Research progress of SARS-CoV-2 main protease inhibitors / 药学学报

As a common protease with high similarity among coronavirus species, the main protease (Mpro) of SARS-CoV-2 is responsible for the catalytic hydrolysis of viral precursor proteins into functional proteins, which is essential for coronavirus replication and is one of the ideal targets for the development of broad-spectrum antiviral drugs. This paper reviews the main protease inhibitors of SARS-CoV-2, including their molecular structures, potencies and drug-like profiles, binding modes and structure-activity relationships, etc.

Efficacy of vitrectomy for highly myopic macular hole with or without foveoschisis / 中华眼底病杂志

Objective:To observe the clinical effects of pars plana vitrectomy (PPV) combined with internal limiting membrane (ILM) peeling and C 3F 8 tamponade for patients with highly myopic macular hole (HM-MH) with and without foveoschisis. Methods:A retrospective case controlled study. From January 2017 to February 2022, 23 eyes of 23 patients with highly myopic macular hole with and without foveoschisis diagnosed in the Shandong Eye Hospital were included in the study. Among them, 5 males had 5 eyes, and 18 females had 18 eyes, the age was (54.43±12.96) years old. The patients with or without foveoschisis were 12 eyes in 12 cases and 11 eyes in 11 cases. Studies were divided into two groups, depending on the presence of a concomitant myopic foveoschisis or not. The groups are high myopia macular hole with foveoschisis (group A) and high myopia macular hole without foveoschisis (group B). Best-corrected visual acuity (BCVA), B-scan ultrasonography, optical coherence tomography and axial length (AL) measurement were performed in all eyes. Snellen chart was used for BCVA examination, and the visual acuity was converted into logarithm of minimum angle of resolution (logMAR) during statistics. The age of the two groups, sex, macular hole (MH) diameter, logMAR BCVA, AL, posterior scleral staphyloma, there was no significant difference ( P>0.05). PPV combined with ILM peeling and C 3F 8 filling were performed in all eyes. Follow-up was at least 3 months after the last operation. BCVA changes and MH closure were compared between the two groups after surgery. Wilcoxon test was used to compare BCVA before and after operation. Mann-whiteny U test was used to compare preoperative and postoperative BCVA between groups. Results:After initial surgery, MH was closed in 17 of 23 eyes (74%, 17/23). MH was closed in 8 eyes in group A (66.7%, 8/12). Four eyes were not closed (33.3%, 4/12); MH closed in 9 eyes in group B (81.8%, 9/11). There was no significant difference between the two groups after initial operation ( P>0.05). At 1 and 3 months after surgery, the logMAR BCVA of patients in group A and group B were 1.00±0.46, 1.03±0.83 and 0.53±0.63, 0.55±0.41, respectively. Compared with before operation, there was no significant difference at 1 month ( P=0.783, 0.358), but the difference was statistically significant at 3 months ( P=0.012, 0.007). There was no significant difference in logMAR BCVA between group A and group B at 1 and 3 months after operation ( P=0.687, 0.950). Conclusion:PPV combined with ILM peeling and C 3F 8 tamponade can promote MH closure and improve visual acuity in most affected eyes with HM-MH with and without foveoschisis.

Comparison of corneal higher order aberration after femtosecond laser-assisted laser in situ keratomileusis and small incision lenticule extraction / 中华实验眼科杂志

Objective:To analyze and compare the corneal higher order aberration (HOA) after femtosecond laser-assisted laser in situ keratomileusis (FS-LASIK) and small incision lenticule extraction (SMILE).Methods:A non-randomized controlled study was conducted.Sixty myopic patients (60 eyes) who underwent FS-LASIK or SMILE correction at Shandong Eye Hospital from April 2018 to January 2020 were enrolled and the data from the right eye were collected for analysis.Thirty cases (30 eyes) who received FS-LASIK in FS-LASIK group and 30 cases (30 eyes) who received SMILE in SMILE group had a preoperative equivalent spherical diopter of (-5.36±1.11)D and (-4.93±1.03)D, respectively.The HOA of the 6-mm anterior surface, posterior surface, and whole cornea were measured before surgery and at 1, 3, 6, and 12 months after surgery using Pentacam.The root mean square values (μm) of total corneal HOA, spherical aberration, coma and trefoil were obtained.Differences in the above root mean square values at different time points were compared between the two groups.The study protocol adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Shandong Eye Hospital (No.SDSYKYY20180306). Written informed consent was obtained from each subject.Results:The preoperative and 1-, 3-, 6-, 12-month postoperative total HOA of the anterior corneal surface were (0.428±0.126), (0.775±0.169), (0.811±0.194), (0.759±0.214), (0.704±0.199)μm in the FS-LASIK group and (0.409±0.094), (0.656±0.148), (0.681±0.161), (0.668±0.175), (0.648±0.160)μm in the SMILE group, with a significant overall difference ( Fgroup=5.652, P=0.024; Ftime=107.169, P<0.01). Compared with SMILE group, the postoperative total HOA of anterior corneal surface and spherical aberration at different time points were increased in FS-LASIK group, showing statistically significant differences (all at P<0.05). Compared with before surgery, the postoperative total HOA of the anterior corneal surface and spherical aberration at different time points were increased in both groups, showing statistically significant differences (all at P<0.05). In the two groups, the 6- and 12-month postoperative total HOA of the anterior corneal surface were reduced in comparison with the 3-month postoperative ones of the anterior corneal surface, and the 12-month postoperative spherical aberrations of the anterior corneal surface were significantly reduced in comparison with the 1- and 3-month postoperative ones of the anterior corneal surface, showing statistically significant differences (all at P<0.05). There were significant differences in the coma and trefoil of the anterior corneal surface between before and after the operation (coma: Ftime=47.848, P<0.01; trefoil: Ftime=2.497, P=0.046). Compared with before surgery, the postoperative coma was significantly increased in the two groups (all at P<0.05). There were significant differences in total corneal HOA and spherical aberration at different postoperative time points between the two groups (total HOA: Fgroup=8.093, P=0.008; Ftime=125.019, P<0.01.spherical aberration: Fgroup=4.771, P=0.037; Ftime=34.033, P<0.01). Compared with SMILE group, the total corneal HOA and spherical aberration were significantly increased in FS-LASIK group at different postoperative time points (all at P<0.05). Compared with before surgery, postoperative total HOA of the anterior corneal surface and spherical aberration at different postoperative time points were significantly increased in both groups (all at P<0.05). In both groups, the 12-month postoperative corneal spherical aberration was significantly reduced in comparison with the 1- and 3-month postoperative ones (all at P<0.05). There was a significant difference in coma between before and after surgery ( Ftime=30.829, P<0.01). Compared with before surgery, the postoperative coma was significantly increased at different time points in both groups (all at P<0.05). Conclusions:Both FS-LASIK and SMILE increase the HOA of the anterior corneal surface and the whole cornea.Compared with FS-LASIK, SMILE introduces less HOA of the anterior corneal surface and the whole cornea as well as spherical aberrations.

Study on alternative method for vasopressin impurity test of oxytocin injection from biological extraction / 中国药理学通报

Aim To explore the alternative study on rat blood pressure method and HPLC method for vasopressin impurity test of oxytocin injection from biological extraction. Methods The HPLC method for the vasopressin impurity test in vitro was established and validated. The bio-extrac tion oxytocin injection samples and simulated samples were examined for vasopressin impurity by HPLC and rat blood pressure methods respectively. Results Vasopressin and adjacent impurity peaks were successfully separated by the established method. In the range of 210~13 330 IU•L -1the concentration of vasopressin had a good linear relationship with its peak area with r=0.999 9. The results of HPLC method were consistent with the biological examination method-rat blood pressure method in the current standard. Conclusions The method is proved to be specific, sensitive, and accurate, which can be used as a test method for vasopressin impurity to replace the rat blood pressure method in the current standard.

Inhibitory Effect of Kaempferol on Proliferation of KG1a Cells and Its Mechanism / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of kaempferol on proliferation of acute myeloid leukemia (AML) KG1a cells and its mechanism.@*METHODS@#Human AML KG1a cells in logarithmic growth stage were taken and set at 25, 50, 75 and 100 μg/ml kaempferol group, another normal control group (complete medium without drug) and solvent control group (add dimethyl sulfoxide) were also set. After 24 and 48 hours of intervention, the cell proliferation rate was detected by CCK-8 assay. In addition, interleukin-6 (IL-6) combined with kaempferol group (Plus 20 μg/l IL-6 and 75 μg/ml kaempferol) was set up, 48 hours after culture, the cell cycle and apoptosis of KG1a cells were detected by flow cytometry, the mitochondrial membrane potential (MMP) of KG1a cells was detected by MMP detection kit (JC-1 method), and the expression of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway related proteins in KG1a cells were detected by Western blot.@*RESULTS@#The cell proliferation rate of 25, 50, 75 and 100 μg/ml kaempferol group decreased significantly (P<0.05), and with the increase of kaempferol dose (r24 h=-0.990, r48 h= -0.999), the cell proliferation rate decreased gradually (P<0.05). The inhibitory effect of 75 μg/ml kaempferol on cell proliferation reached half of effective dose after 48 hours of intervention. Compared with normal control group, the G0/G1 phase cell proportion and apoptosis rate of cells in 25, 50 and 75 μg/ml kaempferol group increased, while the S phase cell proportion, MMP, phosphorylated JAK2 (p-JAK2)/JAK2 and phosphorylated STAT3 (p-STAT3)/STAT3 protein expression decreased in a dose-dependent manner (r=0.998, 0.994, -0.996, -0.981, -0.997, -0.930). Compared with 75 μg/ml kaempferol group, the G0/G1 phase cell proportion and apoptosis rate of cells in IL-6 combined with kaempferol group decreased, while the S phase cell proportion, MMP, p-JAK2/JAK2 and p-STAT3/STAT3 protein expression increased significantly (P<0.05).@*CONCLUSION@#Kaempferol can inhibit KG1a cell proliferation and induce KG1a cell apoptosis, its mechanism may be related to the inhibition of JAK2/STAT3 signal pathway.

Screening methods of SARS-CoV-2 main protease inhibitors and current applications / 药学学报

COVID-19 epidemic continues to spread around the world till these days, and it is urgent to develop more safe and effective new drugs. Due to the limited P3 biosafety laboratories for directly screening inhibitors of virulent viruses with high infectivity, it is necessary to develop rapid and efficient screening methods for viral proteases and other related targets. The main protease (Mpro), which plays a key role in the replication cycle of SARS-CoV-2, is highly conserved and has no homologous proteases in humans, making it an ideal target for drug development. From two different levels, namely, molecular level and cellular level, this paper summarizes the reported screening methods of SARS-CoV-2 Mpro inhibitors through a variety of representative examples, expecting to provide references for further development of SARS-CoV-2 Mpro inhibitors.

Diagnosis and treatment strategy of periodontitis with diabetes / 中华口腔医学杂志

The number of diabetic patients visiting stomatology for periodontal disease is increasing, and the symptoms are relatively severe, and often complications increase the complexity of periodontal treatment. This article briefly describes the research progress and clinical manifestations of the epidemiology and related pathological mechanisms of periodontitis with diabetes, focusing on the treatment and providing reference for stomatologists in the clinical diagnosis and treatment of patients with diabetic periodontitis.

The impact of extended waiting time on tumor regression after neoadjuvant chemoradiotherapy for locally advanced rectal cancer / 中华外科杂志

Objective: To investigate the influence of extending the waiting time on tumor regression after neoadjuvant chemoradiology (nCRT) in patients with locally advanced rectal cancer (LARC). Methods: Clinicopathological data from 728 LARC patients who completed nCRT treatment at the First Affiliated Hospital, Naval Medical University from January 2012 to December 2021 were collected for retrospective analysis. The primary research endpoint was the sustained complete response (SCR). There were 498 males and 230 females, with an age (M(IQR)) of 58 (15) years (range: 22 to 89 years). Logistic regression models were used to explore whether waiting time was an independent factor affecting SCR. Curve fitting was used to represent the relationship between the cumulative occurrence rate of SCR and the waiting time. The patients were divided into a conventional waiting time group (4 to <12 weeks, n=581) and an extended waiting time group (12 to<20 weeks, n=147). Comparisons regarding tumor regression, organ preservation, and surgical conditions between the two groups were made using the t test, Wilcoxon rank sum test, or χ2 test as appropriate. The Log-rank test was used to elucidate the survival discrepancies between the two groups. Results: The SCR rate of all patients was 21.6% (157/728). The waiting time was an independent influencing factor for SCR, with each additional day corresponding to an OR value of 1.010 (95%CI: 1.001 to 1.020, P=0.031). The cumulative rate of SCR occurrence gradually increased with the extension of waiting time, with the fastest increase between the 9th to <10th week. The SCR rate in the extended waiting time group was higher (27.9%(41/147) vs. 20.0%(116/581), χ2=3.901, P=0.048), and the organ preservation rate during the follow-up period was higher (21.1%(31/147) vs. 10.7%(62/581), χ2=10.510, P=0.001). The 3-year local recurrence/regrowth-free survival rates were 94.0% and 91.1%, the 3-year disease-free survival rates were 76.6% and 75.4%, and the 3-year overall survival rates were 95.6% and 92.2% for the conventional and extended waiting time groups, respectively, with no statistical differences in local recurrence/regrowth-free survival, disease-free survival and overall survival between the two groups (χ2=1.878, P=0.171; χ2=0.078, P=0.780; χ2=1.265, P=0.261). Conclusions: An extended waiting time is conducive to tumor regression, and extending the waiting time to 12 to <20 weeks after nCRT can improve the SCR rate and organ preservation rate, without increasing the difficulty of surgery or altering the oncological outcomes of patients.

Methodological study on the establishment of limit values in the Standards of Drinking Water Quality / 中华预防医学杂志

The establishment of limit values for standards of drinking water quality is an important and complex process. This study systematically introduced the methodology of the establishment of standard limit values for drinking water quality and elaborated on the workflow of setting limit values of water quality indicators, principles and methods of selecting water quality indicators, derivation of safety reference values, and establishment of limit values. It also aimed to provide reference and support for the future revision of relevant standards.

Research on the standard limits for vinyl chloride and trichloroethylene in the "Standards for Drinking Water Quality（GB5749-2022）" in China / 中华预防医学杂志

The usage of vinyl chloride and trichloroethylene in China has been increasing year by year, and they have been detected in both drinking water and environmental water, making them important environmental pollutants. Based on the latest research results on the health effects of vinyl chloride and trichloroethylene, the newly issued, "Standards for Drinking Water Quality (GB5749-2022)" in China has adjusted the standard limit of vinyl chloride from 0.005 mg/L to 0.001 mg/L and the standard limit of trichloroethylene from 0.07 mg/L to 0.02 mg/L. This article analyzed and discussed the relevant technical contents for determining the above standard limits, including the levels and exposure conditions of vinyl chloride and trichloroethylene in the water environment, health effects, derivation of safety reference values, and determination of hygiene standard limits. Suggestions were also made for the implementation of this standard.

Research on the determination of the limit value of perchlorate in the "Standards for Drinking Water Quality（GB5749-2022）" in China / 中华预防医学杂志

Perchlorate is an environmental pollutant that has been a focus of attention in recent years. It has been detected in many environmental water bodies and drinking water in China, with a high level of presence in some areas of the Yangtze River Basin. The human body may ingest perchlorate through exposure pathways such as drinking water and food, and its main health effect is to affect the thyroid's absorption of iodine. The "Standards for Drinking Water Quality" (GB5749-2022) includes perchlorate as an expanded indicator of water quality, with a limit value of 0.07 mg/L. This article analyzes the technical content related to the determination of hygiene standard limits for perchlorate in drinking water, including the environmental presence level and exposure status of perchlorate, main health effects, derivation of safety reference values, and determination of hygiene standard limits.

Comparison of clinical efficacy between closed reduction combined with semi-circular external fixator and minimally invasive percutaneous plate osteosynthesis (MIPPO) in the treatment of middle and distal tibia fractures / 中国骨伤

OBJECTIVE@#To compare the clinical efficacy between closed reduction combined with semi-circular external fixator and minimally invasive percutaneous plate osteosynthesis (MIPPO) in the treatment of middle anddistal tibia fractures.@*METHODS@#The clinical data of sixty patients with middle and distal tibia fractures admitted between January 2019 and November 2022, were retrospectively analyzed. These patients were categorized into external fixation group (n=30) and internal fixation group (n=30). There were 18 males and 12 females in the external fixation group, with an average age of (49.29±2.35) years old. Among them, 14 patients presented with fractures on the left side, and 16 patients presented with fractures on the right side. Closed reduction, arched wire, and semi-circular external fixator were used for treatment. There were 20 males and 10 females in the internal fixation group, with an average age of (48.96±1.87) years old. Among them, 15 patients presented with fractures on the left side, and 15 patients presented with fractures on the right side. MIPPO technique was used for the treatment. Perioperative parameters, including time injury to surgery, surgical duration, incision length, intraoperative bleeding, time to active activity, and incision healing level, were compared between the two groups. Clinical outcomes were also assessed, including Johner-Wruhs scores, time to minimum pain-adapted full weight-bearing, visual analog scale (VAS), SF-36 scale, and complications.@*RESULTS@#The external fixation group exhibited a significantly shorter incision length (1.36±0.86) cm and lower intraoperative bleeding (10.83±5.73) ml compared to the internal fixation group (12.74±3.12) cm and (86.47±8.90) ml, respectively(P<0.05). The postoperative active activity time (1.50±0.54) days and minimum pain-adapted full weight-bearing activity time(108.87±3.43) days in the external fixation group were slightly delayed than the internal fixation group(1.15±0.98) days and (105.27±3.68) days, respectively(P<0.05). Over a mean postoperative follow-up duration of (6.23±1.89) months, both groups showed improved VAS and SF-36 scale scores. There were no statistically significant differences in VAS and SF-36 scale scores 1, 3, 6 months post-operatively between the two groups(P>0.05). The intraoperative surgical time in the external fixation group (35.42±9.31) minutes was shorter than that in the internal fixation group(74.22±7.81) minutes (P<0.05). There was no intraoperative vascular or nerve injury, nor postoperative skin necrosis in the external fixation group. However, skin necrosis was observed in 6 patientsin the internal fixation group, representing a statistically significant difference (P<0.05).@*CONCLUSION@#Both external fixation and plate internal fixation are effective methods for the treatment of middle and distal tibia fractures. External fixation exhibits the advantage of less surgical trauma and a lower incidence of complications.

Effects of exogenous AGR2 on the proliferation and invasion abilities of colon cancer cells / 国际肿瘤学杂志

Objective:To detect the expressions of anterior gradient protein 2 (AGR2) in the cultures of three colon cancer cell lines SW480, SW620 and COLO205, and to investigate the effects of different concentrations of exogenous AGR2 on the proliferation, migration and invasion abilities of SW620 cells.Methods:Western blotting was used to detect the expression levels of AGR2 protein in SW480, SW620 and COLO205 colon cancer cell cultures. SW620 cells were divided into blank control group, anterior gradient protein 2 homologous human recombinant protein (rAGR2) low concentration group (100 μg/ml) and rAGR2 high concentration group (200 μg/ml), and CCK-8 assay, cell scratch assay and Transwell migration and invasion assay were used to detect the effects of different concentrations of rAGR2 on the biological behaviors of SW620 cells.Results:Western blotting results showed that the expression levels of AGR2 protein in SW480, SW620 and COLO205 cells were 0.545±0.097, 0.662±0.040 and 0.882±0.156 respectively, with a statistically significant difference ( F=7.46, P=0.024). The level of AGR2 protein in COLO205 cell line was significantly higher than that in SW480 and SW620 cell lines ( P=0.009; P=0.047). The results of CCK-8 experiment showed that the proliferative activities of SW620 cells in the blank control group, rAGR2 low concentration group and rAGR2 high concentration group were 0.422±0.031, 0.542±0.040 and 0.574±0.033 respectively, with a statistically significant difference ( F=26.35, P<0.001), and the rAGR2 low concentration group and rAGR2 high concentration group were significantly higher than the blank control group (both P<0.001). The results of cell scratching assay showed that the percentage of 36 h cell scratching area was (28.029±2.107)%, (20.642±0.983)% and (16.951±1.608)% for the three groups of cells respectively, with a statistically significant difference ( F=35.85, P<0.001), the rAGR2 low concentration group was higher than the blank control group ( P=0.001), and the rAGR2 high concentration group was higher than the rAGR2 low concentration group ( P=0.032). The results of cell migration assay showed that the number of cells migrated in the three groups was 447.1±32.3, 513.1±55.8 and 632.4±50.3 respectively, with a statistically significant difference ( F=35.62, P<0.001), the rAGR2 low concentration group was more than the blank control group ( P=0.007), and the rAGR2 high concentration group was more than the rAGR2 low concentration group ( P<0.001). The results of the invasion assay showed that the number of cells invaded in the three groups was 369.1±56.1, 505.1±34.4 and 579.0±71.5 respectively, with a statistically significant difference ( F=32.40, P<0.001), the rAGR2 low concentration group was more than the blank control group ( P<0.001), and the rAGR2 high concentration group was more than the rAGR2 low concentration group ( P=0.010). Conclusion:The expression of AGR2 protein varies in the extracellular fluid of different invasive colon cancer cells and increases with the invasive ability. AGR2 protein can increase the proliferation, migration and invasive abilities of colon cancer cells SW620.

CXCL8 Promotes M2 Macrophage Chemotaxis and Infiltration in Colorectal Cancer Microenvironment / 中国生物化学与分子生物学报

CXC chemokine ligand 8 (CXCL8) is highly expressed in many human tumors including colorectal cancer, and it can promote the malignant progression of tumors. It was reported that M2 macrophages were abundant in colorectal cancer microenvironment, but whether CXCL8 affects the infiltration of M2 macrophages and its potential mechanism are not yet clear. The study aimed to investigate the effect of CXCL8 on M2 macrophage infiltration and chemotaxis in the colorectal cancer. Firstly, we analyzed the CXCL8 expression and immune cell infiltration in human colorectal cancer tissues from TCGA RNA-seq data. The expression of CXCL8 was verified by immunohistochemistry in tissues obtained from Shanxi Provincial Cancer Hospital. Then, Western blot and qRT-PCR were employed to detect CXCL8 expression in five colorectal cancer cell lines. THP-1 cells were allowed to differentiate into M2 macrophages via the phorbol myristate acetate (PMA) and IL-4 treatment, followed by detection of the chemotaxis of M2 macrophages towards HCT116, SW480 and CXCL8-HCT116, CXCL8-SW480 cell lines. HCT116 and SW480 cells were treated with interleukin 1β (IL-1β) to detect the expression of CXCL8, and co-cultured with M2 macrophages to analyze the chemotactic activity. The results revealed that the expression of CXCL8 was increased in pairs of CRC tissues versus normal adjacent tissues, and there were more M2 macrophage infiltration in cancer tissues with high expression of CXCL8. The mRNA and protein expression of CXCL8 in HCT116 and SW480 were increased after the IL-1β treatment (P < 0. 05). We confirmed that CXCL8 is a chemotactic factor for M2 macrophages by transwell assays (P<0. 05). In conclusion, CXCL8 in colorectal cancer cells can be induced by IL-1β in colorectal cancer cells and the upregulation of CXCL8 can promote the chemotaxis of M2 macrophages. The massive infiltration of M2 macrophages in colorectal cancer microenvironment may be related with the increased expression of CXCL8.

Siglec-15 Promotes the Proliferation and Migration of Colorectal Cancer Cells and Inhibits the Infiltration of CD4 / 中国生物化学与分子生物学报

Sialic acid binding immunoglobulin type lectin-15 (Siglec-15), one of the Siglec gene family members, is a new immunosuppressive molecule. Siglec-15 is highly expressed in a variety of human tumor cells and tumor-associated macrophages, but the biological function of Siglec-15 in colorectal cancer (CRC) and its effect on the tumor immune microenvironment remains poorly understood. This study aimed to investigate the effects of aberrant expression of Siglec-15 on the biological behaviors of CRC cells and the infiltration of CD4

The method transfer and validation of freeze-dried rabies vaccine pyrogen detection in HL-60 monocyte activation assay / 中国药理学通报

Aim To study the application of HL-60 monocyte activation test in the pyrogen detection of freeze-dried rabies vaccine for human use.Methods The established HL-60 mononuclear cell activation test (MAT) was transferred between laboratories and the method was verified; referring to the interference test in the photometric method of the bacterial endotoxin test method of the Chinese Pharmacopoeia, HL-60- IL- 6 MAT was used to detect the recovery and pyrogen content of 13 batches of freeze-dried rabies vaccine for human use.Results The linearity of the amount of 1L-6 secreted by HL-60 cells, which stimulated by dif¬ferent concentrations of endotoxin standards was above 0.95; the calculated minimum detection limit was not more than 0.125 EU • mL"1 ; the recovery experiment with a solution containing 0.5 and 1.0 EU • mL"1 of endotoxin was performed to cheek the accuracy of the method.HL-60-IL-6 was used to detect 13 hatches of Freeze-dried rahies vaccine for human use, and the re¬covery of endotoxin was between 50 % to 200%.It was consistent that HL-60-IL-6 with pyrogens and en¬dotoxin test for 4 batches of freeze-dried rabies vaccine for human use which pyrogens and endotoxin test failed and the 3 batches of water for injection.Conclusion The HL-60 MAT using IL-6 as a detection indicator is suitable for the detection of pyrogenic substances in freeze-dried rabies vaccine for human use.

Development of bacterial endotoxin standard for insoluble samples / 中国药理学通报

Aim To establish a batch of endotoxin standard for baeterial endotoxin detection of insoluble samples.Methods Candidate A and candidate B were prepared by freeze -drying bacterial endotoxin without excipient.The two batches of candidates were calibrated by three methods, including 13 laboratories for gel method, 9 laboratories for kinetic-turbidimetric assay and 5 laboratories for kinetic chromogenic assay.Results After statistical analysis, the geometric mean values of gel method, kinetic-turbidimetric assay and kinetic chromogenic assay calibration of candidate A were 680.1 EU, 827.0 EU and 800.8 EU, with RSD of 22.4%, 16.2% and 16.7%, respectively.The P value of variance analysis of calibration results of the three methods was 0.067, showing no significant difference.The weighted mean of potency was 774.0 EU (95% confidence interval 721.0 - 831.0, FL% 7.10).The geometric mean values of the calibration of candidate B by gel method, kinetic-turbidimetric assay and kinetic chromogenic assay method were 1 640.6 EU, 1 828.6 EU and 3 224.8 EU, with RSD of 33.9% , 47.0% and 54.4% , respectively.The P val¬ue of variance analysis of the calibration results of the three methods was 0.030, showing significant differ¬ence.Chi-square test was used to correct the weight of each method , and weighted average of the results of the three methods was used to obtain a corrected weighted average efficiency value of 1 822.7 EU (95% confi¬dence interval 1 548.7 -2 145.2, FL% 16.4).Can¬didate B was eliminated based on the results.Conclu¬sion Candidate A has become the first batch of na¬tional standard bacterial endotoxin (for insoluble sam¬ples only) approved by National Standard Substance Committee of China, and the potency is 700 EU.

Study on equivalence between micro kinetic chromogenic assay and kinetic chromogenic assay / 中国药理学通报

Aim To evaluate the equivalence between micro kinetic chromogenic assay anrl kinetic chromogenic assay in order to provide data support for the use of alternative methods.Methods Detection conditions; micro kinetic chromogenic assay and kinetic chromogenic assay limulus reagent were used, sample amount of each well and limulus reagent was 25 jxL ( kinetic chromogenic assay was 100 jxL) , detection wavelength was 405 nm, ONSET OD value was 0.03, and half- well elisa plate was used for detection ( kinetic chromogenic assay was ordinary ELISA plate).The equivalence of the two methods was evaluated by various statistical methods, such as equivalence test, in collaboration with four laboratories in China.Results The results of one-way an OVA, paired T test and equivalence test were consistent, indicating that there were some differences between the existing kinetic chromogenic assay of different manufacturers, while there was no significant difference between the trace or conventional amount of reagent used by each manufacturer.Conclusions Micro kinetic chromogenic assay is e- quivalent to existing reagents in terms of accuracy and recoverv.J.

Study on bacterial endotoxin detection of egg yolk lecithin of Chinese Pharmacopoeia 2020 Edition / 中国药理学通报

Abstract; Aim To solve the problems in the appliea- tion of egg yolk leeithin endotoxin test method, and and to establish the baeterial endotoxin examination method for egg yolk lecithin (for injeetion). Methods The ethanol solution of Tween 80 ( the volume ratio of tween 80 to anhydrous ethanol was 2. 5 • 2. 7, mixed for 4 min) was used to prepare lecithin solution of egg yolk at 0. 1 kg • L 1 , and 10 test water was added to 1 mL lecithin solution of egg yolk (500 EU • mL 1 standard solution of endotoxin IOjxL was added for pos¬itive control). After diluted 20 times with endotoxin test water, the standard curve range was 10 ~0. 01 EU • mL 1 by kinetic-turbidimetrie assay. Methodology of endotoxin test was studied using limulus lysate from two manufacturers and eight hatches of samples. Results The recoveries of eight hatches of samples all met the requirement of interference test between 50% and 200% stipulated in the pharmacopoeia, which solved the problems of the current endotoxin test method in practical application. Conclusions The bacterial en¬dotoxin test method of egg yolk lecithin with good dura-bility is established to provide the basis for the revision of pharmacopoeia.

Interlaboratory method validation of slope ratio determination for anticoagulant activity of leeches / 中国药理学通报

Aim To investigate the slope ratio method for the determination of anticoagulant activity of leeches. Methods Three batches of leeches, four groups of Japanese medical vermiculite yinpian and fifteen groups of leech preparations were chosen, with contrast medicinal leeches herbs and Philippine cattle leech contrast medicinal materials, and different concentrations of leaching solutions were prepared in parallel. APTT value was determined after anticoagulant activity was determined by slope ratio method for the joint validation of laboratory, intermediate precision and accuracy between the linear range. Results The slope ratio method was accurate and accurate in the determination of anticoagulant activity of leeches, with linearity between 64% and 156% relative titer level. Conclusion Slope ratio method can be used to determine the anticoagulant activity of leeches.