Objective We aimed to compared matrix metalloproteinase-13 (MMP-13) and transforming growth factor-β1 (TGF-β1) in synovial fluid,the phosphorylation level of Smad3 in articular cartilage (P-Smad3),and their correlation with traditional Chinese medicine (TCM) patterns in patients with knee osteoarthritis (KOA) of liver-kidney deficiency pattern and pattern of intermingled phlegm and blood stasis.Methods Using a cross-sectional field investigation method,KOA patients hospitalized in the Orthopedics Department of Dongzhimen Hospital,Beijing University of Chinese Medicine from September 2019 to February 2023 were collected. A total of 112 KOA patients were included,among which 63 cases were diagnosed with liver-kidney deficiency pattern,and 49 cases were diagnosed with pattern of intermingled phlegm and blood stasis. The intensity of knee pain,function,and X-ray imaging result were quantified using the Visual Analogue Scale (VAS),Lysholm Knee Scoring Scale,and Kellgren-Lawrence (K-L) Grading Scale,respectively. The TCM pattern was identified and quantified using a TCM Pattern Scoring Scale. Immunohistochemistry was used to determine the phosphorylation characteristics of Smad3 in articular cartilage,and ELISA was used to measure the contents of MMP-13 and TGF-β1 in synovial fluid. The level characteristics and their correlation with the degree of syndrome were analyzed.Results (i) There was no statistically significant difference in VAS scores,Lysholm scores,and K-L grades between KOA patients with different TCM patterns. (ii) Compared with KOA patients with pattern of intermingled phlegm and blood stasis,patients with pattern of liver-kidney deficiency had higher levels of MMP-13 in synovial fluid and lower levels of TGF-β1 in synovial fluid (P<0.05). (iii) In KOA patients with liver-kidney deficiency pattern,there was a positive correlation between the level of MMP-13 in synovial fluid and the score of TCM pattern (r=0.292,P=0.020),while there was a negative correlation between the level of TGF-β1 in synovial fluid and the score of TCM pattern (r=-0.781,P<0.001). In KOA patients with pattern of intermingled phlegm and blood stasis,there was also a positive correlation between the level of MMP-13 in synovial fluid and the score of TCM pattern (r=0.936,P<0.001). (iv) The mean optical density value of P-Smad3 in articular cartilage was lower in KOA patients with liver-kidney deficiency pattern than in pattern of intermingled phlegm and blood stasis (P<0.05).Conclusion KOA patients with liver-kidney deficiency pattern or pattern of intermingled phlegm and blood stasis have different levels of TGF-β1 and MMP-13 in synovial fluid,as well as varying degrees of Smad3 phosphorylation in articular cartilage,which is consistent with the analysis of etiology and pathogenesis under different patterns. The levels of TGF-β1 and MMP-13 in synovial fluid of patients with liver-kidney deficiency pattern can reflect the severity of the pattern to a certain extent,and the mechanism may be related to the inhibition of the activation level of the TGF-β/Smad signaling pathway. This study enriches the research content of the material basis of TCM patterns.

Objective:To identify the chemical constituents of Platycladi Cacumen before and after being carbonized. Method:Chemical constituents in 3 batches of Platycladi Cacumen and its carbonized products were identified and compared by ultra performance liquid chromatography-quadrupole-time of flight-mass spectrometry （UHPLC-QTOF-MS/MS）. Chromatographic separation was performed on an ACQUITY UPLC BEH C₁₈ column （2.1 mm×100 mm， 1.7 μm） with 0.1% formic acid aqueous solution （A）- acetonitrile （B） as mobile phase for gradient elution （0-3.5 min， 5%-15%B； 3.5-6 min， 15%-30%B； 6-6.5 min， 30%B； 6.5-12 min， 30%-70%B； 12-12.5 min， 70%B； 12.5-18 min， 70%-100%B； 18-22 min， 100%B）. The flow rate was 0.4 mL·min^-1 and the injection volume was 5 μL. Mass spectrometry was performed by an electrospray ionization， and the primary and secondary mass spectrometry data were collected with the full scan mode of positive and negative ions， the peaks containing MS/MS data were identified by self-established secondary mass spectrometry database and corresponding fragmentation law matching method. Result:A total of 77 and 76 substances with the same change trend were identified under positive and negative ion modes. After being carbonized， the disappeared components of Platycladi Cacumen were mainly amino acids， ketone aldehydes and other volatile components. Among newly produced components， there were 6 kinds of flavonoid aglycones （rhamnetin， 6，7，3'-trihydroxyflavone， 3，6，3'-trihydroxyflavone， 4'-hydroxy-2'-methyl-3，4，5-trimethoxychalcone， herbacetin and 3'，5'-dimethoxy-3，5，7，4'-tetrahydroxyflavone）， 3 kinds of coumarins （7-hydroxycoumarin， 7，8-dihydroxycoumarin and 8-acetyl-7-hydroxycoum-arin） and 3 kinds of benzoic acids （3-methylcatechol， pyrocatechol and chromone-3-carboxylic acid）. There were a total of 40 flavonoids （quercitrin， quercetin， kaempferol， etc.） among these identified chemical constituents. Conclusion:There are significant quantitative and qualitative changes in the chemical compositions of Platycladi Cacumen after being carbonized. The flavonoids， the identified main active ingredients， can provide data reference for further study on the material basis of efficacy changes of Platycladi Cacumen before and after being carbonized.

Adult ; Asymptomatic Diseases/epidemiology* ; Autoimmune Diseases/etiology* ; China/epidemiology* ; Female ; Humans ; Hypothyroidism/etiology* ; Male ; Metabolic Syndrome/epidemiology* ; Middle Aged ; Obesity/epidemiology* ; Risk Factors ; Thyroid Diseases/etiology* ; Young Adult

OBJECTIVES: To construct a cell line that can stably express human phospholamban(PLN) and initially explore its application in the study of myocardial toxicity mechanism. METHODS: FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expression vector pcDNA5/FRT/TO(hereinafter referred to as pDFT) to construct the pDFT-PLN-Flag plasmid. The Flp-In^TM T-REx^TM 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene. Successfully recombined monoclonal cell lines were screened by hygromycin B resistance. Western blot and indirect immunofluorescence (IFA) were used to examine the expression of the target protein in recombinant cells. After the cell line was exposed to aconitine, it was verified by Western blot to detect changes in PLN protein phosphorylation. RESULTS: After PCR amplification of the recombinant plasmid and DNA electrophoresis, the length of the amplified product is the same as the known PLN gene fragment, which is consistent with the open reading frame (ORF) sequence of the human PLN gene after sequencing. IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation. Preliminary results showed that the phosphorylation level of Thr17-PLN decreased after two hours of exposure to 1 μmol/L aconitine. CONCLUSIONS This human cell line can stably express PLN and can be used to study the mechanism of action of aconitine on the cell at molecular level.
Calcium-Binding Proteins/metabolism* ; Cell Line ; Humans ; Myocardium/metabolism* ; Phosphorylation

In order to effectively solve the over-processing problem of Platycladi Cacumen Carbonisata, which was commonly used as a hemostatic drug in clinical application, we used the quantitative analysis method of multi-components by single marker(QAMS) in this study, with quercetin as internal reference to simultaneously determine the content of six flavonoids which can be used to control the internal quality of Platycladi Cacumen Carbonisata. Based on the comparison of QAMS and external standard method(ESM) results, the limit standards of contents were established as follows: isoquercitroside ≥0.002 0%, quercitroside ≥0.050%, quercetin ≥0.030%, kaempferol and amentoflavone both ≥0.010%, hinokiflavone ≥0.050%. Based on the color detection of Platycladi Cacumen and Platycladi Cacumen Carbonisata with different processing degrees, the law of influence of different processing degrees on the color of Platycladi Cacumen Carbonisata was found. A new external quality standard of Platycladi Cacumen Carbonisata was established by fitting curve of color recognition for the external quality control, based on which the standard ranges of ΔL~*, Δb~* and ΔE were-50.00--44.00, 6.00-11.00 and 45.00-50.00 respectively. Effective combination of established internal and external quality control standards by this study can be used to evaluate the processing degree and quality of Platycladi Cacumen Carbonisata more comprehensively and objectively, which can guarantee its clinical efficacy. At the same time, this study also provides reference and basis for further improving the quality control standard of Platycladi Cacumen Carbonisata.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; Flavonoids ; Hemostatics ; Quality Control

Objective: To investigate the quality of Platycladi Cacumen carbonisata pieces in the market,then to explore control standard of "carbonizing retains characteristics" of Platycladi Cacumen.Method: The properties,water content and alcohol extract content of commercial Platycladi Cacumen carbonisata pieces were determined by the method in the 2015 edition of Chinese Pharmacopoeia.Thin layer chromatography(TLC) identification of quercetin and contents of quercetin and quercitin were determined.Result: The carbonizing degree of 9 batches of Platycladi Cacumen carbonisata pieces were heavier than which is specified in the 2015 edition of Chinese Pharmacopoeia.The conditions of TLC identification in the 2015 edition of Chinese Pharmacopoeia should be revised to avoid stratification;the carbonizing degree could affect the contents of quercetin and quercitin in Platycladi Cacumen carbonisata pieces;the simultaneous detection of quercetin and quercitin can be used as one of the quality control indexes of this carbonisata pieces.Conclusion: Most of commercial Platycladi Cacumen carbonisata pieces have the quality problem of excessive carbonizing degree,the established quality control standard of "carbonizing retains characteristics" can be used to effectively ensure the quality of Platycladi Cacumen carbonisata pieces.

OBJECTIVE: To evaluate the clinical significance of laparoscopic extraperitoneal para-aortic lmphadenectomy(PAL)in high para-aortic lymphadenectomy.METHODS: A non-randomized controlled method was used to analyze the clinical data of laparoscopic extraperitoneal PAL in 35 patients(extraperitoneal group)and laparoscopic transabdominal PAL in 40 patients(transabdominal group)in the Gynecological Department of the First Affiliated Hospital of Xiamen University from March 2018 to April 2019.RESULTS: All the 35 cases of laparoscopic extraperitoneal PAL reached the level of renal vein.Endometrial cancer accounted for the largest proportion,with an average age of 47.57 years and an average body mass index of 23.77.The average operation time was 109 minutes in the first 6 cases and 74.73 minutes in the other 29 cases.The amount of hemorrhage was 15.19 mL,and the average number of para-aortic lymph nodes(PALN)resected was 17.87.There were 5 cases of positive PALN metastasis,and the average gastrointestinal recovery time was13.43 hours.The average postoperative pelvic drainage was 76.57 hours and the average postoperative hospital stay was8.24 days.Intraoperative and postoperative complications included 1 case of inferior vena cava rupture,1 case of chyle leakage,and 1 case of vulvar edema.None of the 40 cases of laparoscopic transabdominal PAL reached the level of renal vein,and endometrial cancer accounted for the largest proportion,with the average age of 46.78 years and the average body mass index of 24.03.The average operation time was 90.55 min;the average intraoperative blood loss was 67.40 mL The average number of para-aortic lymph nodes was 3.30.There was one case of PALN metastasis.The average gastrointestinal recovery time was 22.35 hours,and the average postoperative pelvic drainage time was 75.75 hours.The average length of hospital stay was 8.90 days.There were 2 cases of rupture of the inferior mesenteric artery,3 cases of chyle leakage,and 2 cases of vulvar edema.The number of PALN resection,intraoperative blood loss,and operation time in the extraperitoneal group were not related to the body mass index,but the number of PALN in obese patients in the laparoscopic transabdominal group was significantly reduced,the operation time was longer,the intraoperative blood loss was more,and postoperative gastrointestinal recovery time is longer(P<0.05).CONCLUSION: Laparoscopic extraperitoneal PAL can solve the problems of laparoscopic transabdominal PAL lymph node resection,which is difficult to reach the level of renal vein,intraoperative intestinal tube interference,and the number of lymph nodes resected.It is especially suitable for obese patients.Laparoscopic extra-peritoneal high-grade PAL is safe,feasible,and recommended.

Fingerprints of lipophilic components in the roots of Salvia miltiorrhiza and S.yunnanensis were analyzed by UPLC-DADand UPLC coupled with mass spectroscopy to evaluate the differences and similarities of the lipophilic components in the two kinds of herbs.The UPLC analysis of 18 batches of S.miltiorrhiza and 16 batches of S.yunnanensis was performed on a 25℃Thermo Accucore C_(18)column(2.1 mm×100 mm,2.6μm)by Shimadzu LC-20AD;mobile phase was 0.026%phosphoric acid(A)-acetonitrile(B)with gradient elution;flow rate was 0.4 m L·min~(-1);detection wavelength was set at 270 nm;injection volume was 2μL.The molecular structures of the lipophilic components were analyzed on a 25℃Thermo Accucore C_(18)column(2.1 mm×100 mm,2.6μm)by Thermo U3000 UPLC Q Exactive Orbitrap LC-MS/MS with a mobile phaseconsisting of 0.1%formic acid water(A)and 0.1%formic acidacetonitrile(B).The mass spectrometry was acquired in positive modes using ESI.There are 10 common peaks in the lipophilic components of S.miltiorrhiza.The similarity between the 16 batches of S.miltiorrhiza and their own reference spectra was greater than 0.942,and the average similarity was 0.973.There are 12 common peaks in the lipophilic components of S.yunnanensis.The similarity between the 18 batches of S.yunnanensis and their own reference spectra was greater than 0.937,and the average similarity was 0.976.The similarity between the reference chromatograms of S.miltiorrhiza and S.yunnanensis was only 0.900.There are three lipophilic components in S.yunnanensis,which are not found in S.miltiorrhiza,and one of which isα-lapachone.There is a lipophilic component in S.miltiorrhiza not found in S.yunnanensis,which may be miltirone.The two herbs contain 8 common lipophilic components including dihydrotanshinoneⅠ,cryptotanshinone,tanshinoneⅠ,tanshinoneⅡ_A,nortanshinone in which the content of tanshinoneⅡ_A,dihydrotanshinoneⅠand cryptotanshinone of S.yunnanensisis significantly lower than that of S.miltiorrhiza(P<0.01),and the contents of tanshinoneⅠand nortanshinone are significantly lower than that of S.miltiorrhiza too(P<0.05).There are significant differences in the types and contents of lipophilic components between the roots of S.miltiorrhiza and S.yunnanensis,and the similarity between the fingerprints of interspecies is much lower than that between the same species.Therefore,the roots of S.miltiorrhiza and S.yunnanensis are two kinds of herbs which are quite different in chemical compounds and compositions.
Chromatography, Liquid ; Diterpenes, Abietane ; Molecular Structure ; Plant Roots ; Salvia miltiorrhiza ; Tandem Mass Spectrometry

Objective To investigate the association between the level of polymorphism of APOE gene and cognitive impairment in patients with CNS demyelinating diseases. Methods 56 patients with central nervous system demyelinating disease were applied APOE genotyping,MoCA and expanded disability status (EDSS) scale score. Patients with MOCA scores <26 were divided into cognitive impairment group, and those with MOCA scores ≥26 were divided into normal cognitive preserved group. Results The probability of cognitive dysfunction in patients with central nervous system demyelinating diseases was 53.57%. There was no significant difference in age, gender, and disease duration between the CI group and the CP group(P>0.05), the difference in age and education among groups is statistically significant (P<0.05). There was no statistical significance in the difference in age, sex, education years and EDSS score between APOEε4 gene positive group and APOEε4 gene negative group (P<0.05). The difference of visual space and attention between different cognitive domains is statistically significant(P<0.05). Years of schooling is a risk factor for cognitive dysfunction in patients with central nervous system demyelinating disease(P<0.01). Conclusion The central nervous system demyelinating disease is impaired cognitive function. Patients with APOEε4 gene positive are more severely impaired in visual space and attention than patients with negative APOEε4 gene.Years of education are the risk factors of cognitive dysfunction in patients with central nervous system demyelinating disease. The course of disease and disabled function may not be significant related to cognitive impairment.

Objective To investigate the clinical significance of cortactin antibody in myasthenia gravis (MG).Methods Cortactin antibody in the serum of 100 MG patients, 40 normal controls and 40 other neuroimmune diseases patients was examined by Western blotting and ELISA using purified recombinant human protein cortactin as antigen .Acetylcholine receptor antibody ( AchR-ab ) and muscle specific kinase antibody (MuSK-ab) were parallely measured by ELISA.Results Antibodies to cortactin were found in nine (9％) serum samples of 100 MG patients.Four of the nine cortactin antibody positive sera were also positive for AChR-ab.The rest five MG patients only had antibodies against cortactin ( no detectable AChR-ab or MuSK-ab).None of the control subjects (including 40 normal controls and 40 other neuroimmune diseases patients ) had cortactin antibodies.Most (7/9) of the cortactin antibody positive MG patients presented with early-onset subgroup.Patients only with cortactin antibodies did not appear to have thymoma.Patients with MG who had both AChR and cortactin antibodies showed maximum involvement of muscles and severe Osserman's classification ( three cases of type ⅡB and one case of type Ⅳ) . Conclusion Cortactin antibody may be a new antibody for MG , which can provide clues for further exploring the potential pathogenic mechanisms of the disease .