| Research backgroundPrevious studies based on the traditional Chinese Medicine theory of "spleen controlling saliva" found that salivary alpha-amylase (sAA) activity ratio (after acid stimulation/before acid stimulation) in patients with spleen deficiency syndrome was more significantly decreased than healthy subjects. The index was included in the evaluation index of the curative effect of spleen qi deficiency syndrome by the Ministry of health promulgated the "Guiding principle of clinical research on new drugs of traditional Chinese Medicine" in 1993, for few government departments authorized syndrome microscopic indexes. But the detection rate is about 60%-70% in a variety of diseases in patients with spleen qi deficiency syndrome, the specificity is not high enough, which indicate that the information is not comprehensive enough for single use of the index as microscopic indexes of spleen qi deficiency.ObjectiveFrom the same disease with different syndrome and the same syndrome in different diseases perspective, with sAA activity change as a starting point to explore sAA activity and related indexes’(salivary flow rate, pH value, total protein concentration, etc.) changes in patients with spleen deficiency syndrome, further enrich scientific connotation of Chinese medicine theory of "spleen controlling saliva"; and observe combination sAA activity and related indexes in patients with spleen qi deficiency syndrome on clinical detection rate.Methods1. Fifty-five young healthy volunteers saliva samples before and after acid stimulation were collected by EP tube collecting emulated with natural flow, rotating mouth swab slightly and chewing mouth swab. Compare the changes of sAA activity and salivary flow rate of each saliva sample and its ratio before and after acid stimulation to screen the preferred method for collecting saliva.2.101 Chronic superficial gastritis (CSG) and 60 myasthenia gravis (MG) patients were recruited from the First Affiliated Hospital and Outpatient Department of Guangzhou University of Traditional Chinese Medicine. CSG patients with Spleen-Qi Deficiency, Dampness-Heat due to Spleen Deficiency, or Liver-Stomach Disharmony (36,40 and 25, respectively) were included and their saliva before and after acid stimulation were collected 9:00 to 11:00 in the morning; MG patients with Spleen-Qi Deficiency, Dampness-Heat due to Spleen Deficiency, or Deficiency of Spleen and Kidney (32,23 and 5, respectively) were included and their saliva before and after acid stimulation were collected when waking up within 30 minutes in the morning.3. Young and middle-aged healthy volunteers were recruited and screened. Their saliva before and after acid stimulation were collected when waking up within 30 minutes and 9:00 to 11:00 in the morning (having breakfast after 1 h).4. The subjects’sAA activity, salivary flow rate, pH value, total protein concentration, Ca2+ and Cl- concentration were detected and the detection methods were Bernfeld method, weight method, pH tester, BCA Protein Assay kit, QuantiChromTM Calcium and Chloride Assay kit, respectively.5. The changes of patients’sAA activity and its related indexes were compared with the healthy control group. Multi index parameters of sAA activity and its related indexes evaluated its clinical detection rate.Results1. Optimization of saliva sample collection method:The salivary flow rate ratio, sAA activity ratio and unit time total sAA activity of EP tube collecting emulated with natural flow and rotating mouth swab slightly increased after acid stimulation, reflecting the health of normal stress to acid. The detection rate of the increased indexes was closer between the two methods. Among them, rotating mouth swab slightly had the advantage of objective and paralleled to collect sufficient amount of saliva. However, the results of chewing mouth swab were quite different with the first two methods. The detection rate of each increased index ratio in the chewing mouth swab was significantly lower than the first two, and did not accurately reflect the status of sAA activity increased in healthy people after acid stimulation. So rotating mouth swab slightly as the collection saliva method is reliable for follow-up study.2. Detection of spleen deficiency patients’and healthy subjects’saliva indexes(1) Comparison between CSG patients and healthy subjects: ① After acid stimulation, sAA activity and total activity, salivary flow rate and pH value of healthy subjects increased, reflecting the healthy subjects’normal stress ability to acid stimulation, and the salivary total protein concentration decreased which may because of increased salivary flow rate after acid stimulation. ② sAA activity of CSG and its Spleen-Qi Deficiency, Dampness-Heat due to Spleen Deficiency patients decreased after acid stimulation and sAA activity ratio was lower than healthy subjects, indicating that salivary gland stress ability of CSG and its patients with spleen deficiency to acid decreased which again replicated previous findings. sAA activity and total activity ratios of CSG patients with Liver-Stomach were higher than the patients with Spleen-Qi Deficiency and Dampness-Heat due to Spleen Deficiency, and were not significantly different with healthy subjects. Salivary flow rate, pH value, total protein concentration, Ca2+ and Cl-concentration in different syndrome types were no significant differences, indicating that sAA activity changes were the relative specificity indications for patients with spleen deficiency. ③ Before and after acid stimulation, salivary flow rate of CSG and its three syndrome types patients were lower than healthy subjects, the total protein concentration, Ca2+ and Cl-concentrations were higher than healthy subjects, indicating that patients’ salivary components and concentration had changed relative to healthy subjects and suggesting that it may be related to the dysfunction of autonomic nervous system of patients.④ The number of cases (16 cases) of salivary pH value reduced after acid stimulation in patients with Spleen-Qi Deficiency was higher than that of healthy subjects (2 cases), indicating that the saliva buffer capacity of patients with Spleen-Qi Deficiency had reduced.(2) Comparison between MG patients and healthy subjects: ① In MG and its Spleen-Qi Deficiency patients, sAA activity after acid stimulation decreased and the ratios of sAA activity and total activity ratio were lower than healthy subjects, indicating that salivary gland stress ability of MG and its patients with Spleen-Qi Deficiency to acid decreased which again replicated previous findings. The sAA activity changes of MG patients with Spleen-Qi Deficiency were similar to CSG patients with Spleen-Qi Deficiency, indicating that sAA activity changes were a common phenomenon in patients with Spleen-Qi Deficiency syndrome. Before acid stimulation, the sAA activity of MG and its patients with Spleen-Qi Deficiency and Dampness-Heat due to Spleen Deficiency was higher than healthy subjects, suggesting that in the basal state sympathetic nerve of MG patients with Spleen-Qi Deficiency was hyperactive. ② Before and after acid stimulation, the salivary flow rate of MG and its patients with Spleen-Qi Deficiency were less than healthy subjects, the total protein concentration, Ca2+ and Cl- concentration were higher than healthy subjects, indicating that MG patients’salivary components and concentration had changed relative to healthy subjects and again suggesting that it may be related to the dysfunction of autonomic nervous system of patients. ③ The number of cases (17 cases) of salivary pH value reduced after acid stimulation in MG patients with Spleen-Qi Deficiency was higher than that of healthy subjects (6 cases) and the ratio of pH value was lower than healthy subjects, indicating that the saliva buffer capacity of patients with Spleen-Qi Deficiency had reduced. All of these were similar to the CSG patients with Spleen-Qi Deficiency, while the MG patients with Spleen-Qi Deficiency was more obvious higher that may due to relatively more serious of MG pathological changes. ④ There were no significant differences in each index between Spleen-Qi Deficiency and Dampness-Heat due to Spleen Deficiency syndrome, suggesting that deficiency-weakness of spleen-qi was the basic pathogenesis to MG patients, so syndromes in the index showed no significant difference. ⑤ sAA activity ratio of CSG and MG patients with Spleen-Qi Deficiency syndrome was lower than healthy subjects, which again confirmed the decreased sAA activity ratio may be relative common performance for Spleen-Qi Deficiency patients. The results showed that salivary secretion of the two kinds of diseases with Spleen-Qi Deficiency syndrome patients may have similar pathophysiological changes.GonclusionCollection saliva sample by rotating mouth swab slightly is simple and reliable and can be used for further researchers to choose. According to the traditional Chinese medicine theory of "spleen controlling saliva", this study has confirmed that the CSG and MG patients with Spleen-Qi Deficiency syndrome lies not only in the single index of the abnormal sAA activity, is also reflected in these indicators of sAA total activity, salivary flow rate, pH value, total protein concentration, Ca2+ and Cl- concentration, which provide reference for enriching the connotation of "spleen controlling saliva". The patients with Spleen-Qi Deficiency and Dampness-Heat due to Spleen Deficiency may have pathological manifestations of autonomic nervous system disorders and salivary glands stress ability decreased to acid. In addition to the sAA activity ratio, the change of salivary pH value ratio may also have a certain reference value for Spleen-Qi Deficiency syndrome. Detection rates of Spleen-Qi Deficiency syndrome have increased about 11.00% by combination of sAA activity, salivary flow rate and pH value compared to previous single sAA activity, but due to the limited number of cases, the result of multi index combination remains to be further verified. |
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