb grip strength； moreover， the average fluorescence intensity of VMAT2 protein， DA content， p-ERK/ERK， p-CREB/CREB， and VMAT2 levels in the striatum were all significantly elevated （P＜0.05 or P＜0.01）. ConclusionBHG may restore DA homeostasis and alleviate the damage of dopaminergic neurons by regulating ERK/CREB/VMAT2 signaling pathway.

Objective To study the clinical characteristics and medication patterns of Wangbi Tablets in treating patients with knee osteoarthritis(KOA)in the real world and to analyze the advantages and specific features of Chinese patent medicines in treating advantage disease.Methods A prospective case registry study was conducted,registering 3 000 patients from 30 TCM and Western medicine hospitals across China from April 2019 to December 2021.Data on gender,age,BMI,Chinese medicine staging(CMS),K-L grading,medication duration,VAS score,medical history and combined medication were collected for descriptive analysis.Comparisons were made between different stages and between single-use and combined-use groups.The Apriori algorithm was used for association analysis of combined medications.Results A total of 2 999 patients were included,with 2 063 females(68.79%)and 936 males(31.21%).The average age was(56.89±8.90)years.The average BMI was(23.80±2.88)kg/m2.The proportion of patients in CMS I was 512(17.07%),with a VAS median score of 8,while the proportion of patients in CMS Ⅱ was 2 181(72.73%),with a VAS median score of 6.The proportion of patients in CMS Ⅲ was 306(10.20%),with a VAS median score of 3;316 cases(10.54%)were classified as K-L grade I,2 477 cases(82.59%)as grade Ⅱ,204 cases(6.80%)as grade Ⅲ,and 2 cases(0.07%)as grade Ⅳ.Medication analysis indicated that the single-use group(1507 cases,50.25%)was larger than the combined-use group(1 492 cases,49.75%).In terms of the number of drugs used in combination,one(39.01%)and two(38.14%)were the main types;in terms of medication types,combination therapy with Western medicine(62.27%)and simultaneous use of Western medicine and other Chinese materia medica(26.14%)were the main methods;the top three drugs with the highest frequency of combination use were glucosamine capsules,imrecoxib tablets and sodium hyaluronate injection.The top three drug combinations with the highest support were"Huoxue Zhishang Powder+imrecoxib tablets","glucosamine capsules+imrecoxib tablets"and"glucosamine capsules+sodium hyaluronate injection".Inter-group comparisons showed that the medication duration for Wangbi Tablets in CMS I was longer than in CMS Ⅱ and CMS Ⅲ(P<0.01).The proportion of patients on monotherapy in CMS I(62.11%)was higher than in CMS Ⅱ(46.54%)and CMS Ⅲ(56.86%)(P<0.001).Among patients in CMS I(16.41%)and CMS Ⅲ(21.24%),the highest proportion used one combined medication,whereas in CMS Ⅱ,the highest proportion was for those using two combined medications(20.50%).In all three groups,CMS I(19.53%),CMS Ⅱ(33.70%)and CMS Ⅲ(30.72%),the highest proportion of combined medications was Western medicine.Conclusion More than half of the patients treated KOA with Wangbi Tablets alone.Approximately one-fifth of the patients were in CMS I,with a median pain score of 8.The average duration of medication for patients in CMS I,CMS Ⅱ and CMS Ⅲ decreases,and there is no obvious pattern in the medication method at different stages;combination therapy is represented by"Huoxue Zhishang Powder+imrecoxib tablets"and"glucosamine capsules+imrecoxib tablets".

Objective To study the clinical characteristics and medication patterns of Wangbi Tablets in treating patients with knee osteoarthritis(KOA)in the real world and to analyze the advantages and specific features of Chinese patent medicines in treating advantage disease.Methods A prospective case registry study was conducted,registering 3 000 patients from 30 TCM and Western medicine hospitals across China from April 2019 to December 2021.Data on gender,age,BMI,Chinese medicine staging(CMS),K-L grading,medication duration,VAS score,medical history and combined medication were collected for descriptive analysis.Comparisons were made between different stages and between single-use and combined-use groups.The Apriori algorithm was used for association analysis of combined medications.Results A total of 2 999 patients were included,with 2 063 females(68.79%)and 936 males(31.21%).The average age was(56.89±8.90)years.The average BMI was(23.80±2.88)kg/m2.The proportion of patients in CMS I was 512(17.07%),with a VAS median score of 8,while the proportion of patients in CMS Ⅱ was 2 181(72.73%),with a VAS median score of 6.The proportion of patients in CMS Ⅲ was 306(10.20%),with a VAS median score of 3;316 cases(10.54%)were classified as K-L grade I,2 477 cases(82.59%)as grade Ⅱ,204 cases(6.80%)as grade Ⅲ,and 2 cases(0.07%)as grade Ⅳ.Medication analysis indicated that the single-use group(1507 cases,50.25%)was larger than the combined-use group(1 492 cases,49.75%).In terms of the number of drugs used in combination,one(39.01%)and two(38.14%)were the main types;in terms of medication types,combination therapy with Western medicine(62.27%)and simultaneous use of Western medicine and other Chinese materia medica(26.14%)were the main methods;the top three drugs with the highest frequency of combination use were glucosamine capsules,imrecoxib tablets and sodium hyaluronate injection.The top three drug combinations with the highest support were"Huoxue Zhishang Powder+imrecoxib tablets","glucosamine capsules+imrecoxib tablets"and"glucosamine capsules+sodium hyaluronate injection".Inter-group comparisons showed that the medication duration for Wangbi Tablets in CMS I was longer than in CMS Ⅱ and CMS Ⅲ(P<0.01).The proportion of patients on monotherapy in CMS I(62.11%)was higher than in CMS Ⅱ(46.54%)and CMS Ⅲ(56.86%)(P<0.001).Among patients in CMS I(16.41%)and CMS Ⅲ(21.24%),the highest proportion used one combined medication,whereas in CMS Ⅱ,the highest proportion was for those using two combined medications(20.50%).In all three groups,CMS I(19.53%),CMS Ⅱ(33.70%)and CMS Ⅲ(30.72%),the highest proportion of combined medications was Western medicine.Conclusion More than half of the patients treated KOA with Wangbi Tablets alone.Approximately one-fifth of the patients were in CMS I,with a median pain score of 8.The average duration of medication for patients in CMS I,CMS Ⅱ and CMS Ⅲ decreases,and there is no obvious pattern in the medication method at different stages;combination therapy is represented by"Huoxue Zhishang Powder+imrecoxib tablets"and"glucosamine capsules+imrecoxib tablets".

Objective To explore the impact of pH value of the reaction system on the properties of bovine hemoglobin modified with aldehydeated polyethylene glycol(PEG-bHb).Methods PEG-bHb conjugates were synthesized under varying pH conditions(6.0,6.5,7.4 and 8.0)of the reaction system while consistent molar ratios,temperature,and reaction time were maintained.The structural and functional attributes of PEG-bHb were characterized.Results The proportion of methemoglobin decreased with an increase in pH.In a weakly acidic reaction environment,the PEG-bHb was found to be relatively highly modified.At pH 6.5,the average number of PEG chains attached to the bHb surface was 6.86±0.38.Selective PEG modification of the N-terminal α-NH2 groups was more pronounced under weakly acidic conditions.Specifically,at pH 6.5,the modification efficiency of the N-terminal α-NH2 groups of bHb by aldehyde-activated PEG reached 95.4％for the α-chains and 99.3％for the β-chains.The PEG modification influenced the heme region microenvi-ronment of bHb,with minimal structural impact observed at pH 6.5.After modification,the oxygen affinity of PEG-bHb was enhanced,the Hill coefficient was reduced,and there were significant increases in colloid osmotic pressure,viscosity,and particle size,all of which differed markedly from the unmodified bHb group(P＜0.001).Conclusion The synthesis of PEG-bHb under weakly acidic conditions can result in a high degree of selective modification of the N-terminal α-NH2 groups and an overall high degree of modification.