Experimental Observation On Different Therapeutic Principles Of Chinese Medical Science Of Activating Blood Circulation, Benefiting Gas And Softening Hard Tissue In The Treatment Of Liver Fibrosis In Rats

Objective: Liver fibrosis is the common pathologicalprocess of chronic hepatopathy caused by various etiologicalfactors. It is also the prelude and inevitable link to cirrhosis ofliver. Liver fibrosis mostly resulted from hepatitis B in ourcountry and chronic hepatopathy patients reach above thirtymillion people. 25 to 40 percent of hepatic fibrosis may developinto cirrhosis of liver if not inhibited properly, which will affectthe working capacity and living quality of the patients, to top itoff, result to fatal disease such as hepatic coma, esophagealvarices, liver ascites and hepatocarcinoma and so on. The studyof how to postpone, interdict and even reverse the hepaticfibrosis is becoming the hotspot currently. Hepatic fibrosis isbelong to flank ache, crux category in the Chinese medicalscience. Great hopes are placed on the traditional Chinesemedicine at the present time because western medicine likecolchicine, D-bellacilline and dentoxifylline etc. still have noperfect curative effect on liver fibrosis. The traditional Chinesemedicine compound formula YiGanXian is established in thebasic mechanism—gas deficiency blood stasis, root weaknesssign firmness of liver fibrosis caused by chronic hepatitis andscreened by professor WangJi through many years clinical andexperimental study. It can benefit gas, promote blood circulationto dissipate blood stasis, soften hard lump and dispel nodes andaccordingly alleviate or even reverse the progress of hepaticfibrosis, this is identified by the previous study of ourdepartment. Liver fibrosis model rats were induced by carbontetrachloride and treated with traditional herbs of activatingblood circulation, benefiting gas and softening hard tissuerespectively, then to observe the curative effect on interruptinghepatic fibrosis and also compared with the traditional Chinesemedicine compound formula YiGanXian test group. Newtheories and methods were adopted by this study to compare thecurative effects of different therapeutic principles on liverfibrosis from more angles. Discussing and further demonstratingthe action mechanism of different therapeutic principles ofChinese medical science in the treatment of liver fibrosis in ratsthrough the observation of pathomorphological changes, themensuration of biochemistry and immunohistochemistry, andanalysis of pathological image etc. The success of this study willprovide quantifiable index of dialectical therapy of Chinesemedical science in treating liver fibrosis with new informationsand consequently make reference to the establishment of moreunified and impersonal therapeutic principles. The process ofhepatic fibrosis is likely to be interdicted and numbers ofhepatopathy sufferer got well.Methods: 48 healthy male Wistar rats were randomlydivided into six groups after normal feedings for one week,eight rats per group, that is normal control group (in brief Ngroup), model group (in brief M group), Chinese herbalcompound formula YiGanXian group (in brief C group),activating blood circulation group (in brief A group), benefitinggas group (in brief B group) and softening hard tissue group (inbrief S group). Except N group, the rest five groups weresubcutaneously injected with solutions of 40 percent carbontetrachloride peanut oil inside the thigh of the rats, 0.3 ml per100 gram body weight. Normal group were injected withequivalence of saline, two times a week, continued for totaleight weeks. From the second day at the end of the 8th week ofmaking models, C group were treated with YiGanXiandecoction, 1 ml per 100 gram body weight (contain raw herbs 3gram per ml), one time a day. A group were treated with salviamiltiorrhiza and szechwan lovge rhizome decoction, 1 ml per100 gram body weight (contain raw herbs 0.69 gram per ml),one time a day. B group were treated with membranousmilkvetch root and largehead atractylodes rhizome decoction, 1ml per 100 gram body weight (contain raw herbs 1.38 gram perml), one time a day. S group were treated with turtle, crust andoyster shell decoction, 1 ml per 100 gram body weight (containraw herbs 0.7 gram per ml), one time a day. Normal controlgroup and model group were perfussed stomach with distilledwater, 1 ml per 100 gram body weight, one time a day,continued for total eight weeks. All rats were fed by ordinaryanimal feeds and clean water during the whole period ofexperimentation, moreover, dosage was adjusted by weighingup once a week. All rats were put to death by decollation afterthe last time medication and fasting for 12 hr. At the same time,took out the rat liver rapidly and cut about 1cm×1cm×1cmsize hapatic tissue from the same section of left lobe of liver at atime. HE and Masson stained preparation were fixed with 10%neutral formaldehyde solution while immunohistochemistrystained samples were fixed with mixed liquor of 4% citromintand 0.1% glutaral. Desiccation through alcohol bit by bit,pellucidum through dimethylbenzene, embedded with paraffinroutinely, then sliced up a serial section for 5um thickness. Thesurplus liver tissue samples were preserved freezingly at -70degree centigrade. The reserved specimens were detectedindexes as follows: The changes of pathomorphology of livertissue were observed through routine HE staining and theproliferation degree of collagenous fibers through Massonstaining. The contents of liver homogenate hydropropline (hyp)were examined through chromatometry. The expression andlocalization of TGF-β1 and PAI-1 in liver tissue were detectedwith method of immunohistochemistry staining,semiquantitative image analysis of hepatic histological collagenfibers, TGF-βand PAI-1 were performed by colourful 1multimedia pathological image analyzing system.Results1. The changes of pathomorphology of liver tissue amonggroups: In normal group, the structure of liver tissue wasintegrated and darity, hepatocyte chorda arranged regularly. Noinflammatory cell infiltration at the converging duct districtinside hepatic lobufe and no hyperplasia of fibrous tissue as well.In model group, hepatocyte swelled up obviously, fattydegeneration accompanied with focal necrosis were observed.The normal structure of hepatic lobufe badly damaged, fibroustissue and small bile duct proliferated markedly andsynchronously accompanied with more lymphocyte andmonocyt infiltration. In YiGanXian group, activating bloodcirculation group and benefiting gas group, the degree ofinflammatory cell infiltration, hepatocyte degeneration andnecrosis, fibrous tissue proliferation were remarkably lessenedrespectively compared with those of model group. YiGanXiangroup was the most effective among aforementioned threegroups. No remarkable differences between softening hardtissue group and model group in improving pathologicalchanges of liver tissue. The results of semiquantitative imageanalysis of hepatic histological collagen fibers indicated that thearea consistency of collagen fibers significantly boosted up inmodel group, which had notable difference compared with thatof normal group (P<0.01). However, the collagen aera density inC, A and B treatment group were obviously decreased aftermedication by contrast with that in model group (P<0.01). Theresult in S group showed a slight reduction but no statisticalsignificance compared with model group (P>0.05). The areaconsistency of collagenous fibers stepped up in file from C, A,B to S group and the difference between every two groups wassignificant (P<0.05 or P<0.01).2. The contents of liver homogenate hydropropline (hyp) amonggroups: The contents of Hyp in model liver tissue increasedevidently compared with that of normal group (P<0.01). Thecontents of Hyp in C, A and B group liver tissue showed a sharpdrop after medication and presented a striking contrast to that ofmodel group (P<0.01). C group was the most potent amongabove-mentioned three test groups, whose Hyp contents weremuch lower than that of B group (P<0.01) yet a little lower thanthat of A group (P<0.05). Although the contents of Hyp in Sgroup liver tissue decreased slightly, the difference was notstatistically evident in contrast with that of model group(P>0.05).3. The expression of TGF-β1 protein in liver tissue amonggroups: In normal group, only a small quantity of TGF-β1protein expressed in liver tissue. In model group, thedistribution of TGF-β1 protein in liver tissue was extensive,and mainly fastened on hepatic converging duct area andhyperplastic collagenous interstitial, moreover, the pigmentingtook on colours from symmetrical light-brown to nigger-brown.The positive staining degree of TGF-β1 in each treatment groupliver tissue was distinctly lightened compared with that in modelgroup. The analytical results of immunohistochemical stainingshowed that the positive area consistency of TGF-β1 in modelgroup raised strongly, which formed a striking contrast to thosein normal group (P<0.01). TGF-β1 area density were lesseneddramatically in each treatment group after medication, whichhad remarkable discrepancy compared with model group(P<0.01). The function of C and B group was especiallyprominent and was more potent than that of A or S group(P<0.05 or P<0.01).4. The expression of PAI-1 protein in liver tissue among groups:The positive staining of PAI-1 was brown and yellow. Thepositive expression of PAI-1 in model liver tissue wasconsiderably intensified and pitched in fibrous intervals. Almostno positive expression of PAI-1 was examined in normal groupliver tissue. A very little of it expressed faintly only inperisinusoidal space of disse, blood capillary endothelial celland bile duct cell. The positive staining degree of PAI-1 in C, Aand B group liver tissue were significantly lessened bycomparison with model group, while no evident signs ofalleviation were seen in softening hard tissue group. Theanalytical results of immunohistochemical image indicated thatthe positive area consistency of PAI-1 in model group increasedobviously, which had significance of difference compared withthose in normal group (P<0.01). PAI-1 area density in C, A andB group liver tissue was significantly decreased after medication,which had statistical discrepancy by contrast with those inmodel group (P<0.01). The function of C and A group inlessening the level of PAI-1 protein in liver tissue wasremarkable and the effect was better than that of B group(P<0.01). The expression of PAI-1 in S group liver tissueattenuated slightly, but it was insignificantly different fromthose in model group (P>0.05).Conclusions1. Method of activating blood circulation and removing stasiscan inhibit liver fibrosis and its mechanism possibly contributesto the down-regulation of PAI-1 protein in liver tissue by actingon HSC, so as to elevate the activity of fibrinolysin, which isrelative to accelerating the degradation of collagen fibers. Itreveals that PAI-1 is probably a potential target to alleviate liverfibrosis.2. The antifibrotic action of the method of benefiting gas andstrengthenning spleen might be related to protecting hepatocyte,regulating immune system, changing cytokine activity andfacilitating plerosis of hepatocyte, which can make up thelimitations of single activating blood circulation method,thereby exert a synergistic action on antagonizing hepaticfibrosis.3. Using softening hard tissue method only can not significantlyreverse formed already hepatic fibrosis under experimentalconditions. Whether softening hard tissue method could exert acooperative influence on the complex prescription of Chineseherbs in changing cytokine activity, inducing amelioration of...