Cordyceps cicadae extracts ameliorate renal malfunction in a remnant kidney model.

BACKGROUND AND OBJECTIVES: Chronic kidney disease (CKD) is a growing public health problem with an urgent need for new pharmacological agents. Cordyceps cicadae is widely used in traditional Chinese medicine (TCM) and has potential renoprotective benefits. The current study aimed to determine any scientific evidence to support its clinical use. METHODS: We analyzed the potential of two kinds of C. cicadae extract, total extract (TE) and acetic ether extract (AE), in treating kidney disease simulated by a subtotal nephrectomy (SNx) model. Sprague-Dawley rats were divided randomly into seven groups: sham-operated group, vehicle-treated SNx, Cozaar, 2 g/(kg∙d) TE SNx, 1 g/(kg∙d) TE SNx, 92 mg/(kg∙d) AE SNx, and 46 mg/(kg∙d) AE SNx. Renal injury was monitored using urine and serum analyses, and hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) stainings were used to analyze the level of fibrosis. The expression of type IV collagen (Col IV), fibronectin (FN), transforming growth factor-ß1 (TGF-ß1), and connective tissue growth factor (CTGF) was detected by immunohistochemistry. RESULTS: Renal injury, reflected in urine and serum analyses, and pathological changes induced by SNx were attenuated by TE and AE intervention. The depositions of Col IV and FN were also decreased by the treatments and were accompanied by reduced expression of TGF-ß1 and CTGF. In some respects, 2 g/(kg∙d) of TE produced better effects than Cozaar. CONCLUSIONS: For the first time, we have shown that C. cicadae may inhibit renal fibrosis in vivo through the TGF-ß1/CTGF pathway. Therefore, we conclude that the use of C. cicadae could provide a rational strategy for combating renal fibrosis.

A novel mutation in proprotein convertase subtilisin/kexin type 9 gene leads to familial hypercholesterolemia in a Chinese family.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal disorder associated with elevated plasma low density lipoprotein (LDL) levels leading to premature coronary heart disease (CHD). As a result of long-term hyperlipemia, FH patients will present endarterium thickening and artherosclerosis. In the present study we scanned the related gene of a clinically diagnosed autosomal genetic hypercholesterolemia family for the possible mutations and established eukaryotic expression vector of mutation of proprotein convertase subtilisin/kexin type 9 (PCSK9) gene with gene recombination technique to investigate the contributions of the variation on low density lipoprotein receptor (LDL-R) metabolism and function alternation. METHODS: Mutation detection was conducted for LDL-R, apolipoprotein B(100) (apoB(100)) and PCSK9 gene with nucleotide sequencing in a Chinese FH family. The full-length cDNA of wild type PCSK9 gene (WT-PCSK9) was obtained from Bel-7402. Site mutagenesis was used to establish the recombinant eukaryotic expression vector carrying pathogenic type of PCSK9 gene and the inserted fragment was sequenced. With the blank vector as control, liposome transfection method was used to transfect the Bel-7402 cells with recombinant plasmid. The expression of LDL-R mRNA was examined by RT-PCR. PCSK9 and the expression of LDL-R protein were determined by Western blotting. RESULTS: The G-->T mutation at the 918 nucleotide of PCSK9 gene resulted in the substitution of the arginine by a serine at the codon 306 of exon 6. After sequencing, it was confirmed that the inserted fragment of established expression vector had correct size and sequence and the mutant was highly expressed in Bel-7402 cells. There was no significant variation in the levels of LDL-R mRNA. LDL-R mature protein was decreased by 57% after the cells were transfected by WT-PCSK9 plasmid. Mature LDL-R was significantly decreased by 12% after the cells were transfected by R306S mutant as evidenced by gray scale scanning, suggesting that the new mutant R306S can significantly decrease the expression of mature LDL-R protein. CONCLUSIONS: A novel missense mutation of PCSK9 gene, R306S, was found and the eukaryotic expression vectors of mutant and wild-type of PCSK9 gene were established. There was no significant variation in the levels of LDL-R mRNA. The R306S mutation could significantly lead to the decrease of LDL-R mature protein expression, which might be the pathogenic gene of the FH family.

Functional analysis of low-density lipoprotein receptor in homozygous familial hypercholesterolemia patients with novel 1439 C-->T mutation of low-density lipoprotein receptor gene.

BACKGROUND: Familial hypercholesterolemia (FH), caused by low density lipoprotein (LDL) receptor (LDL-R) gene mutations, is associated with increased risk of premature coronary heart disease. Until now, limited molecular data concerning FH are available in China. The present study described the clinical profiles and cell biological defects of a Chinese FH kindred with novel LDL-R gene mutation. METHODS: The patient's LDL-R gene coding region was sequenced. The patient's lymphocytes were isolated and the LDL-R expression, binding and up-take functions were observed by immunohistochemistry staining and flow cytometry detection. The patient's heart and the major large vessels were detected by vessel ultrasound examination and myocardial perfusion imaging (MPI). RESULTS: The patient's LDL-R expression, LDL binding and up-take functions were significantly lower than normal control (39%, 63% and 76% respectively). A novel homozygous 1439 C-->T mutation of the LDL-R gene was detected in the patient and his family. ECG showed atypical angina pectoris. Echocardiogram showed stenosis of the coronary artery and calcification of the aortic valve and its root. Blood vessel ultrasound examination showed the thickness of large vessel intima, and the vessel lumen was narrowed by 71%. MPI showed ischemic changes. CONCLUSIONS: The LDL-R synthesis dysfunction of FH patients leads to arterial stenosis and calcification, which are the major phenotype of the clinical disorder. The mutation of the LDL-R gene is determined. These data increase the mutational spectrum of FH in China.

[The effects of polygoni multiflori total glycosides on the experimentally atherosclerotic formation in apoE-deficient mice].

OBJECTIVE: To study the effect of polygoni multiflori total glycosides (PMTG) on the expressions of ICAM-1 and VCAM-1 in the apoE-deficienct (ApoE-/-)mice with experimental atherosclerosis (AS) and underlying mechanism. METHOD: Thirty-two female apoE-deficienct mice were randomized into four groups: high dose PMTG group (150 mg x kg(-1) x d(-1)), low dose PMTG group (25 mg x kg(-1) x d(-1)), atorvastatin positive control group (5 mg x kg(-1) x d(-1)) and model group. At the end of the tenth week of treatment, all mice were killed. The serum levels of total cholesterol (TC), triglyceride(TG), high-density lipoprotein-cholesterol (HDL-C) were measured by enzyme dynamics method. Light microscopy were adopted to assess the degree of atherosclerotic plaque of aortic wall and image analysis was performed with computer. The expressions of ICAM-1 and VCAM-1 were studied by SABC imunohistochemistry. RESULT: In comparison with the model group, (1) PMTG reduced the levels of serum TC and TG significantly (P < 0.01), but elevated HDL level obviously (P < 0.01) . (2) PMTG increased the levels of serum NO and the anti-oxidation capacities significantly (P < 0.05 and P < 0.01), but reduced the levels of serum MDA markedly (P < 0.01). (3) PMTG reduced also the extent of atherosclerotic plaque of aorta areas were (P < 0.05). (4) PMTG deregulated the expressions of ICAM-1 and VCAM-1 in aortic wall. CONCLUSION: PMTG could inhibit the occurrence and development of atherosclerotic lesions by the regulating lipid metabolism and anti-oxidation and deregulating the of expressiona of ICAM-1 and VCAM-1 in AopE-/- mice in aortic wall.

[Identification of a novel splice mutation of low density lipoprotein receptor gene in a Chinese family with familial hypercholesterolemia].

OBJECTIVE: To identify the mutation of low density lipoprotein receptor(LDLR) gene in a large Chinese family with familial hypercholesterolemia(F H) and make a discussion on the pathogenesis of FH at the molecular level. METHODS: Investigations were made on a patient with the clinical phenotype of homozygous FH and his parents for mutations of promoter and all 18 exons of LDLR gene. Screening was carried out using Touch down PCR and a g arose gel electrophoresis, combined with DNA sequence analysis. The results were compared with the normal sequences in GenBank and FH database (www.ucl.uk/fh) t o find the mutation. Then the mutation was identified in other members of the family. In addition, the authors screened the apolipoprotein B(100) (apoB(100)) gene f or known mutations (R3500Q) that cause familial defective apoB(100) (FDB) by PCR-RFLP. RESULTS: A novel homozygous IN III 5' GT --> AT mutation in the splice donor of LDLR intron 3 was detected in the homozygote propositus with FH. The mutation was also identified in four heterozygous carriers in his family. No mutations R3500Q of apoB(100)were observed. CONCLUSION: A homozygous G --> A splice mutation in LDLR gene was first reported. The change of the splice donor in LDLR intron 3 may cause skipping of exon 3, which is responsible for FH. Perhaps it is a particular pathogenesis for Chinese people.