OBJECTIVE:To establish a method for the contents determination and cluster analysis of free and combined anthra-quinone of aloe emodin,rhein,emodin,rhubarb phenol and physcion in commercially available Yiqing granule from different man-ufacturers. METHODS:HPLC was performed on the column of Eclipse plus C18 with mobile phase of methanol-0.1%Phosphoricac-id solution (gradient elution) at a flow rate of 0.8 ml/min,the detection wavelength was 254 nm,the column temperature was 25 ℃,and the injection volume was 20 μl. RESULTS:The linear range was 0.001 6-0.128 0 μg for aloe emodin(r=0.999 9), 0.003 4-0.273 6 μg for rhein(r=0.999 9),0.003 7-0.299 2 μg for emodin(r=0.999 9),0.006 7-0.536 0 μg for rhubarb phenol (r=0.999 9)and 0.001 7-0.134 4 μg(r=0.999 9)for physcion;RSDs of precision,stability and reproducibility tests were lower than 3%;recoveries were 97.77%-101.47%(RSD=1.30%,n=6),98.09%-100.26%(RSD=0.76%,n=6),96.42%-100.38%(RSD=1.58%,n=6),96.63%-102.50%(RSD=2.17%,n=6) and 97.10%-103.70%(RSD=2.47%,n=6),respectively;the content range of total anthraquinone was 0.042-0.218 mg/g, 0.029-0.448 mg/g, 0.022-0.167 mg/g, 0.032-0.284 mg/g and 0.006-0.060 mg/g,combined anthraquinone was 0.010-0.111 mg/g,0.013-0.092 mg/g,0.011-0.097 mg/g,0.030-0.246 mg/g and 0.001-0.034 mg/g,and free anthraquinone was 0.022 3-0.143 mg/g,0.015-0.356 mg/g,0.008-0.071 mg/g,0.006-0.075 mg/g and 0.003-0.032 mg/g. Cluster analysis showed there were 4 batches belonged to Category 1,2 batches belonged to Category 2 and 4 batches belonged to Category 3 for the total anthraquinone in 5 components;4 bathes belonged to Category 1,2 batches belonged to Category 2 and 4 batches belonged to Category 3 for the combined anthraquinone;and 6 bathes belonged to Category 1 and 4 batches belonged to Category 2 for the free anthraquinone. CONCLUSIONS:The method is simple and accurate,and can provide reference for improving the quality and diarrhea efficacy control of Yiqing granule;there were great differences in Rheum palma-tum and combined anthraquinone from different manufacturers.

ObjectiveTo observe the clinical efficacy of acupuncture combined with bloodletting and cupping in the treatment of chronic spontaneous urticaria（CSU） and to explore its potential mechanisms of action. MethodsSeventy CSU patients were randomly divided into loratadine group and acupuncture + bloodletting group， with 35 patients in each group. The loratadine group received oral loratadine tablets， 10 mg once daily in the evening. The acupuncture + bloodletting group received acupuncture at Zhongwan （CV 12）， Guanyuan （CV 4）， Tianshu （ST 25）， Zusanli （ST 36）， Sanyinjiao （SP 6）， Xuehai （SP 10）， Quchi （LI 11）， Hegu （LI 4）， Taichong （LR 3）， Baihui （GV 20）， and Shenting （GV 24）， once daily，along with bloodletting and cupping at Dazhui （GV 14） and Geshu （BL 17）， every other day. Both groups were treated for 4 weeks. The 7-day urticaria activity score（UAS7） was assessed before and after the treatment， and levels of serum immunoglobulin E （IgE）， interleukin-4 （IL-4）， interleukin-5 （IL-5）， eosinophil cationic protein （ECP）， plasma tissue factor （TF）， activated factor Ⅶ （FⅦa）， prothrombin fragment 1+2 （F1+2）， D-dimer （D-D） and complement component 5a （C5a） were detected. ResultsA total of 65 patients were included in the final analysis， 32 in the loratadine group and 33 in the acupuncture + bloodletting group. Before treatment， there was no significant difference in UAS7 score， serum IgE， IL-4， IL-5， ECP levels， or plasma TF， FⅦa， F1+2， D-D， C5a levels between groups （P＞ 0.05）. After treatment， both groups showed significant reductions in UAS7 score， serum IgE， IL-4， IL-5， and plasma TF， FⅦa， F1+2， D-D， and C5a levels compared to those before treatment （P＜0.01）. However， after treatment， there was no significant difference in UAS7 score and serum ECP， IgE， IL-4， IL-5 levels between groups （P＞0.05）. The acupuncture + bloodletting group showed lower plasma TF， FⅦa， F1+2， D-D and C5a levels compared to the loratadine group （P＜0.05 or P＜0.01）. ConclusionAcupuncture combined with bloodletting and cupping can effectively improve the skin symptoms of CSU patients and reduce the levels of inflammatory factors. The potential mechanism of action may involve the regulation of the coagulation-complement-mast cell activation axis， thereby inhibiting mast cell degranulation.

Developing new therapeutic agents for cancer immunotherapy is highly demanding due to the low response ratio of PD-1/PD-L1 blockade in cancer patients. Here, we discovered that the novel immune checkpoint VISTA is highly expressed on a variety of tumor-infiltrating immune cells, especially myeloid derived suppressor cells (MDSCs) and CD8⁺ T cells. Then, peptide C1 with binding affinity to VISTA was developed by phage displayed bio-panning technique, and its mutant peptide VS3 was obtained by molecular docking based mutation. Peptide VS3 could bind VISTA with high affinity and block its interaction with ligand PSGL-1 under acidic condition, and elicit anti-tumor activity in vivo. The peptide DVS3-Pal was further designed by d-amino acid substitution and fatty acid modification, which exhibited strong proteolytic stability and significant anti-tumor activity through enhancing CD8⁺ T cell function and decreasing MDSCs infiltration. This is the first study to develop peptides to block VISTA/PSGL-1 interaction, which could act as promising candidates for cancer immunotherapy.