ObjectiveTo evaluate the impact of yang-reinforcing and blood-activating therapy on the long-term prognosis for patients with dilated cardiomyopathy （DCM） of yang deficiency and blood stasis syndrome. MethodsA retrospective cohort study was conducted involving 371 DCM patients with yang deficiency and blood stasis syndrome. The yang-reinforcing and blood-activating therapy was defined as the exposure factor. Patients were categorized into exposure group （186 cases） and non-exposure group （185 cases） according to whether they received yang-reinforcing and blood-activating therapy combined with conventional western medicine for 6 months or longer. The follow-up period was set at 48 months， and the Kaplan-Meier survival analysis was used to assess the cumulative incidence of major adverse cardiovascular events （MACE） in both groups. Cox regression analysis was used to explore the impact of yang-reinforcing and blood-activating therapy on the risk of MACE， and subgroup analysis was performed. Changes in traditional Chinese medicine （TCM） syndrome score， left ventricular ejection fraction （LVEF）， left ventricular fractional shortening （LVFS）， left ventricular end-diastolic diameter （LVEDD）， and Minnesota Living with Heart Failure Questionnaire （MLHFQ） score were compared between groups at the time of first combined use of yang-reinforcing and blood-activating therapy （before treatment） and 1 year after receiving the therapy （after treatment）. ResultsMACE occurred in 31 cases （16.67%） in the exposure group and 47 cases （25.41%） in the non-exposure group. The cumulative incidence of MACE in the exposure group was significantly lower than that in the non-exposure group ［HR=0.559， 95%CI（0.361，0.895）， P=0.014］. Cox regression analysis showed that yang-reinforcing and blood-activating therapy was an independent factor for reducing the risk of MACE in DCM patients ［HR=0.623， 95%CI（0.396，0.980）， P=0.041］， and consistent results were observed in different subgroups. Compared with pre-treatment， the exposure group showed decreased TCM syndrome score and MLHFQ score， reduced LVEDD， and increased LVEF and LVFS after treatment （P＜0.05）； in the non-exposure group， TCM syndrome score decreased， LVEF and LVFS increased， and LVEDD reduced after treatment （P＜0.05）. After treatment， the exposure group had higher LVEF and LVFS， smaller LVEDD， and lower TCM syndrome score and MLHFQ score compared with the non-exposure group （P＜0.05）. ConclusionCombining yang-reinforcing and blood-activating therapy with conventional western medicine can reduce the risk of MACE in DCM patients with yang deficiency and blood stasis syndrome， meanwhile improving their clinical symptoms， cardiac function， and quality of life.

Objective:This study aimed to investigate the role of bone morphogenetic protein 2 (BMP2)/Smad8 signaling pathway in T-2 toxin-induced apoptosis of rat articular chondrocytes.Methods:Primary chondrocytes from SD rats were cultured in vitro and exposed to varying concentrations of T-2 toxin (0.00, 0.32, 1.60, 8.00 ng/ml). The changes in chondrocytes survival rate were determined by CCK8, and the apoptosis changes of chondrocytes were determined by TUNEL assay kit. Using a group design, chondrocytes were cultured in complete culture media and culture media containing T-2 toxin (1.60 ng/ml), BMP2 cytokine (500 ng/ml), or T-2 toxin (1.60 ng/ml) + BMP2 cytokine (500 ng/ml), referred to as the control group, T-2 toxin group, BMP2 group, and T-2 toxin + BMP2 group, respectively. The survival rate and apoptosis changes of chondrocytes in each group were determined. The expression levels of Caspase-3, BMP2, BMP receptor Ⅱ (BMP-R Ⅱ), and Smad1/4/5/8 were determined by quantitative real-time PCR. Results:Compared with the 0.00 ng/ml of T-2 toxin group [(100.00 ± 0.00)%, (4.33 ± 0.32)%], the chondrocyte survival rates [(85.77 ± 2.96)%, (72.79 ± 2.31)%, (48.87 ± 1.83)%] of the 0.32, 1.60, and 8.00 ng/ml of T-2 toxin groups were significantly lower ( P < 0.05), and the apoptosis rates [(5.43 ± 0.32)%, (6.17 ± 0.15)%, (5.07 ± 0.13)%] were significantly higher ( P < 0.05). Compared with the control group, the T-2 toxin group had a lower survival rate and a higher apoptosis rate of chondrocytes ( P < 0.05). Compared with the T-2 toxin group, the T-2 toxin + BMP2 group had a higher survival rate and lower apoptosis rate of chondrocytes ( P < 0.05). Compared with the control group, the T-2 toxin group showed higher expression level of Caspase-3 mRNA in chondrocytes, while the expression levels of BMP2, BMP-R Ⅱ, and Smad1/4/8 mRNA were lower ( P < 0.05). Compared with the T-2 toxin group, the expression level of Caspase-3 mRNA was lower in the T-2 toxin + BMP2 group, while the expression levels of BMP2 and Smad8 mRNA were higher ( P < 0.05). Conclusion:BMP2 may partially block the apoptosis of chondrocytes caused by T-2 toxin by regulating the BMP2/Smad8 signaling pathway.

Objective To evaluate the quality of Shuanghuanglian Oral Liquid after mutual substitution of honeysuckle and wild honeysuckle using total quantum statistical moment(TQSM)and molecular connectivity index(MCI).Methods UPLC fingerprint of Shuanghuanglian Oral Liquid(honeysuckle)and Shuanghuanglian oral liquid(wild honeysuckle)were established,the TQSM parameters and similarity of the fingerprint were calculated;by reviewing relevant literature,as well as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),chemical composition databases for Shuanghuanglian Oral Liquid(honeysuckle)and Shuanghuanglian Oral Liquid(wild honeysuckle)was established,all components were divided into different component groups,and MCI and its similarity were calculated.Results The number of chromatographic peaks and total zero order moment(AUCT)of 15 batches of Shuanghuanglian Oral Liquid(honeysuckle)were higher than those of Shuanghuanglian Oral Liquid(wild honeysuckle),but there was no significant difference in total first order moment(MRTT)and total second order moment(VRTT);the total quantum statistical moment similarity(TQSMS)between 15 batches of Shuanghuanglian Oral Liquid(honeysuckle)was 1.000 0-0.824 6,the TQSMS between 15 batches of Shuanghuanglian Oral Liquid(wild honeysuckle)was 1.000 0-0.659 0,and the TQSMS between 15 batches of Shuanghuanglian Oral Liquid(honeysuckle)and Shuanghuanglian Oral Liquid(wild honeysuckle)was 1.000 0-0.619 8.The MCI similarity of various components between Shuanghuanglian Oral Liquid(honeysuckle)and Shuanghuanglian Oral Liquid(wild honeysuckle)was 1.000 0-0.984 9,with an overall MCI similarity of 0.995 8.Conclusion There is no significant difference in the various components and overall"imprinting template"between Shuanghuanglian Oral Liquid(honeysuckle)and Shuanghuanglian Oral Liquid(wild honeysuckle).It is speculated that the substitution of honeysuckle and wild honeysuckle will not affect the pharmacological properties of Shuanghuanglian Oral Liquid,but there may be differences in the intensity of pharmacological effects,with Shuanghuanglian Oral Liquid(honeysuckle)being the most effective.

ObjectiveTo explore the correlation between the occurrence of cardiovascular events in rheumatoid arthritis（RA） and traditional Chinese medicine（TCM） syndrome. MethodsThe cross-sectional study selected 6713 RA patients from 122 centres nationwide， in which general information such as name， gender， age， height， body weight， and course of disease were collected by completing a questionnaire； patients were classified into eight types of syndrome according to the information of their four examinations，i.e. wind-dampness obstruction syndrome， cold-dampness obstruction syndrome， dampness-heat obstruction syndrome， phlegm-stasis obstruction syndrome， stasis-blood obstructing collateral syndrome， qi-blood deficiency syndrome， liver-kidney insufficiency syndrome， and qi-yin deficiency syndrome. According to the occurrence of cardiovascular events， they were divided into the occurrence group and the non-occurrence group， and the condition assessment data and laboratory examination indexes were recorded. The test of difference between groups was used to analyse the possible risk factors for the occurrence of RA cardiovascular events， and binary logistic regression was used to analyse the correlation between TCM syndromes and RA cardiovascular events. ResultsA total of 6713 RA patients were included， including 256 cases in occurrence group and 6457 in non-occurrence group. There was no statistically significant difference between groups in terms of height， gender， insomnia， appetite， white blood cell（WBC）， hemoglobin（HGB）， platelets（PLT）， rheumatoid factor（RF）， anti-cyclic peptide containing citrulline（CCP）， alanine aminotransferase（ALT）， aspartate aminotransferase（AST）， γ-glutamyl transpeptidase（GGT）， urea creatinine（CREA）， and glucose（GLU）（P＞0.05）. The TCM syndromes between groups showed significant statistic differences（P＜0.05）. Patients in occurrence group had longer disease duration， heavier body weight， and older age； more severe conditions such as disease activity（DAS-28）， number of painful joints（TJC）， number of swollen joints（SJC）， health questionnaire scores（HAQ）， visual analog scores（VAS）， restlessness， and fatigue； higher blood sedimentation rate（ESR）， low-density lipoprotein（LDL-C）， triglyceride（TG）， total cholesterol（TC）， D-Dimer， and lower high-density lipoprotein（HDL-C）（P＜0.05）. The distribution of syndrome types showed that dampness-heat obstruction syndrome accounted for the largest proportion of patients in both groups and was higher in RA cardiovascular events. Logistic regression analysis showed that the occurrence of RA cardiovascular events was strongly associated with dampness-heat obstruction syndrome［OR=5.937， 95%CI （4.434， 7.949）， P＜0.001］. ConclusionThe occurrence of RA cardiovascular events were associated with TCM syndromes， and the probability of cardiovascular events in the RA patients with dampness-heat obstruction syndrome was 5.937 times higher than patients with other TCM syndromes.

Objective:This study aimed to investigate the role of bone morphogenetic protein 2 (BMP2)/Smad8 signaling pathway in T-2 toxin-induced apoptosis of rat articular chondrocytes.Methods:Primary chondrocytes from SD rats were cultured in vitro and exposed to varying concentrations of T-2 toxin (0.00, 0.32, 1.60, 8.00 ng/ml). The changes in chondrocytes survival rate were determined by CCK8, and the apoptosis changes of chondrocytes were determined by TUNEL assay kit. Using a group design, chondrocytes were cultured in complete culture media and culture media containing T-2 toxin (1.60 ng/ml), BMP2 cytokine (500 ng/ml), or T-2 toxin (1.60 ng/ml) + BMP2 cytokine (500 ng/ml), referred to as the control group, T-2 toxin group, BMP2 group, and T-2 toxin + BMP2 group, respectively. The survival rate and apoptosis changes of chondrocytes in each group were determined. The expression levels of Caspase-3, BMP2, BMP receptor Ⅱ (BMP-R Ⅱ), and Smad1/4/5/8 were determined by quantitative real-time PCR. Results:Compared with the 0.00 ng/ml of T-2 toxin group [(100.00 ± 0.00)%, (4.33 ± 0.32)%], the chondrocyte survival rates [(85.77 ± 2.96)%, (72.79 ± 2.31)%, (48.87 ± 1.83)%] of the 0.32, 1.60, and 8.00 ng/ml of T-2 toxin groups were significantly lower ( P < 0.05), and the apoptosis rates [(5.43 ± 0.32)%, (6.17 ± 0.15)%, (5.07 ± 0.13)%] were significantly higher ( P < 0.05). Compared with the control group, the T-2 toxin group had a lower survival rate and a higher apoptosis rate of chondrocytes ( P < 0.05). Compared with the T-2 toxin group, the T-2 toxin + BMP2 group had a higher survival rate and lower apoptosis rate of chondrocytes ( P < 0.05). Compared with the control group, the T-2 toxin group showed higher expression level of Caspase-3 mRNA in chondrocytes, while the expression levels of BMP2, BMP-R Ⅱ, and Smad1/4/8 mRNA were lower ( P < 0.05). Compared with the T-2 toxin group, the expression level of Caspase-3 mRNA was lower in the T-2 toxin + BMP2 group, while the expression levels of BMP2 and Smad8 mRNA were higher ( P < 0.05). Conclusion:BMP2 may partially block the apoptosis of chondrocytes caused by T-2 toxin by regulating the BMP2/Smad8 signaling pathway.

Objective To evaluate the quality of Shuanghuanglian Oral Liquid after mutual substitution of honeysuckle and wild honeysuckle using total quantum statistical moment(TQSM)and molecular connectivity index(MCI).Methods UPLC fingerprint of Shuanghuanglian Oral Liquid(honeysuckle)and Shuanghuanglian oral liquid(wild honeysuckle)were established,the TQSM parameters and similarity of the fingerprint were calculated;by reviewing relevant literature,as well as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),chemical composition databases for Shuanghuanglian Oral Liquid(honeysuckle)and Shuanghuanglian Oral Liquid(wild honeysuckle)was established,all components were divided into different component groups,and MCI and its similarity were calculated.Results The number of chromatographic peaks and total zero order moment(AUCT)of 15 batches of Shuanghuanglian Oral Liquid(honeysuckle)were higher than those of Shuanghuanglian Oral Liquid(wild honeysuckle),but there was no significant difference in total first order moment(MRTT)and total second order moment(VRTT);the total quantum statistical moment similarity(TQSMS)between 15 batches of Shuanghuanglian Oral Liquid(honeysuckle)was 1.000 0-0.824 6,the TQSMS between 15 batches of Shuanghuanglian Oral Liquid(wild honeysuckle)was 1.000 0-0.659 0,and the TQSMS between 15 batches of Shuanghuanglian Oral Liquid(honeysuckle)and Shuanghuanglian Oral Liquid(wild honeysuckle)was 1.000 0-0.619 8.The MCI similarity of various components between Shuanghuanglian Oral Liquid(honeysuckle)and Shuanghuanglian Oral Liquid(wild honeysuckle)was 1.000 0-0.984 9,with an overall MCI similarity of 0.995 8.Conclusion There is no significant difference in the various components and overall"imprinting template"between Shuanghuanglian Oral Liquid(honeysuckle)and Shuanghuanglian Oral Liquid(wild honeysuckle).It is speculated that the substitution of honeysuckle and wild honeysuckle will not affect the pharmacological properties of Shuanghuanglian Oral Liquid,but there may be differences in the intensity of pharmacological effects,with Shuanghuanglian Oral Liquid(honeysuckle)being the most effective.

ObjectiveBased on the network pharmacology system and quantitative spectroscopy of traditional Chinese medicine（TCM） compounds， a topological network analysis method with equilibrium constant as the core was established to further explore the interaction between allergenic components and their network targets in Shuanghuanglian injection（SHLI）， in order to provide new ideas and experimental basis for identifying and screening potential allergens of SHLI. MethodAfter one week of adaptive feeding， 72 SPF-grade SD male rats were randomly divided into blank group， SHLI standard group， Lonicerae Japonicae Flos（LJF） group， Scutellariae Radix（SR） group， Forsythiae Fructus（FF） group， and 7 groups of SHLI matching groups（groups 1-7）， with 6 rats in each group. Rats in each group were administered the drug intravenously and blood samples were taken after steady state， high performance liquid chromatography（HPLC） characterization profiles of the testing drugs and plasma components in each group were established， and the peak area changes of the drugs and plasma components in each group were calculated after the component groups were classified. Enzyme-linked immunosorbent assay（ELISA） was used to determine the changes of immunoglobulin E（IgE）， histamine（HIS）， tryptase（TPS）， total complement（CH50） and terminal complement complex（C5b-9） in animal blood samples. MATLAB R2020b v9.9.0 software was used to calculate the network balance constants of the component groups with the targets， and the eigenvalues of the matrices composed of network equilibrium constants were calculated and ranked according to their values. ResultELISA results showed that， compared with the blank group， groups 1-3 could significantly increase the IgE level， groups 1-2， groups 4-6 and SHLI standard group could significantly increase the HIS level， group 4 could significantly increase the CH50 level， groups 1， 3-4， LJF group and FF group could significantly increase the TPS level， SR group could significantly increase the C5b-9 level， and the differences were all statistically significant（P<0.05）. According to the retention time of chromatographic peaks， it was classified into 6 component groups from C1 to C6 by HPLC. The order of the network balance constants of each component group was C6>C4>C1>C5>C3>C2， indicating that C6 had the greatest effect on the allergic reaction， and was most likely to be the allergen. The sequence of eigenvalues was C2>C5b-9>C3>C1>CH50>C6>C5>IgE>TPS>C4>HIS， indicating that component group C2 had the greatest contribution to the whole network. ConclusionBased on the correlation analysis of SHLI component group and allergy-like target network， this study clarified that component group C6 may be a potential allergen in SHLI， and the component group C2 may be a key node in the mechanism of drug action， which can provide new strategies and methods for the screening of allergens in TCM injections.

ObjectiveTo establish a theoretical system of pharmacokinetic and spectrokinetic dose-time characterization of traditional Chinese medicine（TCM）. By analyzing the pharmacokinetic and spectrokinetic behaviors of Lonicerae Flos， Houttuyniae Herba injection， Lonicerae Japonicae Flosand Buyang Huanwutang， this paper compared the similarities and differences of the three methods for characterizing the dose-time relationship， namely half-life， statistical moment and statistics， in order to find the most suitable method for characterizing the relationship. MethodTen mice were randomly selected from 100 Kunming mice as the blank group， and the remaining mice were coated with xylene in the auricle to establish the acute inflammation model of ear swelling. After successful modeling， the mice were gavaged with aqueous extract of Lonicerae Flos（30 g∙kg^-1）， and the blank group was gavaged with an equal volume of normal saline. The plasma of mice was collected at different time points to determine the content changes of components. At the same time， the pharmacokinetic results of Houttuyniae Herba injection， Lonicerae Japonicae Flos and Buyang Huanwutang were included， and the pharmacokinetic and spectrokinetic parameters were calculated. Then the difference in the time of calculating 95% total component content of metabolism by half-life method， statistical moment method and statistical method was compared. ResultOn the basis of the half-life method， the mathematical expressions of statistical moment method and statistical method suitable for the characterization of dose-time relationship of multi-component system of TCM were established. The results showed that the pharmacokinetic parameters of the individual components in Lonicerae Flos varied， with cryptochlorogenic acid and rutin showing a two-compartment model and the other components showing a one-compartment model. After calculation of spectrokinetic similarity， the metabolic patterns among the components contained in Houttuyniae Herba injection， Lonicerae Japonicae Flos， Lonicerae Flos and Buyang Huanwutang were different and varied greatly in vivo. The time to metabolize 95% of the total components of the four research subjects in vivo was calculated by the half-life method， statistical moment method and statistical method， and it was found that the difference between statistical moment method and half-life method was large， and the difference between statistical moment method and statistical method was small. ConclusionStatistical method not only reflects the characteristics of statistical moment method， characterizes the dispersion degree of each component， but also can be associated with fingerprint to form spectrokinetics， characterizing the dose-time relationship of 95% of drug components， which is a more desirable method to characterize the dose-time relationship of the component groups in TCM.

Methods: This study established the MFSM method. To demonstrate its effectiveness, we applied this novel approach to analyze Danxi Granules (丹膝颗粒, DXG) and its constituent herbal materials. To begin with, the ultra-performance liquid chromatography (UPLC) was applied to obtain the chromatographic fingerprints of DXG and its constituent herbal materials. Next, the MFSM was leveraged to compress and integrate them into a new fingerprint with fewer analytical units. Then, we characterized the properties and variability of both the original and integrated fingerprints by calculating total quantum statistical moment (TQSM) parameters, information entropy and information amount, along with their relative standard deviation (RSD). Finally, we compared the TQSM parameters, information entropy and information amount, and their RSD between the traditional and novel fingerprints to validate the new analytical method. Results: The chromatographic peaks of DXG and its 12 raw herbal materials were divided and integrated into peak families by the MFSM method. Before integration, the ranges of the peak number, three TQSM parameters, information entropy and information amount for each peak or peak family of UPLC fingerprints of DXG and its 12 raw herbal materials were 95.07 − 209.73, 9390 − 183064 μv·s, 5.928 − 21.33 min, 22.62 − 106.69 min2, 4.230 − 6.539, and 50530 − 974186 μv·s, respectively. After integration, the ranges of these parameters were 10.00 − 88.00, 9390 − 183064 μv·s, 5.951 − 22.02 min, 22.27 − 104.73 min2, 2.223 − 5.277, and 38159 − 807200 μv·s, respectively. Correspondingly, the RSD of all the aforementioned parameters before integration were 2.12% − 9.15%, 6.04% − 49.78%, 1.15% − 23.10%, 3.97% − 25.79%, 1.49% − 19.86%, and 6.64% − 51.20%, respectively. However, after integration, they changed to 0.00%, 6.04% − 49.87%, 1.73% − 23.02%, 3.84% − 26.85%, 1.17% − 16.54%, and 6.40% − 48.59%, respectively. The results demonstrated that in the newly integrated fingerprint, the analytical units of constituent herbal materials, information entropy and information amount were significantly reduced (P < 0.05), while the TQSM parameters remained unchanged (P > 0.05). Additionally, the RSD of the TQSM parameters, information entropy, and information amount didn’t show significant difference before and after integration (P > 0.05), but the RSD of the number and area of the integrated analytical units significantly decreased (P < 0.05). Conclusion The MFSM method could reduce the analytical units of constituent herbal materials while maintain the properties and variability from their original fingerprint. Thus, it could serve as a feasible and reliable tool to reduce difficulties in analyzing multi-components within CMMs and facilitating the evaluation of their quality.

Objective To establish a rapid detection method for zika virus based on direct amplification re-al-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)technique.Methods A direct amplification RT-PCR technique for the rapid detection of zika virus in 5 samples(whole blood,serum,saliva,throat swab and urine)was established by using a special function DNA polymerase and a preferred PCR enhancer.Results The detection limits of the 5 samples were 103 PFU/mL in serum,102 PFU/mL in urine,throat swab,and saliva,and 104 PFU/mL in whole blood.The coefficient of goodness-fit of stand-ard curves was above 0.98,and the amplification efficiency was 90％-110％.Zika virus nucleic acid was suc-cessfully amplified,but non-zika virus nucleic acid was not amplified.Based on the repeatable detection of sam-ples from urine,whole blood,and saliva,the variation coefficient of 6 repeated Ct values at 106 PFU/mL and 102 PFU/mL concentrations were all＜5％.The zika virus detection method established by the direct amplifi-cation RT-PCR technique was consistent with the detection results of conventional RT-PCR technique.Only two serum samples were detected in eight zika virus samples,and the remaining 62 non-zika virus samples and 12 negative samples were not amplified.Conclusion A rapid detection method for zika virus based on direct ampli-fication RT-PCR technique is successfully established.The method is simple,rapid,sensitive and specific.