SNHG15-mediated feedback loop interplays with HNRNPA1/SLC7A11/GPX4 pathway to promote gastric cancer progression.

Dysregulation of long noncoding RNA (lncRNA) expression plays a pivotal role in the initiation and progression of gastric cancer (GC). However, the regulation of lncRNA SNHG15 in GC has not been well studied. Mechanisms for ferroptosis by SNHG15 have not been revealed. Here, we aimed to explore SNHG15-mediated biological functions and underlying molecular mechanisms in GC. The novel SNHG15 was identified by analyzing RNA-sequencing (RNA-seq) data of GC tissues from our cohort and TCGA dataset, and further validated by qRT-PCR in GC cells and tissues. Gain- and loss-of-function assays were performed to examine the role of SNHG15 on GC both in vitro and in vivo. SNHG15 was highly expressed in GC. The enhanced SNHG15 was positively correlated with malignant stage and poor prognosis in GC patients. Gain- and loss-of-function studies showed that SNHG15 was required to affect GC cell growth, migration and invasion both in vitro and in vivo. Mechanistically, the oncogenic transcription factors E2F1 and MYC could bind to the SNHG15 promoter and enhance its expression. Meanwhile, SNHG15 increased E2F1 and MYC mRNA expression by sponging miR-24-3p. Notably, SNHG15 could also enhance the stability of SLC7A11 in the cytoplasm by competitively binding HNRNPA1. In addition, SNHG15 inhibited ferroptosis through an HNRNPA1-dependent regulation of SLC7A11/GPX4 axis. Our results support a novel model in which E2F1- and MYC-activated SNHG15 regulates ferroptosis via an HNRNPA1-dependent modulation of the SLC7A11/GPX4 axis, which serves as the critical effectors in GC progression, and provides a new therapeutic direction in the treatment of GC.

STAT3-mediated up-regulation of DAB2 via SRC-YAP1 signaling axis promotes Helicobacter pylori-driven gastric tumorigenesis.

BACKGROUND: Helicobacter pylori (H pylori) infection is the primary cause of gastric cancer (GC). The role of Disabled-2 (DAB2) in GC remains largely unclear. This study aimed to investigate the role of DAB2 in H pylori-mediated gastric tumorigenesis. METHODS: We screened various datasets of GC to analyze DAB2 expression and cell signaling pathways. DAB2 expression was assessed in human GC tissue microarrays. H pylori infection in vivo and in vitro models were further explored. Immunostaining, immunofluorescence, chromatin immunoprecipitation, co-immunoprecipitation, Western blot, quantitative polymerase chain reaction, and luciferase reporter assays were performed in the current study. RESULTS: The bioinformatic analysis verified that DAB2 was 1 of the 8 genes contributed to tumorigenesis and associated with poor prognosis in GC. The median overall survival and disease-free survival rates in DAB2high group were significantly less than those in DAB2low group. These findings demonstrated that H pylori transcriptionally activated DAB2 expression via signal transducer and activator of transcription 3 (STAT3)-dependent pathway. By bioinformatics analysis and knockdown or overexpression of DAB2, we found that DAB2 upregulated Yes-associated protein 1 (YAP1) transcriptional activity. Mechanistically, DAB2 served as a scaffold protein for integrin beta 3 (ITGB3) and SRC proto-oncogene non-receptor tyrosine kinase (SRC), facilitated the phosphorylation of SRC, promoted the small GTPase ras homolog family member A (RHOA) activation and phosphorylation of YAP1, and ultimately enhanced the YAP1 transcriptional activity. CONCLUSIONS: Altogether, these findings indicated that DAB2 is a key mediator in STAT3-regulated translation of YAP1 and plays crucial roles in H pylori-mediated GC development. DAB2 might serve as a novel therapeutic target for GC.

CD47-targeted immunotherapy unleashes antitumour immunity in Epstein-Barr virus-associated gastric cancer.

The aims of this study were to enhance the antitumour immunity in Epstein-Barr virus-associated gastric cancer (EBVaGC). We performed RNA-seq analysis to compare the differential expression genes between EBVaGC and EBV-negative gastric cancer (EBVnGC) patients. The expression levels of CD68, CD163 and CD47 were analyzed by immunohistochemistry. Different subsets of macrophages were investigated by a coincubation model. The effects of CD47 blockade were also detected. The expression levels of CD68, CD163 and CD47 were significantly higher in EBVaGC, and were associated with poor prognoses. Macrophages coincubated with EBV+ AGS cells tended to be immunosuppressed, which could be reversed by CD47 deficiency or blocking CD47. EBV resulted in cGAS-STING pathway activation, which stimulated CD47 expression and inhibited macrophage phagocytosis. Anti-CD47 therapy activated cGAS-STING signaling, which was responsible for production of IFN-ß, resulting in activation of antitumour immunity. Our results provide a promising new strategy for CD47-targeted immunotherapy in EBVaGC.

Novel management indications for conservative treatment of chylous ascites after gastric cancer surgery.

BACKGROUND: Chylous ascites (CA) presents a challenge as a relatively common postoperative complication in gastric cancer (GC). Primary conservative therapy involved total parenteral nutrition, continuous low-pressure drainage, somatostatin, and a low-fat diet. Drainage tube (DT) clamping has been presented as a potential alternative conservative treatment for GC patients with CA. AIM: To propose novel conservative treatment strategies for CA following GC surgery. METHODS: The data of patients with CA after GC surgery performed at the Fudan University Shanghai Cancer Center between 2006 and 2021 were evaluated retrospectively. RESULTS: 53 patients underwent surgery for GC and exhibited postoperative CA during the study period. Postoperative hospitalization and time of DT removal showed a significant positive association (R 2 = 0.979, P < 0.001). We further observed that delayed DT removal significantly extended the total and postoperative hospitalization, antibiotic usage duration, and hospitalization cost (postoperative hospitalization: 25.8 d vs 15.5 d, P < 0.001; total hospitalization: 33.2 d vs 24.7 d, P < 0.01; antibiotic usage duration: 10.8 d vs 6.2 d, P < 0.01; hospitalization cost: ¥9.2 × 104 vs ¥6.5 × 104, P < 0.01). Multivariate analysis demonstrated that postoperative infection and antibiotic usage were independent factors for delayed DT removal. Furthermore, DT removal times were shorter in seven patients who underwent DT clamping (clamped DT vs normal group, 11.8 d vs 13.6 d, P = 0.047; clamped DT vs delayed group, 13.6 d vs 27.4 d, P < 0.001). In addition, our results indicated that removal of the DT may be possible after three consecutive days of drainage volumes less than 300 mL in GC patients with CA. CONCLUSION: Infection and antibiotic usage were vital independent factors that influenced delayed DT removal in patients with CA. Appropriate standards for DT removal can significantly reduce the duration of hospitalization. Furthermore, DT clamping might be a recommended option for conservative treatment of postoperative CA.

Review article: insights into the bile acid-gut microbiota axis in intestinal failure-associated liver disease-redefining the treatment approach.

BACKGROUND: Intestinal failure-associated liver disease (IFALD) increases mortality of patients with intestinal failure (IF), but lacks effective prevention or treatment approaches. Bile acids, gut microbiota and the host have close and complex interactions, which play a central role in modulating host immune and metabolic homeostasis. Increasing evidence suggests that derangement of the bile acid-gut microbiota (BA-GM) axis contributes to the development of IFALD. AIMS: To review the BA-GM axis in the pathogenesis and clinical applications of IFALD, and to explore future directions for effective disease management. METHODS: We conducted a literature search on bile acid and gut microbiota in IF and liver diseases. RESULTS: The BA-GM axis demonstrates a unique IF signature manifesting as an increase in primary-to-secondary bile acids ratio, disturbed enterohepatic circulation, blunted bile acid signalling pathways, gut microbial dysbiosis, and altered microbial metabolic outputs. Bile acids and gut microbiota shape the compositional and functional alterations of each other in IF; collaboratively, they promote immune dysfunction and metabolic aberration in the liver. Diagnostic markers and treatments targeting the BA-GM axis showed promising potential in the management of IFALD. CONCLUSIONS: Bile acids and gut microbiota play a central role in the development of IFALD and make attractive biomarkers as well as therapeutic targets. A multitarget, individualised therapy aiming at different parts of the BA-GM axis may provide optimal clinical benefits and requires future investigation.

MiR-125-5p/IL-6R axis regulates macrophage inflammatory response and intestinal epithelial cell apoptosis in ulcerative colitis through JAK1/STAT3 and NF-κB pathway.

This study explored the effects of miR-125-5p and interleukin-6 receptor (IL-6 R) on ulcerative colitis (UC) cell models and mouse models. The sera derived from UC patients and healthy subjects were collected for expression analysis. UC in vitro models and in vivo model were constructed and used. Expressions of miR-125-5p, IL-6 R, AK1/STAT3 and NF-κB pathways, and inflammatory factors, histopathology and apoptosis were determined by conducting a series of molecular experiments. The relationship between miR-125-5p and IL-6 R was analyzed by TargetScan7.2 and verified by dual-luciferase assay. The disease activity index (DAI) score, weight change, and colon length of the mice were recorded and analyzed. Decreased expression of miR-125-5p in the sera of UC patients was related to the increased expression of its target gene IL-6 R. In vitro, up-regulation of miR-125-5p decreased IL-6 R expression, contents of inflammatory factors in THP-1 cells and cell apoptosis of NCM460, and inhibited the activation of JAK1/STAT3 and NF-κB pathway. However, down-regulation of miR-125-5p produced the opposite effects to its up-regulation. IL-6 R overexpression partially reversed the effects of miR-125-5p up-regulation on UC cell models. In vivo, miR-125-5p overexpression significantly improved the severity of colitis, including DAI score, colon length, tissue damage, apoptosis, and inflammatory response, in the mice in the UC group. In addition, miR-125-5p up-regulation significantly reduced the expression of IL-6 R in the UC mice, and reduced the expression levels of JAK1, STAT3 and p65 phosphorylation. MiR-125-5p targeting IL-6 R regulates macrophage inflammatory response and intestinal epithelial cell apoptosis in ulcerative colitis through JAK1/STAT3 and NF-κB pathway.

Sphingosine kinase 1 regulates HMGB1 translocation by directly interacting with calcium/calmodulin protein kinase II-δ in sepsis-associated liver injury.

Previously, we confirmed that sphingosine kinase 1 (SphK1) inhibition improves sepsis-associated liver injury. High-mobility group box 1 (HMGB1) translocation participates in the development of acute liver failure. However, little information is available on the association between SphK1 and HMGB1 translocation during sepsis-associated liver injury. In the present study, we aimed to explore the effect of SphK1 inhibition on HMGB1 translocation and the underlying mechanism during sepsis-associated liver injury. Primary Kupffer cells and hepatocytes were isolated from SD rats. The rat model of sepsis-associated liver damage was induced by intraperitoneal injection with lipopolysaccharide (LPS). We confirmed that Kupffer cells were the cells primarily secreting HMGB1 in the liver after LPS stimulation. LPS-mediated HMGB1 expression, intracellular translocation, and acetylation were dramatically decreased by SphK1 inhibition. Nuclear histone deacetyltransferase 4 (HDAC4) translocation and E1A-associated protein p300 (p300) expression regulating the acetylation of HMGB1 were also suppressed by SphK1 inhibition. HDAC4 intracellular translocation has been reported to be controlled by the phosphorylation of HDAC4. The phosphorylation of HDAC4 is modulated by CaMKII-δ. However, these changes were completely blocked by SphK1 inhibition. Additionally, by performing coimmunoprecipitation and pull-down assays, we revealed that SphK1 can directly interact with CaMKII-δ. The colocalization of SphK1 and CaMKII-δ was verified in human liver tissues with sepsis-associated liver injury. In conclusion, SphK1 inhibition diminishes HMGB1 intracellular translocation in sepsis-associated liver injury. The mechanism is associated with the direct interaction of SphK1 and CaMKII-δ.

Previous Intestinal Resection Is Associated with Postoperative Complications in Crohn's Disease: A Cohort Study.

BACKGROUND: To assess the influence of a previous intestinal resection on postoperative complications for Crohn's disease (CD). METHODS: Data on patients with CD undergoing surgery in our department from January 2016 through December 2019 were retrospectively reviewed. Information collected included demographic details, surgical data, and postoperative outcome. A cross-sectional study design was employed. Associations between postoperative complications and preoperative clinical indicators were further analyzed. RESULTS: Of the 129 patients with CD studied, 62 patients (48.06%) underwent previous resection. These patients were more likely to be older (P = 0.031), have longer disease duration (P = 0.025), use less 5-aminosalicylic acid/sulfasalazine preoperatively (P = 0.013), have lower body mass index (P = 0.003), and have a higher American Society of Anesthesiologists (ASA) Physical Status Classification System score (P = 0.043). Patients who had previous surgery had a longer duration of operation (P = 0.003), greater estimated blood loss (P = 0.001), and longer hospital stay (P < 0.001) and were more inclined to develop postoperative complications (P = 0.047), particularly anastomotic leak (P = 0.021) and severe (Clavien-Dindo grade III/IV) complications (P = 0.038). After multivariate analysis, previous intestinal resection (P = 0.019), preoperative use of steroids (P = 0.026), and ASA score of more than II (P < 0.001) were determined to be the independent prognostic risk factors for postoperative complications. During the 30-day follow-up period, there was no postoperative mortality or readmission. CONCLUSIONS: Previous intestinal resection in patients with CD is an independent predictor of overall postoperative complications.

MicroRNA-101 suppresses colorectal cancer progression by negative regulation of Rap1b.

Colorectal cancer (CRC) is the fourth most lethal malignancy and is the second most common cause of cancer-associated mortality worldwide. The development of high-throughput sequencing has enabled the identification of potential biomarkers for the diagnosis and treatment of various types of cancer. Although microRNA-101 (miR-101) has been demonstrated to be a potential biomarker of CRC, its detailed mechanisms remain to be fully discovered. In the present study, overall survival analysis was applied to determine the association between miR-101 and CRC prognosis. Reverse transcription-quantitative PCR (RT-qPCR) was used to examine gene expression levels in tissues and cells. Cell proliferative and apoptotic activities were determined by MTT and flow cytometry assays, respectively. Wound healing and Transwell assays were used to examine CRC cell migration and invasion, respectively. In the present study, RT-qPCR analysis indicated that miR-101 was significantly downregulated in CRC tissues and cells. However, clinical data collected from The Cancer Genome Atlas revealed no significant association between the expression levels of miR-101 and the prognosis of CRC. Additionally, miR-101 inhibited the progression of CRC by directly binding to the 3'-untranslated region of Ras-related protein Rap1b (Rap1b). This was associated with downregulation of Rap1b expression. Furthermore, the overexpression of Rap1b promoted miR-101 mimic-attenuated CRC cell progression. The present study demonstrated that miR-101 may be involved in the repression of the CRC progression by forming a negative feedback loop with Rap1b. The findings revealed the interaction between miR-101 and Rap1b during the progression of CRC, which could aid the development of therapeutic strategies.

Integrated Gene Expression Profiling Analysis Reveals Probable Molecular Mechanism and Candidate Biomarker in Anti-TNFα Non-Response IBD Patients.

PURPOSE: To explore the molecular mechanism and search for candidate biomarkers in the gene expression profile of IBD patients associated with the response to anti-TNFα agents. METHODS: Differentially expressed genes (DEGs) of response vs non-response IBD patients in datasets GSE12251, GSE16879, and GSE23597 were integrated using NetworkAnalyst. We conducted functional enrichment analysis of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and extracted hub genes from the protein-protein interaction network. The proportion of immune cell types was estimated via CIBERSORT. ROC curve analysis and binomial Lasso regression were applied to assess the expression level of hub genes in datasets GSE12251, GSE16879, and GSE23597, and another two datasets GSE107865 and GSE42296. RESULTS: A total of 287 DEGs were obtained from the integrated dataset. They were enriched in 14 Gene Ontology terms and 11 KEGG pathways. Polarization from M2 to M1 macrophages was relatively high in non-response individuals. We found nine hub genes (TLR4, TLR1, TLR8, CCR1, CD86, CCL4, HCK, and FCGR2A), mainly related to the interaction between Toll-like Receptor (TLR) pathway and FcγR signaling in non-response anti-TNFα individuals. FCGR2A, HCK, TLR1, TLR4, TLR8, and CCL4 show great value for prediction in intestinal tissue. Besides, FCGR2A, HCK, and TLR8 might be candidate blood biomarkers of anti-TNFα non-response IBD patients. CONCLUSION: Over-activated interaction between FcγR-TLR axis in the innate immune cells of IBD patients might be used to identify non-response individuals and increased our understanding of resistance to anti-TNFα therapy.

Laboratory Calibration of D-dot Sensor Based on System Identification Method.

D-dot sensors can realize the non-contact measurement of transient electric fields, which is widely applied to electromagnetic pulse (EMP) measurements with characteristics of the wide frequency band, high linearity, and good stability. In order to achieve accurate calibration of D-dot sensors in the laboratory environment, this paper proposed a new calibration method based on system identification. Firstly, the D-dot sensor can be considered as a linear time-invariant (LTI) system under corner frequency, thus its frequency response can be characterized by the transfer function of a discrete output error (OE) model. Secondly, based on the partial linear regression of the transfer function curve, the sensitivity coefficient of the D-dot sensor is obtained. By increasing the influence weight of low-frequency components, this proposed method has better calibration performance when the waveform is distorted in the time domain, and can artificially adapt to the operating frequency range of the sensor at the same time.

VHL deficiency augments anthracycline sensitivity of clear cell renal cell carcinomas by down-regulating ALDH2.

The von Hippel-Lindau (VHL) is deficient in ∼70% of clear-cell renal cell carcinomas (ccRCC), which contributes to the carcinogenesis and drug resistance of ccRCC. Here we show that VHL-deficient ccRCC cells present enhanced cytotoxicity of anthracyclines in a hypoxia-inducible factor-independent manner. By subtractive proteomic analysis coupling with RNAi or overexpression verification, aldehyde dehydrogenase 2 (ALDH2) is found to be transcriptionally regulated by VHL and contributes to enhanced anthracyclines cytotoxicity in ccRCC cells. Furthermore, VHL regulates ALDH2 expression by directly binding the promoter of -130 bp to -160 bp to activate the transcription of hepatocyte nuclear factor 4 alpha (HNF-4α). In addition, a positive correlation is found among the protein expressions of VHL, HNF-4α and ALDH2 in ccRCC samples. These findings will deepen our understanding of VHL function and shed light on precise treatment for ccRCC patients.

REG4 promotes peritoneal metastasis of gastric cancer through GPR37.

Being the major reason of recurrence and death after surgery, peritoneal metastasis of gastric cancer dooms the prognosis of advanced gastric cancer patients. Regenerating islet-derived family, member 4 (REG4) is believed to promote peritoneal metastasis, however, its mechanism is still a moot point at present. In the present study, we show that high expression of REG4 correlates with advanced stage and poor survival prognosis for gastric cancer patients. REG4 overexpression significantly enhances peritoneal metastasis by increasing adhesion ability. Moreover, SP1 is proved to be a transcription factor of REG4 and induce REG4 expression upon TGF-alpha stimulation. Also, G protein-coupled receptor 37 (GPR37) is identified to be in the same complex of REG4, which mediates REG4's signal transduction and promotes peritoneal metastasis of gastric cancer cell. Interestingly, we also discover a positive feedback loop triggered by REG4, amplifying itself through EGFR transactivation, consisting of GPR37, ADAM17, TGF-alpha, EGFR, SP1 and REG4. In conclusion, REG4 promotes peritoneal metastasis of gastric cancer through GPR37 and triggers a positive feedback loop.

Mitochondrial aldehyde dehydrogenase 2 protects gastric mucosa cells against DNA damage caused by oxidative stress.

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is a member of the aldehyde dehydrogenase superfamily and is involved with the metabolic processing of aldehydes. ALDH2 plays a cytoprotective role by removing aldehydes produced during normal metabolism. We examined the cytoprotective role of ALDH2 specifically in gastric mucosa cells. Overexpression of ALDH2 increased the viability of gastric mucosa cells treated with H2O2, while knockdown of ALDH2 had an opposite effect. Moreover, overexpression of ALDH2 protected gastric mucosa cells against oxidative stress-induced apoptosis as determined by flow cytometry, Hoechst 33342, and TUNEL assays. Consistently, ALDH2 knockdown had an opposite effect. Additionally, DNA damage was ameliorated in ALDH2-overexpressing gastric mucosa cells treated with H2O2. We further identified that this cytoprotective role of ALDH2 was mediated by metabolism of 4-hydroxynonenal (4-HNE). Consistently, 4-HNE mimicked the oxidative stress induced by H2O2 in gastric mucosa cells. Treatment with 4-HNE increased levels of DNA damage in ALDH2-knockdown GES-1 cells, while overexpression of ALDH2 decreased 4-HNE-induced DNA damage. These findings suggest that ALDH2 can protect gastric mucosa cells against DNA damage caused by oxidative stress by reducing levels of 4-HNE.

Tumor suppressor miR-24 restrains gastric cancer progression by downregulating RegIV.

BACKGROUND: microRNAs are small noncoding RNAs that modulate a variety of cellular processes by regulating multiple targets, which can promote or inhibit the development of malignant behaviors. Accumulating evidence suggests miR-24 plays important roles in human carcinogenesis. However, its precise biological role remains largely elusive. This study examined the role of miR-24 in gastric cancer (GC). METHODS: The expression of miR-24 in GC tissues compared with matched non-tumor tissues and GC cells was detected by qRT-PCR. Synthetic short single or double stranded RNA oligonucleotides and lentiviral vectors were used to regulate miR-24 expression in GC cells to investigate its function in vitro and in vivo. RESULTS: miR-24 was significantly downregulated in GC tissues compared with matched non-tumor tissues and was associated with tumor differentiation. Ectopic expression of miR-24 in SGC-7901 GC cells suppressed cell proliferation, migration and invasion in vitro as well as tumorigenicity in vivo by inducing cell cycle arrest in G0/G1 phase and promoting cell apoptosis. Furthermore, we identified RegIV as a target of miR-24 and demonstrated that miR-24 regulated RegIV expression via binding its 3' untranslated region. CONCLUSIONS: miR-24 functions as a novel tumor suppressor in GC and the anti-oncogenic activity may involve its inhibition of the target gene RegIV. These findings suggest the possibility for miR-24 as a therapeutic target in GC.

Biglycan enhances gastric cancer invasion by activating FAK signaling pathway.

Biglycan (BGN) is an important member of small leucine-rich proteoglycans family, and has been implicated in oncogenesis and development of various human cancer types. Here we report that BGN promotes tumor invasion and metastasis of gastric cancer both in vitro and in vivo. BGN expression is significantly higher in gastric cancer tissues and associated with lymph node metastasis, depth of tumor invasion and TNM stage. BGN enhances gastric cancer cell wound healing, migration and invasion ability as well as the tube formation ability of endothelial cells in vitro. Animal experiments results in vivo are consistent with outcomes in vitro. BGN induces increased phosphorylation of FAK (Tyr576/577, Tyr925 and Tyr397) and Paxillin. These results indicate that BGN is upregulated, and plays an oncogenic role, in gastric cancer metastasis by activating the FAK signaling pathway.

[Study on correlations between total saponins content in rhizome or mycorrhizal infection rate of Pairs polyphylla var. yunnanensis and soil factors].

OBJECTIVE: Through correlation and path analysis between total saponins content in rhizome/mycorrhizal infection rate in roots of Pairs polyphylla var. yunnanensis and soil factors, to make an inquiry into the role of soil factors in the quality formation of P. polyphylla var. yunnanensis. METHOD: Tested total saponins in rhizome, mycorrhizal fungal infection rate in root and physical and chemical properties in rhizosphere soil in 25 different growth areas, and statistically analyzed the relationship between total saponins in rhizome/mycorrhizal infection rate in roots of P. polyphylla var. yunnanensis and soil factors by using correlation and path analysis. RESULT: The symbiosis relationship between AM mycorrhizal and roots of P. polyphylla var. yunnanensis were better established under natural condition, of which the infection ratio between 36.41%, 83.37%. There were significantly positive correlation between total saponins content in rhizome and urease activity or alkaline phosphatase activities or organic matter in soil, but there was significantly negative correlation between total saponins content and bulk density. There was significantly positive correlation between AM infection ratio and alkaline nitrogen. Path analysis indicated that total saponins of rhizome mainly affected by alkaline nitrogen in soil rhizosphere, secondly by soil organic matter and soil urease activity. While the mycorrhizal fungal colonization ratio was mainly affected by soil pH, secondly by alkaline nitrogen, urease activity, and available phospherus in soil. CONCLUSION: There is closed relationship between quality formation of P. polyphylla var. yunnanensis and soil factors. Path analysis is better for reflecting the contribution of soil factors to total saponins and mycorrhizal infection ratio.