Effect of Huan-shuai Recipe (缓衰方) on Expression of Extracellular Matrix in 5/6 Nephrectomized Rats

Ｈｕａｎ ｓｈｕａｉＲｅｃｉｐｅ（ＨＳＲ，缓衰方），ａＣｈｉｎｅｓｅｈｅｒｂａｌｍｅｄｉｃｉｎａｌｐｒｅｐａｒａｔｉｏｎｅｓｔａｂｌｉｓｈｅｄｂｙｔｈｅａｕ ｔｈｏｒｓ，ｐｏｓｓｅｓｓｅｓｔｈｅｍａｉｎｆｕｎｃｔｉｏｎｏｆｂｅｎｅｆｉｔｉｎｇｖｉｔａｌｅｎｅｒｇｙ，ｎｏｕｒｉｓｈｉｎｇｂｌｏｏｄ，ｐｒｏｍｏｔｉｎｇｂｌｏｏｄｃｉｒｃｕｌａ ｔｉｏｎ，ｐｒｏｍｏｔｉｎｇｄｉｕｒｅｓｉｓｔｏｅｌｉｍｉｎａｔｅｅｖｉｌＷｅｔｎｅｓｓａｎｄｐｕｒｇｉｎｇｔｈｅＴｕｒｂｉｄｎｅｓｓ，ｈａｓｂｅｅｎｕｓｅｄｔ…     

Fuzheng Huayu Formula (扶正化瘀方) Prevents Rat Renal Interstitial Fibrosis Induced by HgCl2 via Antioxidative Stress and Down-regulation of Nuclear Factor-Kappa B Activity

Objective: To investigate the mechanism of action of Fuzheng Huayu Formula (扶正化瘀方, FZHY) against renal interstitial fibrosis (RIF) relating to oxidative injury and nuclear factor-kappa B (NF-κB) activity. Methods: Thirty-two Sprague-Dawley rats were randomly divided into 3 groups: normal group, model group and FZHY treatment group. The RIF model was induced by oral administration of HgCl2 at a dose of 8 mg/kg body weight once a day for 9 weeks. Meanwhile, rats in FZHY treatment group orally took FZHY at a dose of 4.0 g/kg rat weight for 9 weeks. The content of hydroxyproline (Hyp) and collagen deposition in kidney were observed. The activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), the content of glutathione (GSH) and malondialdehyde (MDA) of kidney were tested. The expressions of inhibitor-κappa B (IκB), phospho-IκB (p-IκB), tumor necrosis factor-α (TNF-α), matrix metalloproteinase-2 (MMP-2) and α-smooth muscle actin (α-SMA) were analyzed by Western blot. α-SMA expression was also observed by immunofluorescent staining. MMP-2 activity was measured by gelatin zymography. NF-κB activation was determined by electrophoretic mobility shift assay. Results: Renal interstitial fibrosis was induced by HgCl2, demonstrated by remarkably increased Hyp contents and excessive collagen deposition in kidney (P<0.01). FZHY significantly inhibited renal interstitial collagen deposition and reduced Hyp content of the HgCl2-treated rats (P<0.01). GSH content decreased obviously, and MDA content increased significantly in HgCl2-treated rats compared with that of normal rats (P<0.01). FZHY significantly increased GSH content and decreased MDA content in the model rats (P<0.01). The expression α-SMA was increased in model rats compared with that of normal rats, FZHY significantly decreased its expression (P<0.01). The expressions of p-IκB and TNF-α and MMP-2, MMP-2 activity, and NF-κB activation were increased in model group compared with that in normal group (P<0.01), FZHY significantly decreased NF-κB activation, MMP-2 activity and p-IκB and TNF-α expressions (P<0.01). Conclusions: FZHY could protect kidney from oxidative injury intoxicated by HgCl2, and antagonized oxidative stress-stimulated NF-κB activity through inhibition of IκB phosphorylation in the interstitial fibrotic kidney, these effects importantly contributed to FZHY action mechanism against renal interstitial fibrosis.     

Effects of Huogu I Formula (活骨I方) on correlated factors of bone regeneration in chickens with steroid-induced necrosis of femoral head

Objective:To study the mechanism of HuoguⅠFormula（活骨Ⅰ方） in treating osteonecrosis of femoral head.Methods:Forty-eight healthy female Leghorn chickens were randomly divided into control group,model group and HuoguⅠgroup,and each group consisted of 16 chickens.At the meantime of model establishment,chickens of the HuoguⅠgroup were administrated with decoction,while the model and control group with distilled water by gavage.At the 8th and 16th week after medication,blood samples were obtained for blood lipid detection while both sides of femoral head were harvested for the rest of examinations.Specifically, expressions of bone morphogenetic protein-2（BMP2）,transforming growth factor betal（TGFβ₁）,Smad4 and Smad7 were evaluated by immunohistochemistry,while expression of osteoprotegerin/receptor activator of nuclear factor kappaB ligand（OPG/RANKL） mRNA was detected by in situ hybridization.Results:Compared with the control group,serum levels of total cholesterol（TC）,triglyceride（TG） and low-density lipoprotein cholesterol （LDL-C） in the model group rose significantly.Positive cell counting of BMP2,TGFβ₁,Smad4 and OPG in femoral head of the model group dropped prominently.Positive cell counting of Smad7 and RANKL increased dramatically. In contrast with the model group,levels of TC,TG and LDL-C in Huogu I group reduced significantly.Positive cell counting of BMP2,TGFβ₁,Smad4 and OPG in femoral head of the Huogu I group increased prominently. Indices of Smad7 and RANKL both decreased significantly.Especially at the 8th week,these variations were more significant.Conclusion:HuoguⅠFormula is effective in promoting repair of necrotic femoral head by regulating the expressions of BMP2,TGFβ₁,Smads and OPG/RANKL of osteoclast in femoral head.     

Effects on the Tumor Specific Growth Factor and Tumor Necrosis Factor α in Rats' Precancerous Lesion of Primary Hepatocellular Carcinoma by Direct Moxibustion at Ganshu(BL 18) Acupoint

Objective: To investigate the effect of direct moxibustion at Ganshu(BL18) on the serum concentrations of tumor specific growth factor(TSGF) and tumor necrosis factor α(TNF-α) in a rat model with precancerous lesion of primary hepatocellular carcinoma(HCC), so as to explore the mechanism of moxibustion underlying improvement of HCC. Methods: Sixty male Wistar rats were randomly divided into control group(n=10), model group(n=20), prevention group 1(n=15) and prevention group 2(n=15). The normal rats were injected with physiological saline as blank control. At the same time, the rats of other three groups were injected with diethylnitrosamine to establish the HCC model. Direct moxibustion with grain-sized moxa was applied to bilateral Ganshu acupoint of the rats in the prevention group 1(1 treatment course, 20 days) and prevention group 2(2 treatment courses, 40 days), 5 doses for each acupoint, 0.5 mg/dose, once every other day. At each time point(before model establishment, the end of 1st course prevention, the end of 2nd course prevention and the end of model establishment), serum levels of TSGF and TNF-α were detected using enzyme-linked immunosorbent assay. Results: Compared with the control group, there was a remarkably increase of serum TSGF and TNF-α contents in the model group at the end of the experiment(P0.05). At the end of the 1st course of direct moxibustion, the contents of serum TSGF and TNF-α of rats in the prevention group 1 were significantly increased compared with that of the model group(P0.05). At the end of the 2nd course of direct moxibustion, serum TSGF and TNF-α levels of rats in the model group were higher than the normal group with significantly difference(P0.05), and the levels of TSGF and TNF-α in the prevention group 2 were significantly reduced in comparison with the model group(P0.05). Conclusion: It was possible that direct moxibustion could inhibit precancerous lesion and postpone hepatocarcinogenesis, and the therapeutic effect of two courses were better than one course.     

Protective Effect of Zengye Decoction(增液汤)on Submandibular Glands in Nonobese Diabetic Mice

Objective: To investigate the protective effect of Zengye Decoction(增液汤, ZYD) on the submandibular glands(SMGs) in nonobese diabetic(NOD) mice. Methods: Twenty-seven female NOD mice were randomly equally divided into 3 groups: the model group, the hydroxychloroquine(HCQ) group, and the ZYD group. Nine C57/B6 mice served as the normal group. After 1-week acclimation, the HCQ and ZYD groups were intragastrically administered with HCQ and ZYD, respectively, and the normal and model groups were administered with normal saline. Changes in the salivary flow rate were observed. Mice from all 4 groups were sacrificed at the age of 20 weeks. The serum and SMGs were collected. Serum cytokines gamma-interferon(IFN-γ), interleukin-10(IL-10) were detected by enzyme-linked immunosorbent assay. Histological changes in the submandibular glands were examined by hematoxylin and eosin staining. The mRNA expression of IFN-γ, IL-10 and vasoactive intestinal peptide(VIP) in the submandibular glands were measured by realtime polymerase chain reaction. Results: Compared with the model group, the salivary flow of the ZYD group significantly increased(P0.05), the extent of the histological changes was ameliorated(P0.05), and the Th1/Th2 cytokine imbalance was remedied(P0.05). In the ZYD-treated mice, the VIP mRNA was up-regulated(P0.05). Conclusions: ZYD is beneficial in protecting structure and function of SMGs in NOD mice. The mechanism may be associated with the correction of the Th1/Th2 cytokine imbalance, and with the prevention of a progressive decline of the VIP level.     

Effect of Huxin Formula (护心方) on reverse cholesterol transport in ApoE-gene knockout mice

Objective:To observe the effect of Huxin Formula(护心方,HXF)on expressions of the chief reverse cholesterol transport(RCT)associated genes,caveolin-1 and scavenger receptor-BⅠ(SR-BⅠ)in ApoE-gene knockout[ApoE(-/-)]mice.Methods:Thirty ApoE(-/-)mice of 4-6 weeks old were randomly divided into three groups(A-C).After being fed with high-fat diet for 16 weeks,they were treated with HXF(1 mL/100 g),pravachol (0.3 mg/100 g),and saline in equal volume respectively for 16 weeks successively;in addition,a blank group was set up with 10 C57BL76J mice of 6-week old received 16-week high-fat feeding and saline treatment.Animals were sacrificed at the termination of the experiment,their paraffin sections of aortic tissue were used to measure the size of plaque,expressions of cavolin-1 and SR-BⅠwere detected by immunological histochemical method.Results:As compared with the blank group,levels of caveolin-1 and SR-BⅠwere increased in Groups A and B(P<0.01);but the increase in Group A was more significant than that in Group B(P<0.05).The plaque/aorta area ratio decreased significantly in Groups A and B,but showed insignificant difference between the two groups.Conclusion:HXF could obviously increase the expressions of RCT associated genes,caveolin-1 and SR-BⅠ,promote the RCT process, so as to reduce the formation of aorta atherosclerotic plaque in ApoE(-/-)mice.