Compatible Rule Of TongMai Oral Liquid (TMOL) And Pharmacological Study Of Effective Compound Of TMOL

Chronic nephritis(CGN),which as also called chronic glomerulonephritis, is a kind of immunologic disease. It was estimated that the mainly pathological type is mseangial proliferative glomerulonephritis(MsPGN), which occupy up to 66.6%. The chronic nephritis is not easy to cure and it is on of chiefly causes to renal failure.Adrenocortical hormone or Cytotoxic medicine have good effect in treating chronic nephritis, but both of the have side-effects. So the effective and safety medicine to be found from the traditional Chinese medicine become more and more important.According to a long-term clinical observation, we found the chiefly syndrome of patients with CGM was Qi-deficiency and blood stasis. Deficiency of Qi caused the happenness of CGM, and blood stasis was the chiefly factor which lead Chronic nephritis not easy to be cured. Under the guidance of this view, invigorating Qi and promting blood flow was regarded as the primary ways in treating CGM. TMOL which has the effects of invigorating Qi and promting blood flow was widely used to treat chronic nephritis. The pharmacological study showed that TMOL had the effects of reducing the 24-hour urinary protein contents, FDP, blood urea nitrogen(BUN)and serum creatinine(Scr) .It could also markedly alleviated blood hypercoaguability and delayed the progression of pathological changes to glomerular.The recipe of TMOL is composed of Astragalus root and Panax notoginseng. The proportion between Astragalus root and Panax notoginseng was 3:1. To found out the best proportion between Astragalus root and Panax notoginseng and the effective compound of TMOL, the follow experimental tests had been carried on.Part I The study on the culture condition of GMC and the concentration of plus Lipoplysaccharide(LPS)The mainly objective of the study was to find out the best concentration of plus Lipoplysaccharide(LPS) which could activate the proliferation of GMC. Different concentration of LPS(0.1, K 1(K 100ug/ml) acted on GMC at 12, 24, 48, 72 th to observe the proliferation of glomerular mesangial cells. The result showed LPS 100ug/ml could activate the proliferation of glomerular mesangial cells at 12th and 24th,and LPS 1, 10 ug/ml could activate the proliferation of glomerular mesangial cells at 24th,48th and 72th, but 0.1ug/ml could not activate the proliferation of GMC. The best concentration was LPS 10ug/ml.Part II The study on concentration of drug serum taken from the rats given TMOLDifferent concentration of serums(5%> 10%, 20%) taken from the rats given TMOL were added to culture solution of GMC to observe the proliferation of GMC. The result showed 5%, 10%, 20% could inhibit the proliferation of GMC. The inhibit effect had little difference among them. To avoid the cytotoxicity, we selected 10% concentration of drug serum as the best concentration during the following experimental studies.Part III The study on proliferation of GMC about different compatible proportion between Astragalus root and Panax notoginsengDifferent medicine including TMOL, different proportion(6:3, 3:1, 1.5:1) of TongMai (6:1, 3:1, 1.5:1 are the proportion between Astragalus root and Panax notoginseng) were given to the rats for 7 days firstly, and then the rat's serums were separated and added into the culture solution of GMC. Afterwards, an observation on the proliferation of GMC was mace. The result showed that TMOL, different proportion of TongMai could inhibit the proliferation of GNC cultured both in fetal cow serum(FCS) and those cultured in FCS plus lipoplysaccharide (LPS). The difference was markedly compared with contral group. The inhibit effect of TongMai(3:1) is as well as the effect of TMOL, better than TongMai (6:1)and TongMai (1.5:1). So the best compatible proportion of tongmai is 3:1Part IV The study on proliferation of GMC of TongMai extractDifferent drugs including TongMai (3:1), extract of TongMai (3:1), total polysaccharide % total saponin and noneffective extract were given to the rats for 7 days first, and then the rat's serum was separated and added into the culture solution of GMC, Afterwards, an observation on proliferation of GMC was made. The result showed that extract of TongMai (3:1) .. total polysaccharide and total saponin could inhibit the proliferation of GMC cultured both in fetal cow serum(FCS) and those cultured in FCS plus lipoplysaccharide (LPS). The difference was markedly compared with contral group. But noneffective extract could not inhibit the proliferation of GMC. The inhibit effect of extract of TongMai (3:1) is better than that of total polysaccharide or total saponin. So total polysaccharide and total saponin were the chiefly effective compounds.Part V The study on proliferation of GMC with differentcompatibility of effective compoundThe objective was to find out the best compatibility of effective compoundswhich is composed of different dose of total polysaccharide and total saponin. L₉ (³4) orthogonal experiment was designed.Level total polysaccharide (A) total saponin (B)1320mg/kg80mg/kg2640mg/kg160mg/kg3960mg/kg240mg/kgThe result showed A3B3 group could markedly inhibit the proliferation of GMC than all the other groups. That means the best compatible proportion between total polysaccharide and total saponin is 960:240mgPart VI The preparation of rabbit anti-rat thymocyte serum(ATS) and the measuration of ATS titerThe objective was to prepare rabbit anti-rat thymocyte serum(ATS) throughimmunizing the New Zealand(NZ) Rabbit with rat's thymocytes cells(3×10⁷) suspension . The emulsified mixture of rat's thymocytes cells suspension and Completed Freund's adjuvant immunized rabbits in subcutaneous multi-spot firstly, then the mixture were used to consolidate immunization every two weeks for 3 times . ATS titer was measured by indirect agglutination method. The immunizing ways could induce rabbits to produce high-titer ATS. The ATS titer was 1:640Part VII The pharmacological study on effective compound of TongMai in treating MsPGN of ratsObjective: To observe the therapeutic effects of effective compound which were exected from TongMai on the rat experimential model with MsPGN induced by rabbit anti-rat thymocyte serum(ATS).Methods: 72 SD rats were randomly divided into 6 groups: the normal control group, the model control group, Jinshuibao group(treated by Jinshuibao) high ,dose treatment groups(treated by effective compound of TongMai ). SD rats experimential model with MsPGN were induced by subcutaneously injecting rabbit anti-rat thymocyte serum(ATS) with the dose of 5ml/kg for 4 weeks once a week. Then the rats were forced to swim for one week at the end of the first and the third week. After subcutaneously injecting ATS for the first time, the rats were respectively treated with different dose of effective compound of TongMai for 4 weeks. The dose was respectively 1200mg/kg (the proportion between the total polysaccharide and the total saponinis 960mg:240mg ) , 600mg/kg(the proportion between the total polysaccharide and the total saponinis 480mg:120mg ), 300mg/kg(the proportion between tie total polysaccharide and the total saponinis 240mg:60mg ). 24-hour urinary protein contents, hematuria, red-cell count ,blood urea nitrogen (BUN) , creainine(Scr), albumin(ALB) and total protein(TP) were detected at week 2 end 4. The blood rheology and the value of T-lymphocyte subgroups (including CD₃, CD₄, CD₈, CD₄/CD₈)function were tested by the end of experiment .At the same time, the proliferative degree of mesangium was examined by HE stain , PAS stain and immunfluorescent. mesagium area proportion in glomerular capillaries area is calculated using compute picture analyzing system. The expression of PCNA in the glomeruli was examined...