| Objective With the dramatic development of people's living standards, more and more people are suffering from non-alcoholic steatohepatitis or NASH as time goes by. Its mechanism is complex and still unknown. Several factors such as extra free fatty acids or FFA, oxidative stress, injuries caused by cytokines or CKs are considered to be pathologically relative.Some existed clinical and experimental researches have shown that steatohepatitis is in correlation with NF-κB as well as PPAR r, but pertinent literature is still not enough. It is necessary to do further research to clarity the relations between NASH and NF-κB as well as PPAR r. At present, it seems to be hard to choose some effective medications to treat NASH. TCM which has some predominances can be turned to concerning its treatment. According to his long term of clinical experiences, my tutor, professor Sun Guo-jie claimed that NASH might be caused by intaking excess fat and sweet foods for a long time, which lead to phlegm and blood stasis generating from within, constrained liver qi, heat caused by permanent stagnation, Yin deficiency due to internal heat. All in all, all these pathological factors lead to such a pathological characteristic as being insufficient in nature but the signs and symptoms are seemed to be excess. Its therapeutical principle should be supplementing the kidney and liver, strengthening the spleen and drain the excess water and phlegm, expelling the blood stasis. Pro. Sun Guo-jie chose Gan-shu acupoint, Zu Sanli acupoint, Feng Long acupoint and Tai Chong acupoint to treat NASH. We needled the acupoints mentioned above to treat patients who suffered NASH and gained significant effects concerning relieving the symptoms, improving the liver function, decreasing the blood fat and resisting fibrosis. In this research, we used classical animal models which are accepted worldwide, armed with the technologies in the fields of pathology, biochemistry, molecular biology, aiming to probe the activity of NF-κB and the expression of PPARrmRNA, make a clear explanation on the therapeutical mechanism of electric acupuncture on NASH in order to offer strong proofs for the clinical use of electric acupuncture in treating NASH.Methods 48 SD rats were divided them into 2 groups, normal group with 12 rats and modeling group with 36 rats according to random digits table. The rats of the normal group were fed with common food;while the rats of the model group were fed with fat-rich diet, i.e,normal diet plus 13% lard oil and 2% cholesterol to rebuild NASH animal models according to the classical method offered by Fan Jian-gao. All the rats took food and drank water in a free way and the dairy staffs were changed on a daily basis. They were fed for 12 weeks in total. After 12 weeks, 2 rats of the normal group and 6 rats of the modeling group were picked out randomly. Pathological sections were made to show that animal model had been built successfully. Then the rest 30 rats of the modeling group were randomly divided into3 groups: model group, electric acupuncture group and the western Medicine group.The rats of both the normal group and the model group were treated as before continuously for another 4 weeks;the rats of the acupuncture group were punctured with electric acupuncture at such acupoints as Ganshu (BL 18), Zusanli (ST 36), Fenglong (ST 40) and Taichong (LR 3) bilaterally and also fed with a fat-rich diet continuously for another 4 weeks; the rats of the western medicine group were lavaged with UDCA 250mg/kg.d and also fed with a fat-rich diet continuously for another 4 weeks.All the rats were anesthetized with 2% Pentobarbitalum Natricum through peritoneal injection with a regular standard dose after a night at the end of the 16th week. Blood was collected from the inferior vena and Serum was separated with regular methods. The weight, appetite, behavior, state,hair and death rate of the rats were closely observed.Liver index numbers were calculated by humid weight/body weight x 100%.Serum alanine aminotransferase or ALT, aspartate aminotransferase or AST, triglyceride or TG, total cholesterol or TC, Free fatty acid or FFA, fasting blood glucose or FBG were tested through Olympus Au560 Automatic biochemistry analyzer. Tumor necrosis factor-αor TNF-αand fasting Insulin or FINS were tested by radio immunoassay.As a result, Insulin resistance index or IR index could be calculated through the formula of FBGx FINS/22.5. Fresh liver tissue was made into tissue homogenate. Malondialdehyde or MDA and superoxide dismutase or SOD were tested by way of tissue homogenate biochemistry. NF-kappa B Binding Activity detected by electrophoretic mobiLity shift assay (EMSA): According to the procedure in the manual of the kits, took 1.75μmol/L unlabelled double-stranded oligonucleotide probe, T4polynucleotide kinase 1μ1,γ<sup>32p ATP (0.37 GBq/ml) 1μ1 to llabel. Took 20μg ribonucleoprotein and put into 10μl gel retardation binding buffer 10mINS at 37℃, added 1μ132p-labelled oligonucleotide probe 30miNS at 37℃, then carried out 6% native polyacrylamide gel electrophoresis and autoradiography 72 hs after gel vaccum dehydration, Observed the results and gray scale scanning was carried out with BIO-PROFIL image analysis system, assayed coloration positive area in comparison with the background negative area and calculated the mean gary scale values. PPAR rmRNA was detected by RT-PCR. Part of the liver tissues were flushed with 4℃normal saline and small scraps of liver tissues were taken at a farmost distance of 5mm to the rim of the liver lobes.Histological specimens were stained with HE after being fixated with 10% netural Formalin and made into slices buried in Paraffin and then studied. 1 mm3 Hepatic slices were fixed with 2.5% Glutaric Dialdehyde and 1% osmic acid and stained with plumbum and uranium and the ultrastructures of the liver tissues was observed through HITACHI H7500 electron microscope.Results1 NASH animal models were replicated successfully. In comparison to the rats of model group, the body weight number of the rates of the normal group were significantly decreased (P<0.05); so were serum ALT and AST.Steatosis, inflammation,necrosis and fibrosis were present in the livers of the rats of all the modelling groups, and were most severe in the liver tissues of the rats of the model group while the conditions mentioned above were significantly improved in the livers of the rates of the electric acupuncture group. The morphosis of the hepatic tissues of the rats of the electric acupuncture group were markedably improved.2 The serum TG, TC and FFA contents were markedly higher in the model group than those of the normal group (P<0.05); There was also a significant difference between the model group and the electric acupuncture group (P<0.05); Concerning the serum TG and TC contents, there was a downward trend in the electric acupuncture group compared with the western medicine group, but there was no difference between them (P>0.05); Concerning the serum FFA contents, there was a downward trend in the western medicine group compared with the electric acupuncture group, but there was no difference between them (P>0.05).3 The MDA contents were markedly higher in the model group than those of the normal group (P<0.05); The MDA contents were markedly lower in the model group than those of the normal group (P<0.05); The MDA and SOD contents were markedly lower and higher respectively in the livers of the electrnic acupuncture group in comparison to the livers of the model group (P<0.05); There was a significant difference between the electric acupuncture group and the western medicine group concerning the MDA contents (P<0.05);Regarding the SOD contents,there was a downward trend in the livers of the electric acupuncture group, but there was no significant difference between the electric acupuncture group and the western medicine group (P>0.05).4 The serum TNF-αcontents were markedly higher in the model group than those of the normal group (P<0.05); There was also a significant difference between the model group and the electric acupuncture group (P<0.05); Concerning the serum TNF-αcontents, there was a downward trend in the western medicine group, but there was no difference between the electric acupuncture group and the western medicine group (P>0.05).5 Concerning the IR levels, IR levels were markedly higher in the model group than those of the normal group (P<0.05); There was also a significant difference between the model group and the electric acupuncture group (P<0.05); there was a downward trend in the western medicine group, but there was no difference between the electric acupuncture group and the western medicine group (P>0.05).6 NF-kappa B binding activity was obviously higher in the model group and the western medicine group than those in the normal group (P<0.01) and the electric acupuncture group(P<0.05).The expression of P P A RrmRNA of the model group, electric group and western medicine group was weaker than that of the normal group (P<0.01) and the level of P P A RrmRNA was lower in the livers of the model group than that of electric acupuncture group(P<0.01).Conclusion Oxidative stress can leads to lipid peroxidation and hepatic injuries as well. In the end, it can cause the appearance of the pre-inflammatory cell factors which might initiate inflammatory reactions in the liver.In the state of NASH, NF-kappa B binding activity will be significantly enhanced while the levels of the expression of PPAR rmRNA will be lowered, which shows the correlatin among NASH, NF-kappa B binding activity and PPAR rmRNA. Electric acupuncture can be used to treat NASH at multiple target sites:It can decrease the liver index of the rats, improve adipoe degeneration, inflammation degree and liver function; it can lower serum lipid, decrese the hepatotoxity caused by FFA, improve the regulation of liver on the metabolim of lipid, relieve the inflammatory reaction; it can strengthen the hepatic tissues against oxidation,relieve lipid peroxidation; it can decrease the expression of TNF-αand as a result it can decrease the injuries caused by cytokines; it can inhibit the binding activity of NF-KB and promote the expression of PPARrmRNA. It is a possibility that electric acupuncture can be used as a therapeutical method in treating NASH through such two ways as the correlaton among acupoints, channels and collaterals and visceras as well as the interaction between nerve, endocrine and immunological network. |
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