Screening Of Herba Proteinase Inhibitor And Its Effect/Mechanism Of ALI Prevention & Treatment

Proteinase inhibitors are ubiquitously distributed in the animal, plant and microorganism kingdoms, and could bind with relevant target proteinase to form specific protein-protein complex, cause competitive inhibiting effect, decrease enzyme activity, form a certain dynamic balance, play key regulatory roles in many biological processes, including the blood coagulation system, the complement cascade ,apoptosis and the hormone processing pathways. According to modern pharmacological research, Herba houttuyniae could strengthen immune function, resist virus, and inhibit cancer;it was mainly clinically applicable to treat respiratory diseases, such as pulmonary abscess, premises, hot sputum, asthma cough, bronchitis, pneumonia, bronchiectasis, and pertussis. Acute lung injury (ALI) was early stage of Acute Respiratory Distress Syndrome (ARDS) or Multisystem Organs Failure (MOF), and was the acute progressive anoxic respiratory failure caused by various factors inside and outside the lung. ALI was of very high morbidity rate and mortality rate, and ARDS occurred annually in about 100,000 American people with a mortality rate of up to 50～70%, and was a focus of recent medical research.This test aimed to find out safe effective vegetable proteinase inhibitor from TCM materials, and explore its effect and mechanism. It screened the activity of 60 TCM proteinase inhibitors, and studied the effect and mechanism of Herba houttuyniae proteinase inhibitor (HHPI) compositions for ALI prevention & treatment, so as to provide scientific basis for clinical drug application. Its main research contents and results were summarized as follows:1. Screen TCM materials with activity of proteinase inhibitorThis test studied the in vitro trypsin inhibiting effect of water and 70% ethanol extract of 60 decoction-free TCM materials granule and 5 TCM materials (Rhubarb, Herba houttuyniae, Ephedra, White peony, and Mongolian snakegourd peel), and the in vivo trypsin inhibitor activity of these 5 TCM materials through the substrate of trypsin and the control of soybean trypsin inhibitor. All these test TCM materials granules and TCM materials were of wider clinical application.(1) TCM materials of stronger proteinase inhibitor activity mainly occurred in wind/cold-dispersing drugs and heat/toxin clearing drugs. Among 60 test TCM materials granules, trypsin inhibiting effect was stronger at 100mg/mL dose in 13 TCM materials (i.e. pseudo-ginseng, Ephedra, Rhubarb, white peony, and Herba houttuyniae from strongest to weakest effect), stronger at 50mg/mL dose in 9 TCM materials (i.e. Tuckahoe, Rhubarb, subprostrate sophora, Ephedra, white peony, Herba houttuyniae, and Forsythia from stronger to weaker effect), and evident at both above concentrations in 5 TCM materials (i.e. Ephedra, Rhubarb, Herba houttuyniae, white peony, and Forsythia).(2) In vitro trypsin inhibiting effect was stronger in water extract of Rhubarb/Herba houttuyniae and 70% ethanol extract of Ephedra/white peony, stronger in both extracts of Rhubarb/Herba houttuyniae/Ephedra, and not evident in Mongolian snakegourd peel.(3) After the gavage of their water extract in rats, compared with that in Ephedra and White peony, trypsin inhibiting effect was stronger in vitro plasma containing Rhubarb and Herba houttuyniae, and not evident in water extract of Mongolian snakegourd peel.2. Separate HHPI compositionsThis test studied, through orthogonal test, the effect of Herba houttuyniae water extract on proteinase inhibiting activity under 4 extracting conditions (i.e. extracting temperature, decoction time, solution volume, and solution pH), and finally found out optimal extraction conditions (i.e. 60℃, water addition by 30 times, 3h decoction, and pH9). Extract Herba houttuyniae under this optimal condition, concentrate water extract at reduced pressure, centrifuge, remove sediment, extract supernatant with chloroform, filter water layer, remove sediment, pass through macroporous resin, elute with deionized water until no sediment with 95% ethanol in elution, concentrate elution at reduced pressure, add 95% ethanol until 75% alcohol content, freeze for overnight, filter, elute sediment repeatedly with ethanol and acetone until colorless elution, dry at low temperature, and obtain Composition 1. Elute macroporous resin with 30%, 50%, 70% and 95% ethanol respectively until colorless elution, recover ethanol at reduced pressure, dry concentrated solution at reduced pressure and vacuum state, and obtain Composition 2, 3, 4, and 5 respectively. Pass chloroform layer through ion exchange resin, elute with ammonia water, collect elution, concentrate at reduced pressure, dry at vacuum state, and obtain Composition 6.By assaying qualitatively through test tube method and TLC, Composition 1 was polysaccharides and glycosides, Composition 2, 3, 4 and 5 were flavones, and Composition 6 was alkaloids. As shown by assaying the inhibition of 1mg/mL crude extract of each composition on trypsin activity, the proteinase inhibitor activity occurred in Composition 6 (alkaloid), Composition 1 (polysaccharide), and Composition 2, 3, 4 and 5 (flavones) respectively in the order from strongest activity to weakest activity.3. Test on prevention & treatment of LPS-induced ALI with HHPI compositions in ratsThis test studied the prevention & treatment effect of HHPI compositions on ALI through LPS-induced ALI rat model. As shown by the test results, HHPI could alleviate pulmonary mesenchyme thickening, reduce hemorrhage/ edema/exudates in pulmonary mesenchyme and alveolar cavity, decrease collagen fiber content in pulmonary mesenchyme, alleviate fibrous proliferation of lung tissue, improve lung injury symptoms, decrease significantly MDA content in bronchoalveolar lavage fluid and IL-6, IL-8 and TNF-αlevel in serum, tend to significantly decrease HYP content in lung tissue, and down-regulate MMP-2 and MMP-9 content and protein expression in lung tissue. Thus, HHPI had a certain effect of ALI prevention & treatment, possibly by blocking the lipid peroxidation process at early stage of ALI, reducing the release of oxygen free radical and inflammatory factor, decreasing MMPs activity, and reducing injury of lung tissue.4. Test of HHPI effect on gene expression spectrum of A549 cells (gene chip technique)This test detected the effect of HHPI compositions on gene expression spectrum of A549 cells through gene chip technique by replacing Type II alveolar epithelial cells with A549 cancer cells strain in alveolar epithelium. As shown by the test results, among all 15552 detected genes, there were 204 genes (1.3%) of significantly different expression (including 139 up-regulated ones and 65 down-regulated ones);and among apoptosis-related genes, there were 25 up-regulated ones and 7 down-regulated ones. Apoptosis-related genes at up-regulated expression mainly existed in the following functional types: signal transduction, tumor inhibition, protein transcription factor, cell division/cell apoptosis, cell skeleton/movement protein, body defense/heat shock protein, DNA injury/repair, metabolism/proteolysis, cell factor/chemical factor, membrane protein, enzyme/cancer gene/cell cycle, transcription-related protein, and proteinase inhibitor. Apoptosis-related genes at down-regulated expression mainly existed in the following functional types: cell division/cell apoptosis, signal transduction, cell division, cell cycle, and cancer gene. HHPI could regulate various genes related to cell structure/function, cell signal transduction, cell metabolism, transcription, transcription factor, and protein translation/synthesis.To sum up, TCM materials of stronger proteinase inhibitor activity mainly occurred in wind/cold-dispersing drugs and heat/toxin clearing drugs. (i.e.pseudo-ginseng, Ephedra, Rhubarb, white peony, Herba houttuyniae ,Tuckahoe, subprostrate sophora, and Forsythia). As shown by assaying the inhibition of each composition on trypsin activity, the Herba houttuyniae proteinase inhibitor activity occurred in alkaloid, polysaccharide, and flavones respectively in the order from strongest activity to weakest activity. HHPI had a certain effect of ALI prevention & treatment, possibly by blocking the lipid peroxidation process at early stage of ALI, reducing the release of oxygen free radical and inflammatory factor, decreasing MMPs activity, and reducing injury of lung tissue. HHPI could regulate various genes related to cell structure/function, cell signal transduction, cell metabolism, transcription, transcription factor, and protein translation/synthesis.