Genomics Of Hepatorenal Fibrosis Induced By Apoptosis And Anti-fibrotic Effect Of Hongbeiyegen Based On Collateral Disease

BackgroundFibrosis is in general the consequence of the chronic activation of wound healing response to reiterated organ damage and is the pathology change; Pro-gressive organ fibrosis is the main cause of organ failure in chronic diseases of any aetiology, including myocardial fibrosis, pulmonary fibrosis, liver fibro sis, renal fibrosis, skin sclerosis and so on. however, the therapeutic effect of antifibrotic medicine is not satisfied with our demand.Among those diseases, Hepatic fibrosis and renal interstitial fibrosis are relatively important.Hepatic fibrosis is the result of chronic liver injuries due to hepatitis viru--s, alcohol, medicine, abnormal metabolism, genetic disease and so on damaged hepatocytes release injurious or proinflammatory contents which can attract macrophages, Kupffer cells, lymphocytes etc, promote the activation of hepatic stellate cells (HSC) into a myofibroblast-like phenotype, produce lar--ge extracellular matrix deposite liver.Renal interstitial fibrosis is the result of chronic renal injuries due to bact erium, virus, rays, medicine, poison and so on, the damaged renal inhere-nt cells induce the activation of interstitial fibroblasts and tubular epithelial ce -11s into a myofibroblast-like phenotype, produce large extracellular matrix dep--osite renal interstitium, which lead to the progressive renal failureThe coincidence of patho-process, adjusted mechanism, final product, anti-fibrotic medicine such as gene or cytokine inhibition etc between liver and re nal fibrosis fit the idea of "homotherapy for heteropathy" in traditional Chinese medicine (TCM)."treat disease depend on" is the essence of "homotherapy for heteropathy" in TCM, the "symptom-complex" decide the therapyThe "symptom-complex" is the Summary of the symptoms and signs at a certain stage in a certain disease, according to "the whole concept" and the TCM theory.the symptoms and signs is products deciding by gene expression, thus, the "symptom-complex" is also the Summary of "gene expression produ cts"Therefore, to study the common molecular mechanism between hepatic and renal fibrosis depending on gene chip technology and bio-information tech nology, together with TCM whose advantages lying in "target set",which is important in reversing fibrosis, retrieving organ function from failure.Liver and renal fibrosis is not belong to TCM diseases, but we treat fibr-osis according to classical theory of traditional Chinese medicine basing on cli-nical symptoms and signs, so, liver fibrosis always "zheng ji", "ji jv", "ty mpany", "gan zhe" etc; renal fibrosis often belong to be summarized as "ede ma", "uroschesis", "guan ge" etc. In modern "collateral disease theory", the Hepatic and renal fibrosis belong to "Yin Collateral diseases—the changes in entrails'collaterals", the "Blood-stasis Blocking Collaterals" is the common pa-thological mechanism. most of the anti-fibrotic medicine have "activating blo od circulation to dissipate blood stasis" effect Hongbeiyegen is the root of Hongbeishanmagan which belong to euphorbi a plants branching from dicotyledon, it belongs to "rattan" in Collateral Disea-ses, it has "Sweet taste and cool feeling", "detoxification, dehumidification, stop the bleeding" effect. Previous studies have shown that Hongbeiyegen by inhibiting tumor necrosis factor-a, interleukin-1, transforming growth factor-p, tissue inhibitor of metalloproteinase-1 expression, protecting liver from Bacill-us Calmette-Guerin (BCG) and lipopolysaccharide (LPS)-induced damage, resist rat liver fibrosis induced by alcohol and carbon tetrachloride. Whether Hong--beiyegen has "Huo Xue Tong Luo" effect? Whether it can resists rat renal inte-rstitial fibrosis? Whether it can be used as a member of anti-fibrosis drugs in "target set" TCM? are important in invest of Hongbeiyegen ObjectiveTo observe the common changes in expressed genes,between Hepatic and renal fibrosis, study the fibrosis molecular mechanisms; To observe effects of Hongbeiyegen on hemorheology and microcirculation in immunological rat hepatic fibrosis and unilateral ureteral obstruction (UUO) rat renal interstitial fi--brosis, attempt to use "luo bing xue shuo" explain the mechanism of Hongb eiyegen's anti-fibrotic effect MethodsFirst, Rat liver fibrosis Model induced by pig serum preparation24 specific pathogen free (SPF) SD rats,100-120g weight half male and half female, were randomly assigned into three groups after one week's adap-tation living, with eight in each, as follows:the normal control group, the liver fibrosis model group (the model group), Hongbeiyegen groupAt 10am, rats in the model and the drug group received 0.5 ml of pig serum by intraperitoneal injection twice weekly for 12 weeks, normal control group received 0.5 ml of saline; at 3pm, Hongbeiyegen decoction were giv en Hongbeiyegen groups, model and normal group was given saline (1ml/100 g per day). All rats were allowed food and water ad libitumSecond, Rat renal interstitial fibrosis Model induced by unilateral ureteral obstruction preparation24 specific pathogen free (SPF) SD rats,180-200g body weight, half male and half female, were randomly assigned into three groups after one we-ek's adaptation living, with eight in each, as follows:the sham operation group, the renal interstitial fibrosis model group (the model group), Hongbeiy egen group.At 9am, renal fibrosis model and Hongbeiyegen group received lorinal by intraperitoneal injection (350mg/kg), then open left lateral region of abdom-en, separate left ureter use ophthalmological pliers, deligate left ureter betwe-en renal calices and inferior pole of kidney by No 5 operative line, but not cut off ureter and protect the renal amicula and the amph-tissue, at last, pla-ced kidney to its initial location, sew up muscle and skin in order; except without left ureter deligation, the other operate process was alike in sham op-eration group; the whole operation demand asepsis. at 3pm, Hongbeiyegen decoction were given Hongbeiyegen groups, model and normal group was giv en saline(lml/100g per day). All rats were allowed food and water ad libitum.Third, Observe rat hypoglottis and auricle thread by stereomicroscopeThe day before the end of experiment, at 8pm, rats were deprived from diet; the next day at 8pm rats received lorinal by intraperitoneal injection (35 Omg/kg), at supine position, gently pull up the whole tongue using nipper C lamping its tip, the angle between tongue and body range from 0°to 15°, observed rat hypoglottis thread by stereomicroscope tuning in 80 times; then at lateral position, let left ear on "ear-phore", unfold its dorsum, observed rat auricle thread, all images import into Legend computer, observe by ACDsee v3.2 or windows Picture and Fax ViewerFourth, Detect fibrotic and hemorheological indexesAfter collecting images, open abdomen, part of the liver and renal tissue were stained with hematoxylin and eosin (HE), the pathological changes and t-he collagen's proliferation of the liver or renal tissue were observed under the light microscope, and set the quantitation standard of the collagen's prolife de-gree according to literatures. take 1ml blood, HCT was measured using or-dinary Packed Volume Tubes, take the upper animal serum. The activity of B lood viscosity at different shear rate was measured using TM-STEELLEX R-80 A blood rheometer; the activity of glutamate-pyruvate transaminase (ALT) and glutamic oxalacetic transaminase (AST) was measured using ECHO LCD auto matic biochemical analyzer; the activity of the hyaluronic acid (HA), the la-minin (LN), precollagen typeⅢ(PCⅢ) type, collagen type IV(CIV) was eval-uated using radio-immunity r counter, according to radioimmunoassay method.Fifth, Analysis of the mutual expressed genes by gene chip and bioinfor--mation technologyDifferent expressed genes were detected and select by Oligonucleotide mic-roarray of Beijing Capital Bio company; we analysis these genes and draw signal pathway by Gene Ontology, MILANO, Panther and Genomatix software. ResultsFirst, the pathological changes in liver and renal fibrosisThe liver fibrosis model group was filled with obvious collagen deposition, inflammatory cells infiltration, the thin and long fibrous septa isolated the hep atic parenchyma and extend into hepatic lobule; the collagen's prolife degree was severer than those in normal group(P<0.01)The renal interstitial fibrosis was filled with obvious collagen deposition, inflammatory cells infiltration, renal tubule atrophy and disappearance; the collagen's prolife degreeSecond, bio-information analysis of the mutual expressed genesThrough analysis by Capital Bio company, there were 1205 different expressed genes in rat liver fibrosis and 2550 different expressed genes in rat renal interstitial fibrosis, by selecting,there are 311 co-expressed genes, among which 134 were up-regulated and 177 were down-regulated.After bio-information analysis, we got the gene signaling pathway mainly relevant to apoptosis (p53 pathway, Integrin signalling pathway, PI3 kinase pathway, G-protein signaling pathway etc), metabolism of extracellular matrix (Integrin signalling pathway, Cytoskeletal regulation by Rho GTPase, Cadherin signaling pathway, EGF receptor signaling pathway, FGF signaling pathway, Toll receptor signaling pathway, Insulin/IGF pathway, Notch signaling pathway etc), inflammation (oxidative stress response, Inflammation mediated by chemokine Interleukin signaling pathway etc) etc; form the gene function network and the core is caspase3, which linked with two gene cluster through Nfkb1, Tp53 respectively.Third, effects of Hongbeiyegen on hemorheology and microcirculation in immunological rat hepatic fibrosisHongbeiyegen could reduce the indexes of Hematocrit, Blood viscosity at different shear rate, Hyaluronic Acid(HA),Laminin (LN),Procolagen TypeⅢ(PCⅢ), Collagen TypeⅣ(CⅣ), glutamic-pyruvic transaminase(ALT) and glutamic-oxalacetic transaminase (AST) in hepatic fibrosis, and improve the microcirculation of hypoglottis and auricle in model rats. Fourth, effects of Hongbeiyegen on hemorheology and microcirculation in renal interstitial fibrosis ratHongbeiyegen could reduce the indexes of Hematocrit, Blood viscosity at different shear rate, Hyaluronic Acid(HA), Laminin (LN), Procolagen TypeⅢ(PCⅢ), Collagen TypeⅣ(CⅣ), serum creatinine (SCr) and blood urea nitrogen (BUN) in renal interstitial fibrosis, and improve the microcirculation of hypoglottis and auricle in model rats. ConclusionFirst, Cell apoptosis is at the beginning of the fibrosis process, p53 pathway maybe the major molecule mechanism in those process.Second, Lyvel maybe is one of the key genes in fibrosis process, because it high expressed in liver and renal fibrosis, and linked apoptosis with the metabolic disorder of ECM.Third, Hongbeiyegen has "promoting blood circulation to remove meridian obstruction" effect.Fourth, Hongbeiyegen maybe involve in principal anti-fibrosis TCM, which has "target point" advantage, due to Hongbeiyegen can resist rat liver fibrosis induced by pig serum and rat renal interstitial fibrosis induced by unilateral ureteral obstruction.