Objective:To explore the influence of tumor cells on CD4+CD25+Treg cells via TLRs.Methods:The numbers changes of CD4+CD25+Treg cells in the co-culture system of Lewis lung cancer cells and splenic lymphocytes were detected by flow cytometry;The expression of Foxp3 and TLR1-9 mRNA after co-culture were detected by RT-PCR;TLR9 expression on Lewis lung cancer cells was blocked by TLR9 receptor antagonist chloroquine.Results:Compared with control group,the number of CD4+CD25+Treg cells and Foxp3 mRNA expression were significantly increased in the co-culture group(P

Objective:To explore the influence of tumor cells on CD4~+ CD25~+ Treg cells via TLRs.Methods:The numbers changes of CD4~+ CD25~+ Treg cells in the co-culture system of Lewis lung cancer cells and splenic lymphocytes were detected by flow cytometry;The expression of Foxp3 and TLR1-9 mRNA after co-culture were detected by RT-PCR;TLR9 expression on Lewis lung cancer cells was blocked by TLR9 receptor antagonist chloroquine.Results:Compared with control group,the number of CD4~+ CD25~+ Treg cells and Foxp3 mRNA expression were significantly increased in the co-culture group (P＜0.05).The expression of a variety of TLRs were affected by the co-culture of lymphocytes with Lewis lung cancer cells,and TLR9 mRNA expression was significantly increased compared with that of the control group (P＜0.05);Blocking TLR9 of Lewis lung cancer cells significantly reduce CD4~+ CD25~+ Treg cells and Foxp3 mRNA (P＜0.05).Conclusion:Lewis lung cancer cells could increase both number and function of CD4~+ CD25~+ Treg cells through inducing TLR9 expression in immunocells.This might be one of mechanisms of tumor-induced immune tolerance,and by which to contribute to the occurrence and progression of tumors.

Objective:To reveal the effect of β-sitosterol on cerebral ischemia-reperfusion injury (CIRI) in rats and whether its mechanism is related to endoplasmic reticulum stress (ERS).Methods:Fifty-three CIRI rats (CIRI models established by modified Longa method) were randomly divided into model group ( n=14), β-sitosterol low-dose group ( n=13), β-sitosterol medium-dose group ( n=13) and β-sitosterol high-dose group ( n=13); 12 rats underwent the same operation without blocking the middle cerebral artery were selected as sham-operated group. Rats in the sham-operated group and model group were given intragastric administration of 1 mL 5 g/L sodium carboxymethyl cellulose daily. Rats in the β-sitosterol low-dose group, β-sitosterol medium-dose group and β-sitosterol high-dose group were given intragastric administration of 1 mL β-sitosterol at 10, 20 and 40 mg/kg/d (dissolved in 5 g/L sodium carboxymethyl cellulose), respectively, for 14 consecutive d. Neurological function was evaluated according to Zea Longa 5 method. Rats were sacrificed and brain tissues were collected. Volume of cerebral infarction was measured by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Brain injury and neuronal apoptosis were evaluated by HE staining, Nissl staining and TUNEL. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) contents were detected by water-soluble tetrazolium 1 (WST-1) method, colorimetric method or thiobarbituric acid (TBA) method, respectively. The mRNA and protein expression levels of protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE-1), activated transcription factor-6 (ATF-6), glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP) and Caspase-12 in the brain tissues were detected by qRT-PCR or Western blotting. Results:Compared with the sham-operated group, the model group had significantly increased neurological function score, cerebral infarction volume and TUNEL positive rate, decreased SOD and GSH-Px content, increased MDA content, and increased mRNA and protein expressions of PERK, IRE-1, ATF-6, GRP78, CHOP and Caspase-12 ( P<0.05). Compared with the model group, the β-sitosterol low-dose group, β-sitosterol medium-dose group and β-sitosterol high-dose group had significantly decreased neurological function score, cerebral infarction volume, and TUNEL positive rate, increased SOD and GSH-Px content, and decreased MDA content ( P<0.05); the β-sitosterol low-dose group, β-sitosterol medium-dose group and β-sitosterol high-dose group had significantly decreased mRNA and protein PERK expressions (mRNA: 2.17±0.17, 1.79±0.07 and 1.33±0.07; protein: 5.11±0.52, 2.91±0.26 and 1.98±0.17), IRE-1 expressions (mRNA: 1.75±0.18, 1.65±0.08 and 1.32±0.08; protein: 5.00±0.31, 4.05±0.27 and 1.98±0.14), ATF-6 expressions (mRNA: 2.24±0.12, 1.77±0.14 and 1.37±0.13; protein: 4.93±0.45, 4.04±0.30 and 3.10±0.20), GRP78 expressions (mRNA: 2.67±0.16, 2.11±0.16 and 1.69±0.11; protein: 5.02±0.38, 2.97±0.26 and 2.05±0.22), CHOP expressions (mRNA: 2.01±0.16, 1.70±0.19 and 1.40±0.10; protein: 4.92±0.39, 4.02±0.27 and 3.08±0.22) and Caspase-12 expressions (mRNA: 1.85±0.09, 1.61±0.09 and 1.30±0.09; protein: 3.03±0.20, 2.19±0.11 and 1.82±0.11) compared with the model group (mRNA: 2.99±0.28, 2.27±0.12, 2.57±0.21, 3.46±0.20, 2.50±0.23 and 2.35±0.16; protein: 6.98±0.48, 6.03±0.58, 5.98±0.63, 7.10±0.45, 6.00±0.53 and 5.02±0.43, P<0.05). Conclusion:β-sitosterol attenuates CIRI in rats, whose mechanism may be related to inhibition of ERS signal pathway.

OBJECTIVE To establish HPLC characteristic chro matogram of Jianpi yifei biyan prescription standard decoction ， to select the quality control index components and determine their contents. METHODS HPLC method combined with Similarity Evaluation System of TCM Chromatographic Fingerprint （2004 edition）were used to establish the characteristic chromatogram of 10 batches of Jianpi yifei biyan prescription standard decoction ；the similarity evaluation and common peaks identification were also carried out. Using common peak area of characteristic chromatogram as variables ，SPSS 26.0 software and SIMCA 14.1 software were used to perfor m cluster analysis （CA），principal component analysis （PCA） and orthogonal partial least squares-discriminant analysis （OPLS-DA）；differential components with variable important i n pro jection（VIP）value greater than 1.5 were screened；the contents of cimifugin and differential components were determined by the same method. RESULTS A total of 24 common characteristic peaks were identified ， and the similarities of 10 batches of samples were higher than 0.960；eight characteristic peaks were identified by comparison with reference substance. CA and PCA results revealed that the samples were classified into 3 categories.OPLS-DA analysis showed that 3 components with VIP value greater than 1.5， which were prim-O-glucosylcimifugin （peak 2），calycosin 7-O-β-D-glucopyranoside （peak 4） and 5-O-methylvisammioside （peak 6） in descending order. The linear ranges of prim- O- glucosylcimifugin，calycosin 7-O-β-D-glucopyranoside，cimifugin and 5-O-methylvisammioside were 0.010 7-0.213 0，0.007 8- 0.156 0，0.008 0-0.160 0，0.009 8-0.195 0 μg（r＞0.999），respectively. RSD values of precision ，repeatability and stability tests （24 h） were all less than 2％. Average recoveries were 105.98％（RSD＝1.75％，n＝6），98.06％（RSD＝3.87％，n＝6），96.38％（RSD＝ 4.03％ ，n＝6） and 104.17％（RSD＝1.27％ ，n＝6）. The contents of the above 4 components in 10 batches of samples were 12.12-18.87，3.86-6.40，3.10-4.27 and 11.17-15.79 μ g/mL，respectively. CONCLUSIONS The established HPLC characteristic chromatographic method is stable and feasible ，it can be used for the quality control of Jianpi yifei biyan prescription standard decoction. Prim- O-glucosylcimifugin，calycosin 7-O-β-D-glucopyranoside，cimifugin and 5-O-methylvisammioside can be used as the index components for quality control of the standard decoction.