Microsatellite Polymorphism In The Heme Oxygenase-1 Gene Promoter Is Associated With Susceptibility To Damp-heat Syndrome In Guangdong Han Chinese

Lingnan belongs to subtropical zone, near the South China Sea, with low-lying land and Nanling Mountains, forming a unique geographical environment. Lingnan region is in low latitude, close to the equatorial zone in China, sunshine and solar radiation is strong. The climate is hot, humid and rainy, the average temperature between 20.4 and 23.1℃, the annual rainfall is from 1500 to 2000 mm, it is a famous rainy region in China. Four Seasons division is not obvious, with long summer and warm winter, and the duration of high temperature in a day is long, the average temperature is high, resulting in heat body constitution of Lingnan people. The annual average relative humidity of Guangzhou and Pearl River Delta zone is 78%, and it can reach 85% between March and August. The hot and humid climate exceeds the regulation limit of the body leads to the formation of damp-heat pathogenic factors easily. Furthermore, people in Guangdong like swimming, bathing, eating fish, shrimp, snail and oyster as well as many other wet feminine goods, with the addition of drinking raw frozen things, affecting the individual digestion and absorption function of spleen and stomach. Wet obstructs spleen and stomach, leading to damp-heat body constitution. The geographical environment, climate factors, eating habits, living custom, body constitution in Lingnan leads to the occurrence of damp-heat syndrome.Body constitution refers to the relatively stable complex of specific physical quality and psychological quality in aspects of shape, structure, function, metabolism, which formed in the process of growth and development as well as under the effect of heredity and the environmental factors. It is not only affected by genetic factors, but also by acquired environmental factors. From the point of TCM theory, body constitution is important material foundation for the development of syndrome. The particularity of the individual body constitution usually determines the susceptibility to certain kind of pathogenic factors and tendentiousness of lesion type. To a certain extent, body constitution determines the occurrence and development as well as prognosis of the disease, determines the type, conversion, and outcome of the syndrome.Individual characteristic of body constitution gradually developed based on hereditary factor, under the influence of acquired disposition, through influence and change of nature, society, situation, catering and many other factors. Body constitution difference determined by the genetic background is an important condition for maintaining relative stability of individual body constitution feature.Human genome sequence variation is one of the basic characteristics of the genome. It is the gene polymorphisms, genomic sequence diversity that constitutes the genetics foundation of susceptibility to diseases, reaction to drug and environmental factors of different individuals and groups. The appearance of gene polymorphisms is the inevitable result of human evolution and adaptation to the environment. Meanwhile, the diversity of it in the population is the internal basis of different susceptibility to diseases for different individual.The climate of Guangdong region is hot and rainy, thus, forming a high temperature and humidity (damp-heat) climate characteristics. The body in such an environment can occur heat stress response. Heme oxygenase-1 (HO-1) is a highly conserved stress protein during the evolution, play an important role to maintain the stability of the body. Its main function is to improve the cellular tolerance capability to stress factors, so that cells maintain normal physiological function, increase defense and adaptation capability to lethal stimuli. It catalyzes the degradation of heme, a potent oxidant, into biliverdin, carbon monoxide (CO) and free iron. HO-1 is a protective factor which exerts anti-inflammatory, antioxidant, anti-proliferative effect.(GT)n dinucleotide repeat sequence length polymorphism in the HO-1 gene promoter influences the susceptibility to pulmonary diseases, cardiovascular diseases, cerebrovascular diseases, tumor and many other diseases. After suffering external and internal stimuli as well as injury, different responses occur in carriers with different lengths (GT)n repeats, which affects the individual adaptive capacity, formation of the body constitution, the susceptibility and tolerance to many diseases, influence the reaction to heat stress response, the adaptive capacity to high dampness and high heat as well as the susceptibility to damp-heat syndrome. HO-1 gene promoter microsatellite polymorphism not only reflects the genetic factors, but also is closely related with environmental factors. HO-1 gene may be candidate gene for the research of body constitution, susceptibility to syndrome. Thus, we investigated whether there was any association between heme oxygenase-1 (HO-I) gene promoter microsatellite polymorphism and susceptibility to damp-heat syndrome. Total antioxidant capacity (TAOC) in the serum could reflect the individuals' capacity against oxidant, and malondialdehyde (MDA) could reflect the extent of peroxidation reaction in the body.Objective:To investigate whether there is any association between heme oxygenase-1 (HO-I) gene promoter microsatellite polymorphism and susceptibility to damp-heat syndrome, geographical environment, oxidative stress; investigate the relationship between damp-heat syndrome and oxidative stress.Methods:With informed consent signed, refering to the standards of normal body constitution and damp-heat body constitution, we selected unrelated Han Chinese volunteers without positive diseases.Inclusion criteria:We conducted health survey of family members of Guangdong Han family members that were more than 18 years old (mainly 30-40 years old), who has been living in Guangdong Province, and both parents of the surveyed subjects were Cantonese. Non-Guangdong Han Chinese who have lived in Guangdong province less than five years; both parents of the surveyed subjects were non-Guangdong Chinese. NO positive organic disease was found in the last year. All the volunteers participated in the project voluntarily, and signed the informed consent.Exclusion criteria:Non-Guangdong Han Chinese who have lived in Guangdong province more than five people; less than 18 years old; other ethnic groups except the Han Chinese; one parents of the volunteer was Guangdong native; volunteer suffered from respiratory, digestive, cardiovascular, urinary, nervous, reproductive system diseases; was in the fever (including the common cold); routine laboratory of heart, liver, lung, kidney, blood lipids, blood sugar, etc was abnormal; pregnant women, lactating women; subjects did not sign informed consent. The criteria of damp-heat body constitution refers to Classification and Determination of TCM body constitution, Chinese Medicine Clinical Research Drug guiding principle (Provisional Regulations) issued in 2002 and the related criteria draw up by splenogastric research institution of Guangzhou University of Chinese Medicine. Cardinal manifestations:yellow tongue fur, greasy tongue fur, thick and foul fur, slippery and fast pulse. Minor manifestations:dirty and oily complexion, acne prone, bitter and sticky mouth, feeling thirst but do not want to drink, heavy feeling of limbs and trunk, epigastric distension and depression, chest distress, viscid and bad stool, yellow and little urine. Volunteers with one cardinal manifestation and three minor manifestations (or more than that), or with two cardinal manifestations and two minor manifestations (or more than that) belonged to volunteers with damp-heat body constitution.Selection criteria of control group:with moist and lustrous complexion and skin, dense, dense and shiny hair, bright eyed, energetic, good appetite, sleep well, normal urine and stool, medium body, usually less illness, light red tongue, thin white fur, mild and strong pulse.There were twenty Cantonese volunteers with damp-heat body constitution in group A, twenty volunteers with damp-heat body constitution who came from other provinces in group B, fifteen healthy control volunteers came from Guangdong province in group C, fifteen healthy control volunteers came from other provinces in group D. Group A and group B belonged to damp-heat group, 40 people in total; group C and group D belonged to control group,30 people in total. People in group A and group C were Cantonese,35 people in total; people in group B and group D were from other province,35 people in total. All the volunteers signed the informed consent for the present study. The study was approved by the Guangzhou University of Chinese Medicine Ethics Committee. There is no difference in gender and age between the two groups.The survey site was Guangzhou city; the time of the investigation was from spring equinox to the autumnal equinox (3-9 months).Fasting venous blood sample was extracted from volunteers met the inclusion criteria in each group, blood extracting time was from spring equinox to the autumnal equinox (3-9 months). Genomic DNA was extracted by using whole blood genomic DNA extraction kit (centrifugal column), dissolved DNA fully at 4℃overnight, stored at -20℃for use. The 5'-flanking region containing (GT)nrepeats of the HO-1 gene was amplified by PCR according to the published procedure (The primer was synthesized by Shanghai Invitrogen Biological Engineering Company.),sense primer,5'-AGAGCCTGCAGCTTCTCAGA-3',antisense primer,5'-ACAAAGTCTGGCCATAGGAC-3',PCR reaction program:total 50ul, 10×bufer 5ul,25 mmol/L MgCl23ul,2.5 mmol/L dNTP 4ul,50umol/L sense primer and anti-sense primer 0.25ul respectively,5U/uL TaqDNA polymerase 0.3ul, genomic DNA 2ul, using sterile distilled water to make up to 50ul.Reaction conditions:denaturation at 94℃in advance for 3 minutes, then started the cycle, denaturation at 94℃for 30 seconds, annealing at 58℃for 30 seconds, and elongation at 72℃for 1 minute. Altogether 35 cycles were performed, elongation at 72℃for 5 minutes at last. The PCR amplification product was analyzed by using agarose gel electrophoresis (100 V,35 minutes) together with DNA ladder (size range 100-600bp). The PCR products was analyzed with Automatic Gel Imaging System (G:BOX). The allele frequency of (GT)n repeats in HO-1 gene promotor was analyzed by the GeneTool Analysis software (PE Applied Biosystems). The allelic repeats were divided into subclasses: short repeats (S) (27 repeats), L-allele.5ml peripheral venous blood was extracted from each volunteers, serum was separated after centrifugation 3 OOOr/min,15 min, frozen at-80℃, preparing to be tested. Malondialdehyde (MDA) and total antioxidant capacity (TAOC) were measured by spectrophotometer. MDA was measured through thiobarbituric acid method, TAOC was measured by using Fe3+/Fe2+reduction method.Results1. The frequency of L allele of HO-1 was higher in group A than that in group C, while the frequency of S allele was lower (P<0.05). The frequency of L allele of HO-1 was higher in group B than that in group D, while the frequency of S allele was lower (P<0.05). The frequency of L allele of HO-1 was higher in the damp-heat syndrome group than that in healthy control group, while the frequency of S allele was lower (P<0.01).2. The frequency of L allele of HO-1 was higher in Guangdong Han Chinese than that in non-Guangdong Han Chinese, while the frequency of S allele was lower (P<0.05). 3. TAOC of the L allele carriers was lower compared with that of S allele carriers (15.97±0.89 vs 18.75±1.34, P<0.01). While the MDA content of the L allele carriers was higher compared with that of S allele carriers (6.49±0.59 vs 5.13±0.47, P<0.01)4. Total antioxidant capacity (TAOC) was lower in the damp-heat syndrome group compared with that in non-damp-heat syndrome group (16.80±1.41 vs 18.01±2.04, P<0.01). Meanwhile, the MDA content was higher in the damp-heat syndrome group compared with that in non-damp-heat syndrome group (6.09±0.95 vs 5.48±0.60, P<0.01).Conclusion:The distribution of L allele between damp-heat syndrome group and non-damp-heat syndrome group, between Guangdong Chinese and non-Guangdong Chinese, was statistically different. This indicated that microsatellite polymorphism in the HO-1 gene promoter was geography different. L allele in the HO-1 gene promoter increased the susceptibility to damp-heat syndrome. L allele in the HO-1 gene promoter was damp-heat body constitution related gene and predisposing gene of damp-heat syndrome, or part of the predisposing gene. It was one of the genetic basis of damp-heat body constitution and susceptibility to damp-heat syndrome of Guangdong Han Chinese. In addition, the total antioxidant capacity of L allele carriers in the HO-1 gene promoter reduced, meanwhile, the reduction of antioxidant capacity was one of the pathological basis of damp-heat syndrome, which indicated that L-allele in HO-1 gene promoter might increase the susceptibility to damp-heat syndrome by affecting individual antioxidant stress injury response.