Lentivirus-mediated short hairpin RNA interference of CENPK inhibits growth of colorectal cancer cells with overexpression of Cullin 4A.

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignant tumors worldwide. The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative. Immunohistochemical analysis has revealed high expression of centromere protein K (CENPK) in CRC. However, the role of CENPK in the progression of CRC is not well characterized. AIM: To evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A (CUL4A) in RKO and HCT116 cells. METHODS: Human colon cancer samples were collected and tested using a human gene expression chip. We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis. In vitro experiments verified the function of this gene. We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction (qPCR), western blot, and flow cytometry. The effect of short hairpin RNA (shRNA) virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging. To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells, we performed a series of in vitro experiments, using qPCR, western blot, MTT assay, and flow cytometry. RESULTS: We demonstrated overexpression of CENPK in human colon cancer samples. CENPK was an independent risk factor in patients with CRC. The downstream genes FBX32, CUL4A, and Yes-associated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells. Significantly delayed xenograft tumor emergence, slower growth rate, and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group. The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo. The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells, with overexpression of the CUL4A. CONCLUSION: We indicated a potential role of CENPK in promoting tumor proliferation, and it may be a novel diagnostic and prognostic biomarker for CRC.

Epithelial-mesenchymal transition and metastasis of colon cancer cells induced by the FAK pathway in cancer-associated fibroblasts.

OBJECTIVE: The role and mechanism of tetrathiomolybdate (TM) in cancer-associated fibroblasts (CAFs) in colon cancer using three-dimensional (3D) culture were investigated, and the associations between the focal adhesion kinase (FAK) pathway and epithelial-mesenchymal transition (EMT) in CAFs were explored. METHODS: A 3D co-culture model of colon cancer LOVO cells with CAFs and normal fibroblasts (NFs) was established using Matrigel as a scaffold material. The differential expression of LOXL2 (lysyl oxidase-like 2) in the supernatant of CAFs and NFs was determined using ELISA, and expression levels of EMT-related proteins and FAK signaling pathway-related proteins were determined using western blot. RESULTS: LOXL2 levels secreted by CAFs were higher compared with that secreted by NFs. In the CAF + LOVO group, compared with the LOVO group, E-cadherin expression decreased significantly, while N-cadherin and F-PAK expression increased significantly. TM results were opposite compared with the above results. CONCLUSIONS: CAFs stimulate EMT in human colon cancer LOVO cells by secreting LOXL2 to activate the FAK signaling pathway, thereby promoting tumor metastasis. TM inhibited the occurrence of EMT in the CAF-induced colon cancer LOVO cell line, thereby reducing the invasion and metastasis of colon cancer cells.

The Synergistic Antitumor Effect of Tanshinone IIA Plus Adriamycin on Human Hepatocellular Carcinoma Xenograft in BALB/C Nude Mice and Their Influences on Cytochrome P450 CYP3A4 In Vivo.

OBJECTIVE: Hepatocellular carcinoma is one of the most common diseases that seriously threaten human life and health. In this study, we evaluated the inhibitory effect of tanshinone IIA (Tan IIA) combined with adriamycin (ADM) on human hepatocellular carcinoma and developed a platform to assess the function if Chinese herbal ingredients combined with chemotherapy drugs have synergistic antitumor effects in vivo. METHODS: Established animal model of human hepatocarcinoma HepG2 cell in nude mice. Mice were divided into model control group, Tan IIA group, ADM group, and Tan IIA + ADM group. The changes from general condition, weight, tumor volume, and inhibition rate were observed. The data were gathered from serum AST level and histopathological changes. The content and activity of cytochrome P450 were determined by spectrophotometric analysis. CYP3A4 protein expression was analyzed by western blotting. The binding model crystal structure of Tan IIA and ADM with pregnane X receptor (PXR) was evaluated by Discovery Studio 2.1. RESULTS: A combination of Tan IIA with ADM could improve life quality by relieving ADM toxicity, decreasing tumor volume, declining serum AST level, and improving liner pathological section in tumor-bearing mice. The inhibitory rates of Tan IIA, ADM, and cotreatment were 32.77%, 60.96%, and 73.18%, respectively. The Tan IIA group significantly enhanced the content of cytochrome b5, P450, and erythromycin-N-demethylase activity. CYP3A4 protein expression was enhanced obviously by the Tan IIA + ADM group. Virtual molecular docking showed that both Tan IIA and ADM could be stably docked with the same binding site of PXR but different interactions. CONCLUSIONS: Tan IIA in combination with ADM could improve the life quality in tumor-bearing mice and enhance the antitumor effect. The Tan IIA group increased the concentration of cytochrome P450 enzymes and activity. Combined Tan IIA with ADM could upregulate the CYP3A4 protein expression and make relevant interaction with protein PXR by virtual docking.

RRS1 silencing suppresses colorectal cancer cell proliferation and tumorigenesis by inhibiting G2/M progression and angiogenesis.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Ribosome biogenesis regulatory protein homolog (RRS1) is an essential factor involved in ribosome biogenesis, while its role in CRC remains largely unclear. Here, we found that RRS1 expression was significantly higher in CRC tissues compared with adjacent normal tissues. RRS1 High expression also predicted poor overall survival of CRC patients. Knockdown of RRS1 induced the G2/M cell cycle arrest, apoptosis and suppressed the proliferation of RKO and HCT-116 CRC cells. Furthermore, angiogenesis was also reduced in CRC cells after RRS1 knockdown. In addition, suppression of RRS1 blunted the tumor formation of CRC cells in nude mice. At the molecular level, silencing of RRS1 decreased the expression of M-phase inducer phosphatase 3 (CDC25C), Cyclin-dependent kinase 1 (CDK1), antigen KI-67 (KI67) and increased the protein level of cyclin-dependent kinase inhibitor 1 (CDKN1A) and tumor suppressor p53 (p53). Taken together, our findings provide evidence that RRS1 may promote the development of colon cancer. Therefore, targeting RRS1 may be a promising therapeutic strategy for CRC patients.

Metastasis-Associated in Colon Cancer-1 Associates With Poor Prognosis and Promotes Cell Invasion and Angiogenesis in Human Cervical Cancer.

OBJECTIVE: The aim of this study is to investigate the clinicopathologic significance and potential role of metastasis-associated in colon cancer-1 (MACC1) in the progression of cervical cancer. METHODS: MACC1 expression was examined in cervical cancer cell lines, 6 matched cervical cancer tissues, and adjacent noncancerous tissues using Western blotting and real-time reverse transcriptase polymerase chain reaction. MACC1 protein expression and localization were determined in 181 paraffin-embedded archived cervical cancer samples using immunohistochemistry. Statistical analyses were applied to evaluate the clinicopathologic significance. The effects of MACC1 on cell migration, invasion, and angiogenesis were examined using migration assay, wound healing assay, 3-dimensional morphogenesis assay, and chicken chorioallantoic membrane assay. Western blotting was performed to examine the impact of MACC1 on the Akt and nuclear factor κB signaling pathways. RESULTS: Both protein and messenger RNA levels of MACC1 was up-regulated in cervical cancer cell lines and cervical cancer tissues, as compared with normal tissues. High MACC1 expression was detected in 96 (53%) of 181 of the cervical cancer tissues. In addition, high MACC1 expression correlated significantly with aggressiveness of cervical cancer, including International Federation of Gynecology and Obstetric stage (P = 0.001), pelvic lymph node metastasis (P = 0.004), recurrence (P = 0.037), and poor survival (P = 0.001). Moreover, enforced expression of MACC1 in cervical cancer cell lines significantly enhanced cell migration, invasion, and angiogenesis. Conversely, knockdown of MACC1 caused an inhibition of cell migration, invasion, and angiogenesis. Up-regulation of MACC1 increased, but knockdown of MACC1 decreased the expression of matrix metalloproteinase-2 and matrix metalloproteinase-9. Furthermore, enforced expression of MACC1 could enhance, but knockdown of MACC1 could reduce AKT and nuclear factor κB pathway activity. CONCLUSIONS: Our findings suggest that MACC1 protein, as a valuable marker of cervical cancer prognosis, plays an important role in the progression of human cervical cancer cells.

Genetic polymorphisms of DNA repair pathways influence the response to chemotherapy and overall survival of gastric cancer.

We aimed to evaluate the clinical response to platinum-based chemotherapy and treatment outcome of gastric cancer patients in the present of ERCC1, ERCC2, NBN, RAD51, and XRCC3 gene polymorphisms. A number of 415 patients of gastric cancer that received platinum-based chemotherapy were enrolled in the present study. The presence of ERCC1 rs11615 and rs2298881, ERCC2 rs1799793 and rs13181, NBN rs1805794, rs709816, and RAD51 rs1801321 and XRCC3 rs1799794 were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Conditional regression analysis identified that CC genotype of ERCC1 rs11615 and AA genotype of ERCC2 rs1799793 was associated with a better response to chemotherapy in gastric cancer patients, and the odds ratio (ORs)(95% confidence interval (CI)) were 2.70(1.33-5.70) and 3.12(1.52-6.84), respectively. By the Cox analysis, the CC genotype of ERCC1 rs11615, AA genotype of ERCC2 rs1799793, and CC genotype of NBN rs1805794 were significantly associated with a longer overall survival (OS) of gastric cancer. In conclusion, our results suggest that ERCC1 rs11615, ERCC2 rs1799793, and NBN rs1805794 polymorphisms in the DNA repair pathways may influence the response to chemotherapy and OS of gastric cancer.

Osteopontin knockdown suppresses the growth and angiogenesis of colon cancer cells.

AIM: To investigate the effects of osteopontin (OPN) gene expression knockdown on colon cancer Lovo cells in vitro. METHODS: Four candidate small interfering RNA (siRNA) constructs targeting the OPN gene and a scrambled control sequence (NC-siRNA) were synthesized and inserted into a pGPU6/GFP/Neo expression vector. After confirmation by restriction enzyme digestion and DNA sequencing, the recombinant plasmids were subsequently transfected into a human colon cancer cell line (Lovo) using a liposome transfection method. Stably transfected cells were maintained with G418 selection and referred to as Lovo-OPN-1, -2, -3, -4, and Lovo-NC cells. Knockdown efficiency of each of the four siRNA constructs was determined by real-time reverse transcription polymerase chain reaction assays and western blotting, and the construct with the most effective silencing was used for subsequent experiments. Cell proliferation, adhesion, and Matrigel invasion assays were performed to analyze the effects of OPN knockdown in stably transfected Lovo cells. The levels of four angiogenic factors, namely vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, MMP-9 and urokinase plasminogen activator were detected by enzyme-linked immunosorbent assays (ELISA). RESULTS: Recombinant vectors containing OPN-specific and scrambled siRNA sequences were successfully constructed and stably transfected into Lovo cells. Compared with the control Lovo and Lovo-NC cells, the levels of OPN mRNA and protein expression in Lovo-OPN-1, -2, -3, and -4 were significantly reduced (all P < 0.05), with the most efficient reduction observed in Lovo-OPN-4 cells (P < 0.05). Relative to untransfected Lovo cells, OPN mRNA expression levels in Lovo-NC and Lovo-OPN-4 cells were 1.008 ± 0.067 and 0.160 ± 0.023, respectively. The relative OPN protein expression levels in Lovo, Lovo-NC, and Lovo-OPN-4 cells were 3.024 ± 0.211, 2.974 ± 0.630, and 0.121 ± 0.008, respectively. Moreover, transfection with the scrambled sequence had no effect on the expression of OPN. After 24, 48, 72, and 96 h of cultivation, absorption values at 450 nm to assess proliferation of Lovo-OPN-4 cells were 0.210 ± 0.017, 0.247 ± 0.024, 0.314 ± 0.037, and 0.359 ± 0.043, respectively, which were significantly lower than those of Lovo (0.244 ± 0.031, 0.313 ± 0.024, 0.513 ± 0.048 and 0.783 ± 0.051) and Lovo-NC cells (0.241 ± 0.029, 0.309 ± 0.022, 0.563 ± 0.023, and 0.735 ± 0.067) (all P < 0.05). The absorption values at 595 nm, which were measured in a cell adhesion assay, showed that adhesion of Lovo-OPN-4 cells (0.215 ± 0.036) was significantly decreased compared to Lovo (0.490 ± 0.037) and Lovo-NC cells (0.462 ± 0.043) (P < 0.05). The number of invasive Lovo-OPN-4 cells (16.1 ± 1.9) was also significantly decreased compared to Lovo (49.9 ± 5.4) and Lovo-NC cells (48.8 ± 4.5) (P < 0.05). ELISA assays showed significant reductions in Lovo-OPN-4 cells compared to Lovo and Lovo-NC cells with regard to the expression of VEGF (1687.85 ± 167.84 ng/L vs 2348.54 ± 143.80 ng/L and 2284.39 ± 138.62 ng/L, respectively), MMP-2 (2966.07 ± 177.36 µg/L vs 4084.74 ± 349.54 µg/L and 4011.41 ± 424.48 µg/L, respectively), MMP-9 (3782.89 ± 300.64 µg/L vs 5062.90 ± 303.02 µg/L and 4986.38 ± 300.75 µg/L, respectively) and uPA (1152.69 ± 120.79 µg/L vs 1380.90 ± 147.25 µg/L and 1449.80 ± 189.92 µg/L, respectively) (all P < 0.05). CONCLUSION: Knockdown of OPN gene expression suppresses colon cancer cell growth, adherence, invasion, and expression of angiogenic factors.

[Effect of human colon carcinoma-associated fibroblasts on biological behavior of colon carcinoma Lovo cells].

OBJECTIVE: To investigate the effects of human colon carcinoma-associated fibroblasts (CAFs) on proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells. METHODS: The co-culture models among colon CAFs, NFs and Lovo cell were established by conditioned medium (CM) of human colon CAFs and colon normal fibroblasts (NFs). Lovo cells in the blank control group was treated with serum-free culture medium. The effects of human colon CAFs on proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells were detected by cell proliferation assay, adhesion assay, migration assay and Transwell invasion assay. RESULTS: After co-culture with colon CAFs, the absorbance (A) value of Lovo cells was (0.667±0.059) in 48 h and (0.709±0.030) in 72 h. The A value of Lovo cells adhesion to fibronectin was (0.588±0.067). The cell mobility rates were (35.2±8.7)% in 12 h and (64.6±7.1)% in 24 h. The number of invasive cell was (56.2±4.8). All the above parameters were increased compared with those in the blank control group and NFs group (all P<0.01). CONCLUSION: Human colon CAFs can promote the proliferation, adhesion, migration and invasion of colon carcinoma Lovo cells.

Antidiarrheal properties of different extracts of Chinese herbal medicine formula Bao-Xie-Ning.

OBJECTIVE: Bao-Xie-Ning (BXN), a traditional Chinese herbal medicine (CHM) formula composed of Fructus Evodiae, Flos Caryophylli and Cortex Cinnamomi, and used for the treatment of infant diarrheal illness, was subject to systematic assessment for its putative multiple pharmacodynamic effects and pharmacological antidiarrheal mechanisms. METHODS: High-performance liquid chromatography-diode array detector-electrospray ionization-mass spectrometric/mass spectrometry was developed and validated for identification and quantification of the main constituents in different extracts of BXN. Male Kunming mice weighing 20 to 25 g were used for detecting the antidiarrheal activity of the extracts. Ethanolic extract (EE), volatile oil extract (VOE), and aqueous extract (AE) of BXN were respectively subjected to pharmacodynamic and pharmacological comparison in assessing antidiarrheal effects with senna-induced diarrhea, castor oil-induced diarrhea, acetic acid-induced writhing assay, and isolated duodenum test. RESULTS: The highest yields of three detected components of BXN, rutaecarpine, eugenol and cinnamaldehyde were observed in EE. EE showed the most remarkable antidiarrheal activity in dose-dependent and time-dependent manners in both senna- and castor oil-induced diarrhea models, and presented dose-dependent analgesic activity in acetic acid-induced algesthesia model. In addition, EE extract of BXN also exhibited strong antimobility action on the intestine and strongest depression on spontaneous contraction of isolated duodenum. CONCLUSION: Ethanol extraction is an efficient method to extract the active constituents of BXN. BXN extract demonstrated multiple pharmacological activities affecting the main mechanisms of diarrhea, which validated BXN's usage in the comprehensive clinical treatment of diarrhea.

Scutellaria extract decreases the proportion of side population cells in a myeloma cell line by down-regulating the expression of ABCG2 protein.

BACKGROUND AND AIMS: Scutellaria is one of the most popular traditional Chinese herbal remedies against various human diseases, including cancer. In this study, we examined the active effects of Scutellaria extract and its main flavonoid constituents on the proportion of side population cells within human multiple myeloma cell line RPMI8226 in vitro and explored the potential molecular mechanisms involved. MATERIALS AND METHODS: The contents of flavonoids in ethanolic extract of Scutellaria baicalensis Georgi were determined using high performance liquid chromatography. The antiproliferative effect of the ethanolic extract on RPMI-8226 was determined by CCK assay. Apoptosis was measured by annexin combining with propidium iodide in a flow cytometer. Cell cycle analysis was performed by propidium iodide staining in combination with flow cytometry analysis. Hoechst 33342 exclusion assay was used for the identification of side population within RPMI8226 cells. The expression of ABCG2 protein was assessed by Western blotting assay. RESULTS: The content of major flavonoids constitutents of Scutellaria extract was baicalin (10.2%), wogonoside (2.50%), baicalein (2.29%), and wogonin (0.99%), respectively. The crude Scutellaria extract did not show significant anti-proliferative effect, apoptosis induction and cell cycle arrest in RPMI-8226 within the concentrations of 1-75µg/mL. However, the ethanolic extract, baicalein, wogonin and baicalin reduced the side population cells in RPMI-8226, and data showed that baicalein and wogonin had stronger inhibitory effects. Correspondingly, they also exhibited significant effects on decreasing the expression level of ABCG2 protein in RPMI-8226 in vitro. CONCLUSIONS: Our results for the first time demonstrated a novel mechanism of action for Scutellaria extract and its main active flavonoids, namely targeting SP cells by modulating the expression of ABCG2 protein. This study provides an insight for new therapeutic strategies targeting cancer stem cells of multiple myeloma.

Association between the GSTP1 codon 105 polymorphism and gastric cancer risk: an updated meta-analysis.

OBJECTIVE: The current meta-analysis was performed to address a more accurate estimation of the association between glutathione S-transferase P1 (GSTP1) codon 105 polymorphism and risk of gastric cancer (GC), which has been widely reported with conflicting results. METHODS: A comprehensive literature search was conducted to identify all the relevant studies. Fixed or random effect models were selected based on the heterogeneity test. Publication bias was estimated using Begg's funnel plots and Egger's regression test. RESULTS: A total of 20 studies containing 2,821 GC cases and 6,240 controls were finally included in the analyses. Overall, no significant association between GSTP1 polymorphism and GC risk was observed in worldwide populations. However, subgroup analysis stratified by ethnicity showed that GSTP1 polymorphism was significantly associated with increased risk of GC in Asians (G vs. A, OR = 1.273, 95%CI=1.011-1.605; GG vs. AA, OR=2.103, 95%CI=1.197- 3.387; GG vs. AA+AG, OR =2.103, 95%CI=1.186-3.414). In contrast, no significant association was found in Caucasians in any genetic models, except for with AG vs. AA (OR=0.791, 95%CI=0.669-0.936). Furthermore, the GSTP1 polymorphism was found to be significantly associated with GC in patients with H. pylori infection and in those with a cardiac GC. Subgroup analysis stratified by Lauren's classification and smoking status showed no significant association with any genetic model. No studies were found to significantly influence the pooled effects in each genetic mode, and no potential publication bias was detected. CONCLUSIONS: This meta-analysis suggested that the GSTP1 polymorphism might be associated with increased risk of GC in Asians, while GSTP1 heterozygote genotype seemed to be associated with reduced risk of GC. Since potential confounders could not be ruled out completely, further studies are needed to confirm these results.

[Clinical study on Shen Shuai Fang in treating chronic renal failure].

OBJECTIVE: To explore the effect of Shen Shuai Fang in treating Chronic Renal Failure (CRF) of deficiency of spleen and kidney with turbid damp and blood stagnation symptom. METHODS: 125 patients with CRF of deficiency of spleen and kidney with turbid damp and blood stagnation symptom were randomly divided into two groups, 75 patients in treatment group were treated with Shen Shuai Fang adding western medicine and the other patients in control group were treated only with western medicine. We observed the effect and indexes change including renal function, Hb, proteinuria, lipid before and after treatment. RESULTS: After six months' treatment, the general effective rate in treatment group was 77.33%, which was higher than that in control group obviously (44.00%, P < 0.01). So treatment group was obviously better than control group on decreasing proteinuria, improving renal function, increasing Hb, ameliorating lipid metabolism (P < 0.05 or P < 0.01). CONCLUSION: Shen Shuai Fang is effective to treat CRF of deficiency of spleen and kidney with turbid damp and blood stagnation symptom.

[A clinical research of Naoxinqing tablet's effects on blood fat and viscosity].

OBJECTIVE: To investigate the effects on blood fat and viscosity of Naoxinqing tablet. METHODS: 66 patients of primary hyperlipoidemia were randomly divided into three groups. The first group was treated by Naoxinqing tablet, the second group was treated by Naoxinqing and combined with half dose Simvastatin, the third group was treated by full dose Simvastatin. Then the clinical effect, fasting plasma lipid, the index of blood viscosity and side effect of the three group' patients were compared after 8 weeks. RESULTS: The index of blood fat improved obviously after treatment of the three groups ( P < 0.05, P < 0. 01), group 2 and 3 were significantly superior to group 1 (P < 0.05), and no significance between group 2 and 3 (P > 0.05). CONCLUSIONS: The Naoxinqing tablet can improve the metabolism of blood fat, decrease blood viscosity obviously and treat hyperlipoidemia effectively with little side effect.

[Clinical study on Dan Shao Tang in treating diabetic nephropathy of deficiency of Yin with damp-heat symptom].

OBJECTIVE: To explore the effect of Dan Shao Tang (DST) in treating diabetic nephropathy (DN) of deficiency of Yin with damp-heat symptom. METHODS: The 72 patients with DN of deficieny of Yin with damp-heat symptom were randomly divided into two groups. Among them, the 42 patients in treatment group were treated with DST and western medicine and the 30 patients in control group were treated only with western medicine. Before and after treatment, the effect and indexes change including blood glucose, renal function, proteinuria, microproteins, lipid metabolism and hyperviscosity were observed. RESULTS: After three months treatment, the general effective rate in treatment group was 76.19%, which was markedly higher than that in control group (50.00%, P < 0.01). Moreover, treatment group was obviously better than control group on decreasing blood glucose proteinuria and microproteins, improving renal function, ameliorating lipuid metabolism and hyperviscosity (P < 0.01 or P < 0.05). CONCLUSION: DST is effective ton treatment on DN of deficiency of Yin with damp-heat symptom.

[Clinical observation on colquhounia root tablet in treating lipid metabolism disturbance secondary to nephrotic syndrome].

OBJECTIVE: To study the effect of Colquhounia root tablet (CRT) in treating nephrotic syndrome with sequential lipid metabolism disorder (NS-LMD). METHODS: The 96 patients with NS-LMD were randomly divided into two groups, the 60 cases in the treated group treated with CRT and the 36 cases in the control group treated with hormone or cytotoxic medicine. The curative effect and the related indexes were determined before and after treatment. RESULTS: After six months treatment, the general effective rate in the treated group was 88. 33%, which was markedly higher than that in the control group (69.44%, P < 0.05). The levels of the treated group in ameliorating lipid metabolism disorder and renal dysfunction were also higher than those in the control group (P < 0.05). CONCLUSION: CRT could improve NS-LMD, improve renal function, eliminate urinary protein and increase plasma albumin. It is highly effective with low toxicity and safe.