ObjectiveTo explore effect of Xianlian Jiedu prescription on the recurrence of colorectal cancer （CRC） and investigate the related mechanisms. MethodsA postoperative recurrence model was established in 25 Balb/c mice by injecting CT26 cells subcutaneously into the armpit， followed by surgical removal of 99% of the subcutaneous tumor. The mice were randomly divided into model group， low-dose Xianlian Jiedu prescription （XLJDP-L） group （6.45 g·kg^-1·d^-1）， medium-dose Xianlian Jiedu prescription （XLJDP-M） group （12.9 g·kg^-1·d^-1）， high-dose Xianlian Jiedu prescription （XLJDP-H） group （25.8 g·kg^-1·d^-1）， and 5-fluorouracil （5-FU） group （1×10^-3 g·kg^-1·d^-1）. The mice were euthanized after 14 days of continuous intervention， and recurrent tumor tissue was harvested. Hematoxylin and eosin （HE） staining was used to observe pathological and morphological changes in the recurrent tumor tissue. Immunohistochemistry （IHC） was employed to assess the expression of proliferating cell nuclear antigen （Ki67）， vascular endothelial growth factor （VEGF）， and platelet-endothelial cell adhesion molecule （CD31） in recurrent tumor tissue. The Western blot was used to detect the protein expression levels of angiopoietin-2 （ANG-2）， VEGF， phosphorylated-protein kinase B （p-Akt）， protein kinase B （Akt）， phosphorylated-phosphatidylinositol 3-kinase （p-PI3K）， and phosphatidylinositol 3-kinase （PI3K） in recurrent tumor tissue. ResultsBefore treatment， there were no statistical differences in tumor volume， tumor weight， and body mass among the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group compared to the model group， indicating model stability. After treatment， compared with those in the model group， the tumor volume and tumor weight in the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group were significantly reduced （P<0.01）， showing dose dependency. Meanwhile， there were no significant differences in body weight among the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group compared to the model group. HE staining showed that compared with that in the model group， tumor tissue in the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group had loosely arranged cells， increased intercellular spaces， small and shriveled nuclei， light staining， fewer mitotic figures and atypical nuclei， and increased necrotic areas. IHC showed that compared with those of the model group， the positive rates of Ki67， VEGF， and CD31 in the recurrent tumor tissue of the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group were significantly reduced （P<0.01） in a dose-dependent manner. Western blot results showed that compared with those of the model group， the protein expression levels of ANG-2 and VEGF in the recurrent tumor tissue of the XLJDP-L， XLJDP-M， and XLJDP-H groups and the 5-FU group were significantly downregulated （P<0.05， P<0.01）， and the p-Akt/Akt and p-PI3K/PI3K ratios were significantly decreased in a dose-dependent manner （P<0.05， P<0.01）. ConclusionXianlian Jiedu prescription significantly inhibits the recurrence of CRC in mice after subcutaneous tumor surgery. The mechanism may involve regulating the PI3K/Akt pathway and downregulating key angiogenic proteins such as ANG-2， VEGF， and CD31.

Pheretima,also called"earthworms",is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edi-tion).However,its zoological origin is unclear,both in the herbal market and CPMs.In this study,a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing al-gorithms(restricted search,open search,and de novo)was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima,including Pheretima aspergillum(PA),Pheretima vulgaris(PV),and Metaphire magna(MM).We identified 10,477 natural peptides in the PA,7,451 in PV,and 5,896 in MM samples.Five specific signature peptides were screened and then validated using synthetic peptides;these demonstrated robust specificity for the authentication of PA,PV,and MM.Finally,all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills,revealing the inconsistent Pheretima species used in these CPMs.In conclusion,our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines,especially non-model species with poorly annotated protein databases.

ObjectiveTo investigate the mechanism by which Shenbai Jiedu prescription （SBJDF） inhibits the proliferation of colorectal cancer （CRC） HCT116 cells. MethodAfter 48 h treatment of HCT116 cells with SBJDF （0， 0.25， 0.5， 1， 2， 4 g·L^-1）， the viability of HCT116 cells were determined by methyl thiazolyl tetrazolium （MTT） colorimetry. Following the classification of cells into blank control group and SBJDF （1， 2， 4 g·L^-1） groups， the effect of SBJDF on HCT116 cell morphology was observed under an inverted microscope. The effects of SBJDF on the proliferation of HCT116 cells and mitochondrial membrane potential （Δψm） were detected by colony formation assay and JC-1 probe， respectively. The flow cytometry was then performed for determining cell cycle distribution and apoptosis. The effects of SBJDF on cell cycle-， apoptosis-， and nuclear factor kappa-B （NF-κB） signaling pathway-related proteins were determined by Western blot. ResultSBJDF effectively inhibited the vitality of HCT116 cells and changed their morphology in a concentration-dependent manner. Compared with the blank control group， SBJDF at 1， 2， 4 g·L^-1 significantly reduced cell colony formation （P<0.05， P<0.01），and SBJDF at 2 and 4 g·L^-1 arrested the HCT116 cell cycle at G₀/G₁ phase （P<0.05， P<0.01）. Compared with the blank control group， SBJDF at 1， 2， 4 g·L^-1 remarkably down-regulated the protein expression of CyclinD₁ （P<0.05， P<0.01）. SBJDF at 2 and 4 g·L^-1 lowered the CyclinA₂ and cyclin-dependent kinase 4 （CDK4） （P<0.05， P<0.01）. SBJDF at 4 g·L^-1 reduced the cyclin-dependent kinase 1 （CDK1） （P<0.01）. Compared with the blank control group， SBJDF at 2 and 4 g·L^-1 induced HCT116 cell apoptosis， down-regulated the protein expression of anti-apoptosis-related proteins Bcl-2 and Bcl-xl as well as the NF-κB signaling pathway-related proteins IκB kinase α （IKKα），inhibitor α of NF-κB （IκBα），and phospho-NF-κB p65 （p-p65） （P<0.05， P<0.01）， and diminished the mitochondrial membrane potential of HCT116 cells. ConclusionSBJDF inhibits the proliferation of HCT116 cells， which may be related to its inhibition of the activation of NF-κB signaling pathway and the induction of cell cycle arrest and apoptosis.

ObjectiveTo explore the influence of Xiaoai Jiedu prescription （XJP）-containing serum on natural killer （NK） cells′ lethal effect on colon cancer cells and the molecular mechanism. MethodXJP-containing serum （0.1%， 0.5%， 1%， 5%， 10%） was used to treat HCT-116 cells and NK-92MI cells respectively for 24 h， and methyl thiazolyl tetrazolium （MTT） assay was employed to detect cell proliferation. Then， low-concentration （0.1%， 0.5%， 1%） XJP-containing serum was selected to treat co-cultured HCT-116 cells and NK-92MI cells for 24 h and calcein acetoxymethyl ester/propidium iodide （Calcein-AM/PI） was applied to detect the killing effect of NK cells on colon cancer cells. Flow cytometry was used to detect apoptosis of colon cancer cells， Western blot the expression of apoptosis-related proteins and signal transducer and activator of transcription 4 （STAT4） pathway-related proteins， and enzyme-linked immunosorbent assay （ELISA） the secretion of interferon （IFN）-γ. ResultHigh-concentration （5%， 10%） XJP-containing serum inhibited the proliferation of HCT-116 and NK-92MI cells （P<0.01）， while low-concentration （0.1%， 0.5%， 1%） XJP-containing serum had no obvious influence on cell proliferation compared with the blank group. As compared with the blank group， low-concentration XJP-containing serum enhanced the killing activity of NK cells against colon cancer cells in a concentration-dependent manner （P<0.01）， and induced apoptosis of colon cancer cells （P<0.01）. Moreover， XJP-containing serum （0.1%， 0.5%， 1%） down-regulated the expression of B-cell lymphoma 2 （Bcl-2） and B-cell lymphoma-extra large （Bcl-xl）， and up-regulated the expression of Bcl-2-associated X （Bax） compared with the blank group （P<0.05， P<0.01）. Compared with the co-culture group， XJP-containing serum （0.1%， 0.5%， 1%） increased the expression of p-STAT4 and IFN-γ （P<0.05）. ELISA result showed that XJP-containing serum （0.1%， 0.5%， 1%） raised IFN-γ secretion （P<0.01）. ConclusionXJP-containing serum can enhance the activity of NK cells to kill colon cancer cells. The mechanism is the likelihood that it activates STAT4 pathway， increases IFN-γ secretion by NK cells， down-regulates the expression of Bcl-xl and Bcl-2， and up-regulates the expression of Bax， thereby promoting the apoptosis of colon cancer cells.

ObjectiveTo study the mechanism of Shenbai Jiedu prescription inhibiting the proliferation of HCT116 colorectal cancer （CRC） cells by regulating the phosphatase and tensin homolog deleted on chromosome ten （PTEN）/phosphatidylinositol 3-kinase （PI3K）/ protein kinase B （Akt） signaling pathway. MethodShenbai Jiedu prescription was extracted by water extraction and alcohol precipitation to prepare freeze-dried powder. HCT116 cells were cultured in vitro， and treated with different concentrations of Shenbai Jiedu prescription （2， 4， 8， 16 g·L^-1）. The inhibitory effect of Shenbai Jiedu prescription on the proliferation of HCT116 cells was tested by methyl thiazolyl tetrazolium （MTT）. Real-time quantitative PCR was used to detect the mRNA expression levels of PTEN， PI3K， Akt， glycogen synthase kinase-3β （GSK-3β）， c-Myc， survivin and Cyclin D₁. Western blot was employed to measure the protein expression levels of PTEN， phosphorylated PTEN （p-PTEN）， PI3K， Akt， phosphorylated Akt （p-Akt）， GSK-3β， phosphorylated GSK-3β （p-GSK-3β）， c-Myc， survivin and Cyclin D₁， β-catenin nuclear import was explored by immunofluorescence assay. ResultCompared with the control group， Shenbai Jiedu prescription inhibited the proliferation of HCT116 cells in a dose-dependent manner （P<0.01）. Compared with the control group， the mRNA expression levels of PTEN and GSK-3β were up-regulated whereas those of PI3K， Akt， c-Myc， survivin and CyclinD₁ were down-regulated after treatment with Shenbai Jiedu prescription （P<0.01）. The protein expression levels of PTEN， p-PTEN and GSK-3β were up-regulated whereas those of PI3K， Akt， p-Akt， GSK-3β， p-GSK-3β， c-Myc， survivin and CyclinD₁ were down-regulated （P<0.05， P<0.01）. Immunofluorescence assay showed that Shenbai Jiedu prescription suppressed β-catenin nuclear import in HCT116 cells. ConclusionShenbai Jiedu prescription inhibited the proliferation of HCT116 cells via the mechanism of regulating the PTEN/PI3K/Akt signaling pathway.

Colorectal cancer (CRC), a malignant tumor worldwide consists of microsatellite instability (MSI) and stable (MSS) phenotypes. Although SHP2 is a hopeful target for cancer therapy, its relationship with innate immunosuppression remains elusive. To address that, single-cell RNA sequencing was performed to explore the role of SHP2 in all cell types of tumor microenvironment (TME) from murine MC38 xenografts. Intratumoral cells were found to be functionally heterogeneous and responded significantly to SHP099, a SHP2 allosteric inhibitor. The malignant evolution of tumor cells was remarkably arrested by SHP099. Mechanistically, STING-TBK1-IRF3-mediated type I interferon signaling was highly activated by SHP099 in infiltrated myeloid cells. Notably, CRC patients with MSS phenotype exhibited greater macrophage infiltration and more potent SHP2 phosphorylation in CD68⁺ macrophages than MSI-high phenotypes, suggesting the potential role of macrophagic SHP2 in TME. Collectively, our data reveals a mechanism of innate immunosuppression mediated by SHP2, suggesting that SHP2 is a promising target for colon cancer immunotherapy.

Objective:To investigate the resistance and transmission mechanism of carbapenem-resistant Enterobacterales (CRE), so as to provide the scientific evidence for the treatment and prevention of CRE infection.Methods:Seventy-six isolates of CRE isolated from Shaoxing Second Hospital between May 2016 and August 2018 were included. The isolates were re-identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The minimum inhibitory concentrations (MICs) of colistin, tigecycline, ceftazidime-avibactam, fosfomycin and other antibacterial drugs were determined using broth microdilution or agar dilution methods. PCR and sequencing analysis were performed to detect carbapenemase encoding genes ( blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48). Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used to analyze homology of strains. S1-PFGE combined with Southern blot hybridization were used to locate the carbapenamase genes. Filter mating test were performed to determine the horizontal transfer ability of plasmids harboring carbapenamase genes. Results:Among the 76 isolates of CRE, 51 isolates were Klebsiella pneumoniae; 10 isolates were Escherichia coli; 15 isolates were other Enterobacterales. The 76 CREs were mainly isolated from urine, sputum and blood samples. The distribution rate of ICU was the highest (55.26%). The 76 CREs showed low resistance rates (0%, 1.33%, 18.42%) to colistin, tigecycline and ceftazidime-avibactam. The resistance rates to amikacin and fosfomycin were <45%, and the resistance rates to other drugs were >97%. The detection rate of KPC-2 carbapenemase was the highest (85.33%). The ST11 CRKP producing KPC-2 carbapenemase accounted for the highest proportion (62.75%), mainly distributed in the ICU (62.50%). Southern blot hybridization showed that blaKPC-2 was mainly located on a plasmid about 90 kb (39/63). Filter mating test showed that blaKPC, blaNDM and blaIMP could be transferred horizontally to recipient bacteria through plasmids. Conclusions:The 76 CRE isolates were only susceptible to a few antibacterial drugs, such as colistin, tigecycline and ceftazidime-avibactam. The production of KPC-2 carbapenemase was the main reason for the resistance of Enterobacterales to carbapenems. KPC-2 carbapenemase-producing ST11 Klebsiella pneumoniae was the main epidemic clone of carbapenem-resistant Klebsiella pneumoniae (CRKP). The 90 kb size plasmid was the main plasmid encoding blaKPC-2 gene. Carbapenemase genes can be transferred horizontally through plasmids. The hospital should strengthen prevention of nosocomial infections to control the clonal prevalence of CRE.

Objective:To explore the clinicopathological and prognostic features of young onset patients with middle-low rectal cancer who received neoadjuvant chemoradiotherapy (NCRT).Methods:After NCRT, a total of 441 patients with primary middle-low rectal cancer treated with radical surgery at the Cancer Hospital, Chinese Academy of Medical Sciences (CHCAMS) from January 2004 to December 2016 were included. According to the age of disease onset, the patients were divided into the young group (51cases) and the middle-old group (390 cases), and the clinicopathological characteristics and survival of these patients were analyzed.Results:In the young group, 68.6% of patients received radical surgery within 7 weeks after NCRT, which was higher than 52.8% in the middle-old group ( P=0.047). The stage ypTNM Ⅲ in the young group was 51.0%, higher than 34.1% in the middle-old group ( P=0.027). The stage ypN+ in the young group was 51.0%, higher than 34.1% in the middle-old group ( P=0.047), The incidence of disease progression in the young group was 39.2%, higher than 25.1% in the middle-old group ( P=0.049). The incidence of distant metastasis in the young group was 35.3%, higher than 21.5% in the middle-old group( P=0.044). Most cases of disease progression occurred in the first 3 years after surgery for the young group, especially in the second year after surgery, the incidence of disease progression in the young group was 55.0%, higher than 26.5% in middle-old group ( P=0.025). The 3-year and 5-year disease-free survival (DFS) rates for the young group were 63.7% and 58.2%, lower than 81.0% and 74.3% in the middle-old group ( P=0.016), respectively. The 3-year and 5-year overall survival in the middle-old group (OS) rates for the young group were 85.4% and 69.2%, lower than 93.6% and 84.1% in the middle-old group ( P=0.033), respectively. The multivariate analysis showed that, response of primary tumor ( HR=4.804, 95% CI: 1.360-16.973) and total number of dissected lymph nodes ( HR=4.336, 95% CI: 1.739-10.809) in the young group were independent prognostic factors related to DFS. The total dissected number of lymph nodes( HR=3.295, 95% CI: 1.076-10.091)was an independent prognostic factor related to OS. In the middle-old group, response of primary tumor ( HR=2.626, 95% CI: 1.354-5.091), ypTNM stage (ypTNM Ⅲ: HR=5.837, 95% CI: 2.968-11.479) and tumor location distance from the anal verge ( HR=0.500, 95% CI: 0.308-0.812) were independent prognostic factors related to DFS. Lymphovascular invasion ( HR=0.500, 95% CI: 0.308-0.812) and ypTNM stage (ypTNM Ⅲ: HR=16.322, 95% CI: 5.049-52.771) were independent prognostic factors related to OS. Conclusions:Young onset rectal cancer patients are associated with shorter operation time interval, advanced pathological stage and poorer prognosis. More intensive adjuvant treatment and post-treatment surveillance should be conducted to young onset rectal cancer with NCRT.

Objective:To explore the risk factors for regional lymph node (RLN) metastasis in colorectal cancer patients with mismatch repair deficiency (dMMR).Methods:The data of 357 dMMR colorectal cancer patients who underwent surgery in National Cancer Center from January 2012 to December 2016 was retrospectively analyzed. Univariate and multivariate analysis were used to identify the risk factors for RLN metastasis.Results:Among the 357 patients, 204 were male and 153 were female, 61.6% (220/357) lesion located in right half colon, while the other 16.2% (58/357) located in rectum. Univariate analysis showed that tumor size, differentiation, lymphovascular invasion, tumor deposit, postoperative pathologic T stage (pT), the number of negative lymph nodes and the expression of the MSH6 protein were significantly associated with RLN metastasis ( P<0.05). All of the patients with well differentiation tumors (15 patients) or staged pT1 (13 patients) had no RLN metastasis. Multivariate analysis showed that tumor differentiation ( OR=2.582, 95% CI=1.567-4.274, P<0.001), pT ( OR=3.778, 95% CI=1.448-12.960, P=0.015) and the expression of MSH6 protein ( OR=2.188, 95% CI=1.159-4.401, P=0.021) were independent risk factors for RLN metastasis. Conclusions:The postoperative pT stage, tumor differentiation and the expression of MSH6 protein are independent risk factors for RLN metastasis of dMMR colorectal cancer. Preoperative assessment of these factors may further improve the accuracy of predicting the risk of RLN metastasis.

Objective:To explore the application value of the conditional disease-free survival (cDFS) analysis in predicting prognosis of stage-specific rectal cancer patients underwent neoadjuvant chemoradiotherapy (nCRT).Methods:Clinicopathologic data of 436 patients with rectal cancer received nCRT and radical operation in Cancer Hospital, Chinese Academy of Medical Sciences between January 2004 and December 2016 were retrospectively reviewed. With reference to conditional probability, the 3-year cDFS of patients at different ypTNM stage after completion of nCRT was estimated using the Kaplan-Meier method.Results:There were 66 patients of ypTNM stage 0 (pathological complete response), 87 patients of ypTNM stage Ⅰ, 135 patients of ypTNM stage Ⅱ and 148 patients of ypTNM stage Ⅲ. The 3-year accumulated DFS of patients with ypTNM stage 0, ypTNM stage Ⅰ, ypTNM stage Ⅱ, and ypTNM stage Ⅲ were 97.0%, 93.1%, 85.2%, and 64.2%, respectively. On the condition of postoperactive disease-free survival for 1 year, 2 years, 3 years, 4 years, and 5 years, the corresponding 3-year cDFS of patients at ypTNM stage 0 were 97.0%, 95.5%, 96.9%, 98.4%, 100.0%, respectively. The corresponding 3-year cDFS of patients at ypTNM Ⅲ were 68.2%, 79.3%, 86.3%, 92.1%, 96.4%, respectively. The more advanced ypTNM staging resulted in the more improvement of 3-year cDFS being acquired.Conclusion:cDFS is a better method to reflect the dynamic changes of the prognosis of rectal cancer patients with nCRT in different ypTNM stage, and it is useful to guide the clinicians to assess the prognosis and propose appropriate surveillance.