CN110702891A - Method for testing harm reduction effect of natural herbal extract on long-term smoking harm - Google Patents
Method for testing harm reduction effect of natural herbal extract on long-term smoking harm Download PDFInfo
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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Abstract
The invention relates to the technical field of research on tobacco exposure biological effect, and discloses a method for testing harm reduction effect of natural herbal extracts on long-term smoking harm, which comprises the following steps: the method comprises the steps of establishing a rat mainstream smoke exposure model by using tested tobacco, observing clinical symptoms, measuring body weight, measuring feed consumption, performing clinical pathology examination, performing histopathology examination and evaluating results.
Description
Technical Field
The invention relates to the technical field of research on tobacco exposure biological effect, in particular to a method for testing harm reduction effect of natural herbal extracts on long-term smoking harm.
Background
Smoking can cause health problems, which have been confirmed in various epidemiological investigations and experimental studies. However, tobacco consumption is increasing worldwide due to the addiction of many smokers to smoking or the desire not to quit smoking. China is a large country for tobacco production and consumption, and a huge smoker team still exists at present and in a long time in the future, so the harm of cigarettes is reduced, and the method has great significance for developing the tobacco industry and protecting the health of smokers.
The materia medica is originally recorded in Han Shu & Pingdi Ji, books of ancient Chinese medicine are mostly called materia medica, and the book says that the herbs and herbs for curing diseases are also mentioned in the book. The ancient people have a large amount of works related to the Chinese herbal medicines named as the Chinese herbal medicines, the Chinese herbal cosmetics are developed along with the continuous improvement of the global position of Chinese economy in recent years, and the application of the Chinese herbal cosmetics in health care products is quite mature, so that the natural Chinese herbal extract has a positive effect on the health of human bodies. At present, cigarettes with the grass fragrance are available in the market, and the research on the harm reducing effect of the natural herbal extract on the cigarettes has an important effect on the mature application and the divergent development of natural products in the field of cigarettes.
Therefore, the present invention intends to study and evaluate the harm reduction effect of natural herbal extracts on the long-term smoking harm from various aspects.
Disclosure of Invention
Based on the problems, the invention provides a method for testing the harm reduction effect of the natural herbal extract on the long-term smoking harm, provides a rational evaluation direction for the effect of the natural herbal extract on reducing the harm of the cigarette to the human health, and provides a new method for reducing the harm of the cigarette.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a method for testing harm reduction effect of natural herbal extracts on long-term smoking harm, which comprises the following steps:
(1) establishing a rat mainstream smoke exposure model by using tested tobacco:
a. weighing the experimental animals, and randomly grouping according to the weight of the experimental animals, wherein the experimental animals comprise a blank control group, a common cigarette model group and a model group, and the model group is added with the herbal extract into cigarettes according to the mass ratio of 1% on the basis of the common cigarette model group;
b. the flue gas exposure method comprises the following steps: exposing the test animal to mainstream smokeIn the cavity, the exposure time of the main stream smoke is 60 min/day and is totally 180 days, and the smoke concentration in the main stream smoke exposure cavity is 990-1210mg/m3The experimental animals of the blank control group do not carry out experimental intervention, the experimental animals of the common cigarette model group adopt the common cigarettes without natural herbal extracts to carry out smoke exposure, and the experimental animals of the model group adopt the cigarettes added with the herbal extracts to carry out smoke exposure;
(2) observation of clinical symptoms: observing the appearance, physical signs, behavior activity, glandular secretion, respiration and fecal characters of the experimental animals once a day;
(3) and (3) measuring the body weight: determining the body weight of the experimental animal once a week;
(4) and (3) determining the feed consumption: the feed consumption is measured once per week and is calculated according to the formula:
(5) clinical pathology examination:
after the smoke is exposed for 180 days, taking part of experimental animals from a blank control group, a common cigarette model group and a model group to perform heart and lung function examination, simultaneously taking blood to perform hematology index detection, blood coagulation detection, serum biochemical index detection, serum antioxidant index detection, immunologic function index detection and genomics detection, taking urine to perform urine routine examination, and performing fasting treatment on the animals 12-16 hours before taking blood;
(6) histopathological examination: the general observation of tissues and organs is carried out on all experimental animals, the brain, the heart, the liver, the spleen, the left lung, the kidney, the adrenal gland, the thymus, the testis and the epididymis are weighed, the ratio of visceral organs to visceral brain is calculated, and the calculation formula of the visceral organs is as follows:
the formula for calculating the ratio of the zang-brain is as follows:
then fixing heart, aorta, liver, spleen, lung, trachea, kidney, brain, adrenal gland, thymus, testis, lymph node and bone marrow with 10% neutral formalin buffer solution, preparing histopathological section, and performing histopathological analysis after HE staining;
(7) clinical symptoms, body weight change, feed consumption, clinical pathology examination results and histopathology examination results of the experimental animals of the blank control group, the common cigarette model group and the model group were compared.
Preferably, the random grouping method for the experimental animals in the step (1) is a piecewise equilibrium random grouping method.
Preferably, the indicators of immune function detected in step (5) include subpopulations of lymphocytes, serum TNF- α, IL-6, IL-1 β, complement C3 and lysozyme.
In summary, compared with the prior art, the invention has the following beneficial effects:
the invention provides an evaluation direction for reducing the harm of the cigarette to human health and provides a new method for reducing the harm of the cigarette; the invention discloses that the weight and the feed consumption of rats can be obviously reduced by smoke exposure, but compared with common cigarettes without natural herbal extracts, the cigarettes added with the natural herbal extracts do not have positive effects in the two aspects, but the MDA level of the cigarettes added with the natural herbal extracts enhances the activity of antioxidant enzymes, and meanwhile, the absolute weight and the relative weight of the thymus of rats in a model group 4 in the embodiment of the invention are increased compared with those of a control group and a common cigarette model group 2; the histopathological examination result indicates that the smoke of the model group 3 can obviously improve the pulmonary atelectasis caused by long-term smoke exposure, and the smoke of the model group 4 has obvious improvement effect on the myocarditis of the animals in the model group.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.
Examples
This example provides a method for testing the harm reduction effect of natural herbal extracts on long-term smoking hazards, comprising the following steps:
(1) rat mainstream smoke exposure model established by using tested tobacco
a. The experimental animals in this example were SD rats, the number of rats was 300 and all rats were male, and the animal grade of the rats was SPF grade; the rats are weighed and then grouped according to the weight of the rats by a piecewise equilibrium random grouping method, the rats are divided into four groups, the four groups comprise a 1# blank control group, a 2# common cigarette model group, a 3# model group and a 4# model group, the 3# model group and the 4# model group respectively use cigarettes added with 3# and 4# natural herbal extracts, the preparation method of the 3# natural herbal extract is prepared according to the preparation method of the embodiment 2 of the patent CN101138432B, the 4# natural herbal extract is prepared according to the preparation method of the embodiment 4 of the patent CN102551207B, and the grouping information of the rats is shown in Table 1.
Table 1 rat grouping information
| Group of | Sex/number of rats | Rat numbering |
| Blank control group # 1 | ♂:60 | M101-M160 |
| Model group of 2# common cigarette | ♂:80 | M201-M280 |
| Model group 3# | ♂:80 | M301-M380 |
| Model set 4# | ♂:80 | M401-M480 |
b. The flue gas exposure method comprises the following steps: placing the rat in the main stream smoke exposure cavity, wherein the main stream smoke exposure time is 60 min/day and is 180 days in total, and the smoke concentration in the main stream smoke exposure cavity is 990-1210mg/m3In this embodiment, the smoke concentration in the mainstream smoke exposure cavity is 1100mg/m3The rats in the No. 1 blank control group are not subjected to experimental intervention, the rats in the No. 2 common cigarette model group are subjected to smoke exposure by adopting common cigarettes without herbal materials, the rats in the No. 3 model group are subjected to smoke exposure by adopting cigarettes containing No. 3 herbal material extracts, and the rats in the No. 4 model group are subjected to smoke exposure by adopting cigarettes containing No. 4 herbal material extracts.
(2) Observation of clinical symptoms: the appearance, physical signs, behavior activity, glandular secretion, respiration and fecal characters of the rats are observed once a day, and in the embodiment, the salivation symptom can be observed on the rats in the 2# common cigarette model group, the 3# model group and the 4# model group at the 1 st and 2 nd weeks of smoking.
(3) And (3) measuring the body weight:
the body weights of the rats were measured once a week and the body weight changes are shown in Table 2, and it can be seen from the table that the body weights of the rats of the 2# plain cigarette model group, the 3# model group and the 4# model group were all lower than that of the 1# blank control group (P < 0.01) on days 8-183 of smoke exposure.
The results of this section show that smoke exposure significantly caused weight loss in rats, and that there was no statistical difference in the body weight of rats in the model group # 3 or model group # 4 compared to the model group #2 (P > 0.05).
TABLE 2 rat body weight changes (
g)
(4) And (3) determining the feed consumption: the feed consumption was measured once a week, and in this example, 200. + -.1 g of feed was placed on each cage of rats the day before the feed consumption was obtained, and the amount of feed remaining was measured at the same time on the day when the feed consumption was obtained, and the feed consumption was calculated by the formula:
the experimental results of this example are shown in table 3, and show that the feed consumption of rats in the 2# common cigarette model group is lower than that of the 1# blank control group on the 4 th to 25 th, 60 th to 67 th and 88 th days of smoke exposure, and the statistical differences (P < 0.05, P < 0.01 and P < 0.05) exist; the feed consumption of rats in the 2# common cigarette model group is lower than that of the 1# blank control group on the 151 th day and 165 th day of smoke exposure, and the feed consumption is higher than that of the 1# blank control group on the 158 th day, so that the statistical difference exists (P is less than 0.01); on the 4 th to 25 th days and 60 th to 74 th days of smoke exposure, the feed consumption of rats in the model group No. 3 is lower than that of the blank group No. 1, and the rats have statistical differences (P is less than 0.01, P is less than 0.05, P is less than 0.01, P is less than 0.05 and P is less than 0.01); the feed consumption of rats in the model group 3 is lower than that of the blank group 1 on the days 102-130 and 172 of smoke exposure, and the statistical difference is generated (P < 0.05, P < 0.01 and P < 0.05); on day 158, the rats in model group # 3 had higher feed consumption than the control group # 1, with statistical differences (P < 0.01); on the 25 th day, the 74 th day and the 102 th day of smoke exposure, the feed consumption of rats in the model group 3 is lower than that of the model group 2# common cigarette, and the rats have statistical differences (P is less than 0.05, P is less than 0.01 and P is less than 0.01). On the 88 th day, 137 th day and 144 th day of smoke exposure, the feed consumption of the 3# model group rats is higher than that of the 2# common cigarette model group, and the statistical difference exists (P is less than 0.05, P is less than 0.05 and P is less than 0.01).
On the 4 th to 11 th days, the 25 th days, the 60 th to 67 th days and the 88 th days of smoke exposure, the feed consumption of the rats of the model group 4# is lower than that of the blank control group 1# and has statistical difference (P is less than 0.01, P is less than 0.05 and P is less than 0.05); on the 102 th, 123 th, 137 th, 144 th, 165 th and 172 th days of smoke exposure, the feed consumption of rats in the model group 4# was lower than that of the blank control group 1# with statistical differences (P < 0.01, P < 0.05); on the 95 th day of smoke exposure, the feed consumption of rats in model group # 4 was higher than that of rats in control group # 1, with statistical difference (P < 0.05); on the 46 th day, the 95 th day, the 123 th day and the 130 th day of smoke exposure, the feed consumption of rats in the model group 4 is higher than that of the model group 2# common cigarette, and the statistical difference exists (P is less than 0.05, P is less than 0.01, P is less than 0.05 and P is less than 0.05); on the 102 th day and the 158 th day of smoke exposure, the feed consumption of rats in the model group 4 is lower than that of the model group 2# common cigarette, and the statistical difference exists (P is less than 0.01 and P is less than 0.01).
The experimental results show that the smoke exposure has certain influence on the feed consumption of rats in the 2# common cigarette model group, the 3# model group and the 4# model group, and the long-term smoke exposure can reduce the feed consumption of the rats; the feed consumption of rats in the model group 3 and the model group 4 is basically consistent with the change of the model group 2 ordinary cigarettes, and is higher/lower than that of the model group 2 ordinary cigarettes only at individual time points, which shows that the 3# cigarettes and the 4# cigarettes have no obvious improvement effect on the feed consumption of rats.
TABLE 3 rat feed consumption Change (
g)
(5) Clinical pathology examination
After 180 days of smoke exposure, 12 rats are respectively taken from a No. 1 blank control group, a No. 2 common cigarette model group, a No. 3 model group and a No. 4 model group, 50mg/kg sodium pentobarbital is used for carrying out intraperitoneal injection anesthesia, then, the heart and lung function examination is firstly carried out, then, blood is taken from an abdominal aorta, the blood taken out is respectively subjected to hematology index detection, blood coagulation detection, serum biochemical index detection, serum antioxidant index detection, immune function index detection and genomics detection, and urine is additionally taken for examination; the rats are fasted 12-16 hours before blood collection.
The hematology index detection is carried out by adopting a full-automatic five-classification rat hematology analyzer of HEMAVET 950, and the used diluent, cleaning solution, hemolytic agent and quality control are all produced by Drew Scientific company.
The blood coagulation index detection mainly measures four blood coagulation indexes, namely Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT), Thrombin Time (TT) and Fibrinogen (FIB), and the used instrument is a semi-automatic four-channel hemagglutination instrument produced by the French STAGO company.
The biochemical index detection of the serum adopts a Konelab PRIME 30 full-automatic biochemical analyzer for detection, the used reagent is produced by Guangzhou Kefang biotechnology, Inc., the clinical chemistry comprehensive quality control serum is produced by RANDOX, and the batch number is as follows: 1199 UN.
The serum antioxidant index detection is carried out according to the instruction of the corresponding kit.
The immune function indexes comprise lymphocyte subsets, serum TNF-alpha, IL-6, IL-1 beta, complement C3 and lysozyme, the lymphocyte subsets are detected by a flow cytometer, 1.25 mu L of CD3 antibody, 0.5 mu L of CD4 antibody, 1 mu L of CD8a antibody and 100 mu L of EDTA anticoagulation are added into a flow tube, RBC lysine Buffer2ml is added after incubation for 15min at room temperature, 800 mu L of PBC is added after Lysis for 7min, and the detection is carried out on a computer.
The genomics detection process is as follows: 3 rats are respectively taken from a No. 1 blank control group, a No. 2 common cigarette model group, a No. 3 model group and a No. 4 model group, the heart, the liver and the lung of the rat are separated, the rat is cleaned by precooled PBS (phosphate buffer solution), the rat is placed into a freezing tube, the rat is rapidly put into liquid nitrogen for freezing for more than 5min, and then the rat is transferred to a refrigerator at the temperature of minus 80 ℃ for storage for later use, and then genomics detection is carried out.
The urine detection process is as follows: urine is taken by a metabolism cage method, and glucose, bilirubin, ketone bodies, urine specific gravity, occult blood, pH, protein, urobilinogen, nitrite and leucocytes of the urine are measured by a test strip method and an H-100 urine analyzer, and the appearance of the urine is observed by naked eyes.
The result of the hematological index detection is shown in table 4, and the result shows that the hematological indexes of rats of the 2# common cigarette model group, the 3# model group and the 4# model group have no statistical difference (P is more than 0.05) compared with those of the 1# blank control group after the smoke is exposed for 180 days; the 3# model group and the 4# model group have no statistical difference in the hematological indexes of rats compared with the 2# common cigarette model group (P is more than 0.05).
TABLE 4 changes in the hematological indices of rats
The serum biochemical index detection results are shown in table 5, and the results show that the smoke is exposed for 180 days, the GLU of the 2# common cigarette model group is lower than that of the 1# blank control group, and the statistical difference exists (P is less than 0.05); the biochemical indexes of each serum of rats in the model group No. 3 and the model group No. 4 are not statistically different (P is more than 0.05) compared with those in the model group No. 2 common cigarette and the blank control group No. 1 blank control group.
TABLE 5 Biochemical index changes in rat serum
The results of the blood coagulation tests are shown in table 6, and show that the smoke exposure time is 180 days, the APTT of the 2# common cigarette model group is lower than that of the 1# blank control group, the statistical difference is generated (P is less than 0.05), and the TT and APTT of the 4# model group are both lower than that of the 1# blank control group, the statistical difference is generated (P is less than 0.05, P is less than 0.01).
TABLE 6 blood coagulation index changes in rats
The classification results of lymphocyte subpopulations are shown in Table 7, the rats of the 2# common cigarette model group, the 3# model group and the 4# model group have no statistical difference (P > 0.05) in comparison with the 1# blank control group in the rats of CD4+, CD8+, CD4+/CD8+ after 180 days of smoke exposure; no statistical difference (P > 0.05) is found between rats in model group # 3 and model group # 4, rat CD4+, CD8+, CD4+/CD8 +.
TABLE 7 rat lymphocyte subpopulation classification
The detection results of the serum antioxidant indexes are shown in table 8, and the results show that smoke is exposed for 180 days, the SOD, MDA and GSH of the 2# common cigarette model group and the 3# model group are higher than those of the 1# blank control group, and have statistical difference (P is less than 0.01, P is less than 0.05, and P is less than 0.01), the SOD and GSH of rats of the 4# model group are higher than those of the control group, and have statistical difference (P is less than 0.01); the MDA of the model group 3 is lower than that of the model group 2 of the ordinary cigarette, the GSH is higher than that of the model group 2 of the ordinary cigarette, and statistical differences exist (P is less than 0.05, and P is less than 0.01); the MDA of the rats in the model group 4 is lower than that in the model group 2 of the common cigarette, and the statistical difference exists (P is less than 0.01); the results show that the long-term smoke exposure can ensure that the level of serum lipid peroxidation products (MDA) of the rats is higher than that of the rats of the No. 1 blank control group, and the oxidation resistance levels (SOD and GSH) are also higher than that of the No. 1 blank control group; meanwhile, the 3# model group and the 4# model group can reduce the MDA level and enhance the activity of antioxidant enzymes.
TABLE 8 changes in antioxidant index in rats
The results of cytokine indexes are shown in Table 9, and show that the results show that the rats of the 2# common cigarette model group, the 3# model group and the 4# model group have no statistical difference (P is more than 0.05) in comparison with the No. 1 blank control group in the aspects of C3, IL-1, IL-6, TNF-alpha and LYZ after being exposed for 180 days; no statistical difference was observed between the rats C3, IL-1, IL-6, TNF- α, LYZ from model # 3 and model # 4 when compared to the normal cigarette model #2 (P > 0.05).
TABLE 9 rat cytokine index Change (x. + -. s)
The detection results of the urine indexes are shown in table 10, and the results show that the urine indexes of rats of the 2# common cigarette model group, the 3# model group and the 4# model group have no statistical difference (P is more than 0.05) compared with the 1# blank control group after the smoke is exposed for 180 days; the urine indexes of rats in the model group No. 3 and the model group No. 4 are not statistically different from those in the model group No. 2 common cigarette (P is more than 0.05).
TABLE 10 variation of index of urine (x. + -. s) for rat
(6) Histopathological examination: the rat is anesthetized and dissected, then tissue and organ visual detection is carried out, the brain, the heart, the liver, the spleen, the left lung, the kidney, the adrenal gland, the thymus, the testis and the epididymis are weighed, the organ coefficient and the ratio of the organ to the brain are calculated, and the calculation formula of the organ coefficient is as follows:
the formula for calculating the ratio of the zang-brain is as follows:
then fixing heart, aorta, liver, spleen, lung, trachea, kidney, brain, adrenal gland, thymus, testis, lymph node and bone marrow with 10% neutral formalin buffer solution, preparing histopathological section, and performing histopathological analysis after HE staining;
the results of the visceral weights are shown in table 11, and the results show that the brain weight of rats in the 2# common cigarette model group is higher than that of the 1# blank control group, the lung weight of rats is lower than that of the 1# blank control group, and statistical differences exist (P is less than 0.05 and P is less than 0.01) after the smoke exposure for 180 days; the lung weight of rats in model group # 3 was lower than that of rats in blank control group # 1, with statistical difference (P < 0.01); the weight of thymus of rats in model group # 4 is higher than that of rats in blank control group # 1, the weight of heart and lung are lower than that of rats in blank control group # 1, and statistical differences exist (P is less than 0.05, and P is less than 0.01); the thymus weight of the rats in the model group No. 4 is higher than that of the model group No. 2 common cigarette, the brain weight of the rats is lower than that of the model group No. 2 common cigarette, and the rats have statistical difference (P is less than 0.05).
TABLE 11 weight changes of rat organs (
g)
The organ coefficient results are shown in table 12, and the results show that the organ coefficients of brain, spleen, kidney, testis and epididymis of rats in the 2# common cigarette model group are higher than those of the 1# blank control group after 180 days of smoke exposure, and have statistical differences (P is less than 0.01, P is less than 0.05, P is less than 0.01 and P is less than 0.05); the brain and testicular organs of the rat in the model group No. 3 are higher than those in the blank control group No. 1, and statistical differences exist (P is less than 0.05 and P is less than 0.01); the brain, thymus, testis and epididymis organ coefficients of the rats in the model group No. 4 are higher than those of the blank control group No. 1, and the lung organ coefficients are lower than those of the blank control group No. 1, and the statistical differences exist (P is less than 0.05, P is less than 0.01, P is less than 0.05, and P is less than 0.01); the brain organ coefficient of the rat in the model group 3 is lower than that of the rat in the model group 2, and the statistical difference exists (P is less than 0.05); the brain organ coefficient of the rats in the model group No. 4 is lower than that of the model group No. 2 common cigarette, and the thymus organ coefficient is higher than that of the model group No. 2 common cigarette, and the statistical difference is realized (P is less than 0.05).
TABLE 12 rat organ coefficient changes (
％)
The results of the visceral-brain ratio are shown in table 13, and the results show that the lung-brain ratio of rats of the 2# common cigarette model group, the 3# model group and the 4# model group is lower than that of the 1# blank control group after 180 days of smoke exposure, and the statistical difference is generated (P is less than 0.01); the thymus brain ratio of the rats in the model group No. 4 is higher than that of the rats in the blank control group No. 1 and the rats in the common cigarette model group No. 2, and the statistical difference is realized (P < 0.05 and P < 0.01).
TABLE 13 rat zang-to-brain ratios
The visceral-cerebral ratio can overcome the influence of weight change on visceral factor, and can reflect the change condition of relative weight of visceral organs more objectively, and the results of the visceral weight, the visceral factor and the visceral-cerebral ratio show that the absolute weight and the relative weight of the lung can be reduced due to long-term smoke exposure; meanwhile, the absolute weight and the relative weight of the thymus of the rats in the model group 4 are increased compared with those of the rats in the control group and the model group 2 of the common cigarette.
Histopathological examination results show (table 14) that the incidence of lung lesions (atelectasis) was increased in the 2# plain cigarette model group and the 4# model group compared to the 1# blank control group and the 3# model group after 180 days of smoke exposure; the incidence of myocarditis of the model group No. 4 is lower than that of the model group No. 1 blank control group, the model group No. 2 common cigarettes and the model group No. 3; the incidence of adrenal fascial cell vacuolation of the 2# common cigarette model group and the 3# model group is higher than that of the 1# blank control group and the 4# model group.
The histopathological examination result indicates that the smoke of the model group 3 can obviously improve the pulmonary atelectasis caused by long-term smoke exposure, and the smoke of the model group 4 has obvious improvement effect on the myocarditis of rats in the model group; the incidence of adrenal fascial cell vacuolation of the 2# common cigarette model group and the 3# common cigarette model group is higher than that of the control group, and the clinical significance of the adrenal fascial cell vacuolation is yet to be further observed.
TABLE 14 rat lesion statistics
The genomics detection result shows that the difference gene expression of the lung, the heart and the liver of each group of rats is shown in table 15 after the rat smoke is exposed for 180 days.
TABLE 15 rat differential Gene expression
The electrolyte index of rats was also studied in this example and the results are shown in Table 16, which shows that the smoke exposure is 180 days, Ca of rats in the 2# common cigarette model group2+The concentration is higher than that of the 1# blank control group, and the statistical difference exists (P is less than 0.05); ca of rats in model No. 3 and model No. 42+The concentration is not statistically different compared with the 1# blank control group (P is more than 0.05); in addition, K in rats of model group # 4+The concentration was lower than that of the model group, with statistical differences (P < 0.05), but no statistical differences (P > 0.05) compared to the No. 1 blank group.
TABLE 16 animal electrolyte index changes
The above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the process of verifying the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all the equivalent structural changes made by applying the content of the specification of the invention should be covered by the scope of the invention.
Claims (3)
1. A method for testing harm reduction effect of natural herbal extracts on long-term smoking harm is characterized by comprising the following steps:
(1) establishing a rat mainstream smoke exposure model by using tested tobacco:
a. weighing the experimental animals, and randomly grouping according to the weight of the experimental animals, wherein the experimental animals comprise a blank control group, a common cigarette model group and a model group, and the model group is added with the herbal extract into cigarettes according to the mass ratio of 1% on the basis of the common cigarette model group;
b. the flue gas exposure method comprises the following steps: placing the experimental animal in a main stream smoke exposure cavity for main stream smoke exposure time60 min/day, and 180 days of exposure, wherein the smoke concentration in the main stream smoke exposure cavity is 990 mg/m and 1210mg/m3The experimental animals of the blank control group do not carry out experimental intervention, the experimental animals of the common cigarette model group adopt the common cigarettes without natural herbal extracts to carry out smoke exposure, and the experimental animals of the model group adopt the cigarettes added with the herbal extracts to carry out smoke exposure;
(2) observation of clinical symptoms: observing the appearance, physical signs, behavior activity, glandular secretion, respiration and fecal characters of the experimental animals once a day;
(3) and (3) measuring the body weight: determining the body weight of the experimental animal once a week;
(4) and (3) determining the feed consumption: the feed consumption is measured once per week and is calculated according to the formula:
(5) clinical pathology examination:
after the smoke is exposed for 180 days, taking part of experimental animals from a blank control group, a common cigarette model group and a model group to perform heart and lung function examination, simultaneously taking blood to perform hematology index detection, blood coagulation detection, serum biochemical index detection, serum antioxidant index detection, immunologic function index detection and genomics detection, taking urine to perform urine routine examination, and performing fasting treatment on the animals 12-16 hours before taking blood;
(6) histopathological examination: the general observation of tissues and organs is carried out on all experimental animals, the brain, the heart, the liver, the spleen, the left lung, the kidney, the adrenal gland, the thymus, the testis and the epididymis are weighed, the ratio of visceral organs to visceral brain is calculated, and the calculation formula of the visceral organs is as follows:
the formula for calculating the ratio of the zang-brain is as follows:
then fixing heart, aorta, liver, spleen, lung, trachea, kidney, brain, adrenal gland, thymus, testis, lymph node and bone marrow with 10% neutral formalin buffer solution, preparing histopathological section, and performing histopathological analysis after HE staining;
(7) clinical symptoms, body weight change, feed consumption, clinical pathology examination results and histopathology examination results of the experimental animals of the blank control group, the common cigarette model group and the model group were compared.
2. The method for testing the harm reduction effect of natural herbal extracts on long-term smoking harm according to claim 1, wherein the random grouping method of the experimental animals in the step (1) is a piecewise equilibrium random grouping method.
3. The method for testing the reducing effect of natural herbal extracts on the harm of long-term smoking according to claim 1, wherein the immune function indicators detected in the step (5) comprise lymphocyte subpopulation, serum TNF- α, IL-6, IL-1 β, complement C3 and lysozyme.
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