ObjectiveThis study aimed to investigate whether Bushen Tongluo prescription inhibits macrophage polarization by regulating the Wnt3a/β-catenin signaling pathway， thereby reducing epithelial-mesenchymal transition and excessive extracellular matrix deposition， in order to elucidate the anti-pulmonary fibrosis mechanisms of Bushen Tongluo prescription and provide a new theoretical basis for the clinical treatment of pulmonary fibrosis. MethodsFifty male Sprague-Dawley （SD） rats were randomly divided into a blank group， model group， pirfenidone group， and high- and low-dose Bushen Tongluo prescription groups. Except for the blank group， the pulmonary fibrosis model was established by intratracheal instillation of bleomycin. Intervention was initiated on day 28 after modeling. The high- and low-dose Bushen Tongluo prescription groups were administered Bushen Tongluo prescription at doses of 30.88， 15.44 g·kg^-1， respectively， by intragastric gavage. The pirfenidone group was administered pirfenidone capsules at 110 mg·kg^-1 by intragastric gavage. The blank and model groups were given an equal volume of normal saline by gavage， once daily for 90 days. After treatment， the level of transforming growth factor-β₁ （TGF-β₁） in bronchoalveolar lavage fluid （BALF） was detected by enzyme-linked immunosorbent assay （ELISA）. Morphological changes in lung tissue and the collagen volume fraction were compared. The protein distribution and expression of E-cadherin， cytokeratin 19， α-smooth muscle actin （α-SMA）， vimentin， collagen type Ⅰ （Col Ⅰ）， and collagen type Ⅲ （Col Ⅲ） in lung tissue were detected by immunohistochemistry. The protein distribution and expression of CD68， arginase-1 （Arg-1）， inducible nitric oxide synthase （iNOS）， Wnt3a， and β-catenin in lung tissue were detected by immunofluorescence. The protein expression of Wnt3a and β-catenin in lung tissue was detected by Western blot， and the mRNA expression of Wnt3a and β-catenin was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultsCompared with the blank group， a large number of inflammatory cells infiltrated the airway walls， alveolar spaces， and interstitial tissue in the model group， with obvious fibrous tissue hyperplasia. The level of TGF-β₁ in BALF was significantly increased. The protein expression of E-cadherin and cytokeratin 19 in lung tissue was decreased， whereas the protein expression of α-SMA， Vimentin， Wnt3a， β-catenin， Col Ⅰ， and Col Ⅲ was increased. The fluorescence-positive area ratios of CD68， Arg-1， iNOS， Wnt3a， and β-catenin in lung tissue were increased. The protein and mRNA expression levels of Wnt3a and β-catenin in lung tissue were significantly increased （P<0.01）. Compared with the model group， all treatment groups showed varying degrees of improvement in inflammatory cell infiltration and fibrous tissue hyperplasia in the airway walls， alveolar spaces， and interstitial tissue， decreased TGF-β₁ levels in BALF， increased protein expression of E-cadherin and cytokeratin 19 in lung tissue， decreased protein expression of α-SMA， Vimentin， Col Ⅰ， and Col Ⅲ， decreased fluorescence-positive area ratios of CD68， Arg-1， iNOS， Wnt3a， and β-catenin in lung tissue， and decreased protein and mRNA expression levels of Wnt3a and β-catenin in lung tissue （P<0.05， P<0.01）. ConclusionBushen Tongluo prescription can improve bleomycin-induced pulmonary fibrosis in rats by inhibiting epithelial-mesenchymal transition and reducing excessive extracellular matrix deposition. The mechanism may be related to inhibition of the Wnt3a/β-catenin signaling pathway and the macrophage polarization mediated by this pathway.

OBJECTIVE T o establish a simple,rapid,highly sensitive and highly specific visualized detection meth-od for Klebsiella pneumoniae based on recombinase polymerase amplification(RPA)technique combined with the lateral flow dipstick(LFD)technique.METHODS The specific primers targeting the khe gene of K.pneumoniae were designed for RPA,the reaction temperature and reaction time for RPA were optimized,the optimal experi-mental conditions for the LFD were determined based on the colorimetric results.The sensitivity,stability,speci-ficity and clinical practicability of RPA-LFD in detection of the K.pneumoniae were evaluated.RESULTS RPA could achieved the optimal detection result for simplification of K.pneumoniae at 34 ℃ for 10 min.The LFD showed the best color development when Milipore HF 135s was used as the nitrocellulose membrane,phosphate buffered saline containing Tween-20(PBST)was used as the sample developing solution,and the concentrations of streptavidin and secondary antibody were 0.8 mg/ml and 0.1 mg/ml,respectively.The detection limit of the RPA-LFD was 5 CFU/ml for detection of K.pneumoniae,the relative standard deviation of the band color inten-sity was less than 5％in six parallel experiments,and there was no cross reactions with other bacterial strains.In addition,the sensitivity of the RPA-LFD in detection of K.pneumoniae was not affected by the serum compo-nents such as proteins and lipids.CONCLUSION The rapid detection method for K.pneumoniae that is established based RPA-LFD is characterized by the simple operation,high sensitivity and high specificity,and it does not need complicated equipment or strict technical requirement for operators and provides a new technique for early diagno-sis and epidemiological survey of the K.pneumoniae.

Objective To investigate the effects and molecular mechanisms of regenerating islet-derived 3-Alpha(REG3A)on the proliferation,apoptosis and cisplatin(DDP)resistance of ovarian cancer(OC)cells.Methods Cancer tissue and adjacent tissue samples of 97 ovarian cancer patients admitted to Qinghai Fifth People's Hospital from January 2021 to December 2022 were collected.Quantitative reverse transcriptase-mediated PCR(qRT-PCR)and immunohis to chemistry(IHC)were used to detect the expression of REG3A in cancer tissues and adjacent tissues,and the relationship between its expression and the clinical pathological characteristics and prognosis of patients was analyzed.QRT-PCR was used to detect the expression of REG3A in human normal ovarian epithelial cell line OSE and human OC cell lines(ES2,HEY,A2780,and SKOV3).SKOV3 and DDP resistant OC cell lines(SKOV3/DDP)were selected and randomly divided into siNC group and siREG3A group.Cell counting kit-8(CCK-8)was used to detect cell proliferation activity.Determination of cell drug resistance by methylthiazolyl tetrazolium(MTT).Flow cytometry was used to detect proliferation cycle and apoptosis.Immunoblotting was used to detect the expression of resistance,proliferation,apoptosis and phsphatidylinostol 3-kinase(PI3K)/protein kinase B(Akt)pathway related proteins in OC cells.Results The expression of REG3A mRNA in ovarian cancer tissues was higher than that in adjacent tissues(1.46±0.43 vs 0.52±0.11),and the difference was statistically significant(t=20.858,P<0.001).The 5-year survival rate of patients with high expression of REG3A was 24.49%(12/49),which was lower than the 91.67%(44/48)of patients with low expression of REG3A,and the difference was statistically significant(χ2=44.841,P<0.001).The proportion of patients with FIGO stages III-IV,moderate to high differentiation and tumor size>3cm in the high expression group of REG3A was higher than that in the low expression group of REG3A,and the differences were statistically significant(χ2=4.537～9.972,all P<0.05).Compared with OSE,the mRNA and protein expression of REG3A in ES2,HKY,A2780 and SKOV3 increased significantly(t=6.725～30.234,all P<0.01).Compared with the siNC group,the expression of REG3A was downregulated in SKOV3 cells in the siREG3A group(0.23±0.02 vs 0.99±0.06),cell activity decreased at 24,48 and 72 hours of transfection,the proportion of G1 phase cells increased,while the proportion of S and G2 phase cells and apoptosis rate,MDR-1,Cyclin D1 and Cleaved caspase 3 protein expression,p-PI3K/PI3K and p-Akt/Akt ratios were decreased,and the differences were statistically significant(t=6.584～22.730,all P<0.01).After treatment with DDP at concentrations of 1.25,2.5,5,7.5 and 15 μg/ml,compared with the siNC group,the survival rates of SKOV3 and SKOV3/DDP cells in the siREG3A group gradually decreased,and the differences were statistically significant(t=2.888～11.135,all P<0.05).Conclusion REG3A is highly expressed in OC tissues and is associated with malignant progression and poor prognosis of OC.Its up-regulation promotes cancer cell proliferation and DDP resistance by activating the PI3K/Akt pathway,and inhibits apoptosis.

Pulmonary sarcoidosis is an immune system disease with an unclear etiology. Guided by the yang-reinforcing method， it is believed that the fundamental pathogenesis of pulmonary sarcoidosis lies in the disharmony between water and fire and the reckless movement of the ministerial fire. The failure of the spleen and stomach to maintain warmth， leading to the production of phlegm and blood stasis， is an important pathogenesis. The invasion of external pathogenic toxins， deeply penetrating into the interior， is considered a triggering factor for the disease. The treatment focuses on supplementing the yang， consolidating the kidney， drawing fire back to its source， warming the yang， benefiting the kidney， and nourishing the spleen to generate metal. It also emphasizes unblocking the yang， transforming turbidity， and eliminating phlegm and blood stasis.

Objective To investigate the effects and molecular mechanisms of regenerating islet-derived 3-Alpha(REG3A)on the proliferation,apoptosis and cisplatin(DDP)resistance of ovarian cancer(OC)cells.Methods Cancer tissue and adjacent tissue samples of 97 ovarian cancer patients admitted to Qinghai Fifth People's Hospital from January 2021 to December 2022 were collected.Quantitative reverse transcriptase-mediated PCR(qRT-PCR)and immunohis to chemistry(IHC)were used to detect the expression of REG3A in cancer tissues and adjacent tissues,and the relationship between its expression and the clinical pathological characteristics and prognosis of patients was analyzed.QRT-PCR was used to detect the expression of REG3A in human normal ovarian epithelial cell line OSE and human OC cell lines(ES2,HEY,A2780,and SKOV3).SKOV3 and DDP resistant OC cell lines(SKOV3/DDP)were selected and randomly divided into siNC group and siREG3A group.Cell counting kit-8(CCK-8)was used to detect cell proliferation activity.Determination of cell drug resistance by methylthiazolyl tetrazolium(MTT).Flow cytometry was used to detect proliferation cycle and apoptosis.Immunoblotting was used to detect the expression of resistance,proliferation,apoptosis and phsphatidylinostol 3-kinase(PI3K)/protein kinase B(Akt)pathway related proteins in OC cells.Results The expression of REG3A mRNA in ovarian cancer tissues was higher than that in adjacent tissues(1.46±0.43 vs 0.52±0.11),and the difference was statistically significant(t=20.858,P<0.001).The 5-year survival rate of patients with high expression of REG3A was 24.49%(12/49),which was lower than the 91.67%(44/48)of patients with low expression of REG3A,and the difference was statistically significant(χ2=44.841,P<0.001).The proportion of patients with FIGO stages III-IV,moderate to high differentiation and tumor size>3cm in the high expression group of REG3A was higher than that in the low expression group of REG3A,and the differences were statistically significant(χ2=4.537～9.972,all P<0.05).Compared with OSE,the mRNA and protein expression of REG3A in ES2,HKY,A2780 and SKOV3 increased significantly(t=6.725～30.234,all P<0.01).Compared with the siNC group,the expression of REG3A was downregulated in SKOV3 cells in the siREG3A group(0.23±0.02 vs 0.99±0.06),cell activity decreased at 24,48 and 72 hours of transfection,the proportion of G1 phase cells increased,while the proportion of S and G2 phase cells and apoptosis rate,MDR-1,Cyclin D1 and Cleaved caspase 3 protein expression,p-PI3K/PI3K and p-Akt/Akt ratios were decreased,and the differences were statistically significant(t=6.584～22.730,all P<0.01).After treatment with DDP at concentrations of 1.25,2.5,5,7.5 and 15 μg/ml,compared with the siNC group,the survival rates of SKOV3 and SKOV3/DDP cells in the siREG3A group gradually decreased,and the differences were statistically significant(t=2.888～11.135,all P<0.05).Conclusion REG3A is highly expressed in OC tissues and is associated with malignant progression and poor prognosis of OC.Its up-regulation promotes cancer cell proliferation and DDP resistance by activating the PI3K/Akt pathway,and inhibits apoptosis.

OBJECTIVE T o establish a simple,rapid,highly sensitive and highly specific visualized detection meth-od for Klebsiella pneumoniae based on recombinase polymerase amplification(RPA)technique combined with the lateral flow dipstick(LFD)technique.METHODS The specific primers targeting the khe gene of K.pneumoniae were designed for RPA,the reaction temperature and reaction time for RPA were optimized,the optimal experi-mental conditions for the LFD were determined based on the colorimetric results.The sensitivity,stability,speci-ficity and clinical practicability of RPA-LFD in detection of the K.pneumoniae were evaluated.RESULTS RPA could achieved the optimal detection result for simplification of K.pneumoniae at 34 ℃ for 10 min.The LFD showed the best color development when Milipore HF 135s was used as the nitrocellulose membrane,phosphate buffered saline containing Tween-20(PBST)was used as the sample developing solution,and the concentrations of streptavidin and secondary antibody were 0.8 mg/ml and 0.1 mg/ml,respectively.The detection limit of the RPA-LFD was 5 CFU/ml for detection of K.pneumoniae,the relative standard deviation of the band color inten-sity was less than 5％in six parallel experiments,and there was no cross reactions with other bacterial strains.In addition,the sensitivity of the RPA-LFD in detection of K.pneumoniae was not affected by the serum compo-nents such as proteins and lipids.CONCLUSION The rapid detection method for K.pneumoniae that is established based RPA-LFD is characterized by the simple operation,high sensitivity and high specificity,and it does not need complicated equipment or strict technical requirement for operators and provides a new technique for early diagno-sis and epidemiological survey of the K.pneumoniae.

This article is based on ZHANG Jingyue's theory of yin-yang holism to explore the etiology， pathogenesis， and treatment of acute exacerbation of rheumatoid arthritis-associated interstitial lung disease （RA-ILD）. It is believed that the foundation of acute exacerbation of RA-ILD lies in real yin insufficiency and yin fail to control yang； external pathogen attacking is a common cause of acute exacerbation of RA-ILD. For treatment， it is important to first suppress ministerial fire by prescribing modified Yinhuo Decoction （引火汤）； if ministerial fire submerged， focus should be on nourishing both yin and yang； if real yin deficiency is the main issue， modified Zuogui Pill （左归丸） should be used； if real yang deficiency is prominent as well， modified Yougui Pill （右归丸） can be chosen. When yin and yang balanced， the disease could be solved.

Objective To investigate the application of organoid technology in colorectal cancer research and analyze the factors influencing the success rate of organoid cultivation. Methods A total of 24 samples of colorectal cancer patients treated at Tianjin Fifth Central Hospital and cultured organoids using specific culture media and Matrigel. The samples were collected within 30 minutes post-excision and stored at 4℃ to minimize contamination and protein degradation. During the cultivation process,the authors recorded instances of bacterial or fungal contamination and the organoid growth,and test for their histological structure and expression of tumor markers. Additionally,by analyzing clinical information from the patients who provided the samples,explore potential factors that may affect the success rate of culturing colorectal cancer organoids. Results A success rate of 70.8％ (17/24) was achieved in the cultivation of organoid. The success rate of organoid culture was significantly different from that of tumor stage (all P<0.05),with significantly higher successful organoid cultivation rate for stage Ⅱ and stage Ⅲ tumor tissues than those for stageⅠand Ⅳ[83.3％ (5/6),90.9％ (10/11) vs. 33.4％ (1/3),25.0％ (1/4)]. Additionally,samples with a Ki-67 positive area proportion of 55％-70％ exhibited the highest success rate (100％). Phenotypic experiments on the organoids indicated that their pathological histological structure and the expression of tumor markers were consistent with those of the primary tissues,suggesting that the organoids retained the histological characteristics of the primary lesions. Conclusions This study successfully established a colorectal cancer organoid model revealing the impact of tumor staging and the proportion of Ki-67 positive areas on the success rate of culture. The organoid model effectively retains the histological characteristics of the primary lesion,providing a powerful in vitro tool for the research and treatment of colorectal cancer.

Objective To investigate the application of organoid technology in colorectal cancer research and analyze the factors influencing the success rate of organoid cultivation. Methods A total of 24 samples of colorectal cancer patients treated at Tianjin Fifth Central Hospital and cultured organoids using specific culture media and Matrigel. The samples were collected within 30 minutes post-excision and stored at 4℃ to minimize contamination and protein degradation. During the cultivation process,the authors recorded instances of bacterial or fungal contamination and the organoid growth,and test for their histological structure and expression of tumor markers. Additionally,by analyzing clinical information from the patients who provided the samples,explore potential factors that may affect the success rate of culturing colorectal cancer organoids. Results A success rate of 70.8％ (17/24) was achieved in the cultivation of organoid. The success rate of organoid culture was significantly different from that of tumor stage (all P<0.05),with significantly higher successful organoid cultivation rate for stage Ⅱ and stage Ⅲ tumor tissues than those for stageⅠand Ⅳ[83.3％ (5/6),90.9％ (10/11) vs. 33.4％ (1/3),25.0％ (1/4)]. Additionally,samples with a Ki-67 positive area proportion of 55％-70％ exhibited the highest success rate (100％). Phenotypic experiments on the organoids indicated that their pathological histological structure and the expression of tumor markers were consistent with those of the primary tissues,suggesting that the organoids retained the histological characteristics of the primary lesions. Conclusions This study successfully established a colorectal cancer organoid model revealing the impact of tumor staging and the proportion of Ki-67 positive areas on the success rate of culture. The organoid model effectively retains the histological characteristics of the primary lesion,providing a powerful in vitro tool for the research and treatment of colorectal cancer.