Nvestigation On The Changes And Mechanisms Of Salivary Alpha-amylase Activity In Pi Deficiency Syndrom Based On N-glycosylation

BackgroundTraditional Chinese medicine think "Pi controlling saliva", salivary amylase is one of the important protein in saliva. there are 40-50% salivary alpha amylase (sAA) in salivary protein. Many scholars studied the nature of Pi deficiency by useing sAA activity. Professor jianhua wang of Guangzhou University of Traditional Chinese Medicine found for the first time that patients with Pi deficiency syndrom (PDS) showed a decline of sAA activity ratio before and after citric stimulation. The study was repeated by a number of domestic medical units, sAA activity ratio become an example syndrome research. But it has some shortcomings, such as the detection rate is about 60% in different diseases, specificity is not high, interference and regulation factors lack of in-depth research. Cann’t reflect the nature of Pi deficiency. So it is necessary to further research the regulation factors of sAA activity change.ObjectivePreliminary discussion on the relevance of pi deficiency syndrome clinical symptoms and sAA activity ratio; By detecting the sAA activity, degree of N glycosylation and N sugar chain composition of MG and CSG, compared with healthy volunteers. Explore mechanism of sAA activity altered, to provide the scientific evidence "Pi controlling saliva" at molecular level.ContentThus this study sets out to investigate the mechanisms of sAA activiy changes in PDS of MG and CSG based on sAA N-glycosylated, and comparison with the change of sAA N-glycosylated in patients with Pi deficiency-heat (PDH) and disharmony syndrome between liver and stomach (DSLT).Methods1. Research objects61 case of Myasthenia gravis (MG) including 33 PDS,23 PDH,5 Spleen and kidney deficiency (SKD); 101 case of chronic non-atrophic gastritis (CSG) including 36 PDS,40 PDH,25 DSLT which conform with inclusionc criteria were included.36 healthy people were classified as the normal group.2. Research methods2.1 Designed performance database of myasthenia gravis and chronic non-atrophic gastritis patients with the syndrome clinical information, analysis the correlation between symptoms and sAA acticity.2.2 Saliva collectionCollecting saliva by useing saliva collection tube. Salivary from MG patients were collected at 6:30-7:30am, CSG patients were collected 9:00-10:30am. Healthy volunteers were collected at the two point. ALL objects hold sitting position, removed the sterile cotton adsorption column from collection tube, rotation 2min in the mouth, spit back cotton column into collection tube, and than put 10% citric acid soaked paper (1×1cm) on the tip of the tongue keeping 30 second, collected salivary as before. Each collection tube after collection are accurately weighed and recorded,. The collection tube was centrifuged at 3000g to 15min, supernatant was retained and stored in -80℃.2.3 Measure sAA activitysAA activity was detected using the method of Bernfeld, and calculated the sAA activity ratio(sAA active after acid stimulated/sAA active before acid stimulated)2.4 Analyze the relationship between clinical symptoms and sAA activity ratioPreliminary study the relationship between clinical symptoms and sAA activity ratio with linear stepwise regression analysis2.5 study of healthy and difference syndromes patients’sAA N-glycosylation changeUsing western blot, Observed sAA glycoprotein expression of difference syndromes, analyzing o sAA N-glycosylated protein of each syndromes patients.2.6 Analysis of different types of sAA N-linked glycanDetect sAA activity before and after binding with conA and WGA. Calculate the binding rate by Binding rate=[(sAA activity before binding with lectin-sAA activity after binding with lectin)/sAA activity before binding with lectin]×100%. And then detect. different types of sAA N-linked glycan by Lectin affinity chromatography2.7 Screening plant lectinsCustomizing lectin microarray to screen plant lectin which have clear distinction with healthy people, MG with PDS and PDH.Results1. Subjects informationThe age of 101 patients with chronic non-atrophic gastritis (44 men and 57 women) ranges from 21 to 60, the average age is 40.1±12.1, including 36 PDS, 40 PDH,25 DSLT;The age of 61 patients with MG (24 men and 37 women) ranges from 20 to 65, the average age is 48.1±14.7, including 33 PDS,23 PDH and 5SKD.2. Clinical symptoms of difference syndromesThe main symptoms of PDS in chronic non-atrophic gastritis is pale tongue, pulse float and weak, lip dim, hiccups, stomach bilges after eating, tired, belch hyperactive bowel sounds, abdominal pain, loose stool; The main symptoms of PDH is red tongue, pulse sinking or quick pulse, lip dim, gasterectasis, stomach bilges after eating, tired, belching, hiccups; The main symptoms of DSLT is wiry pulse, pale tongue, abdominal distension, belch, gloomy mood.The main symptoms of PDS in MG is pulse float and weak, pale tongue, ptosis, face stiff, four limbs weakness, eye movements stiff, sweating, gloomy complexion, tired, loss of appetite; The main symptoms of PDH is pulse sinking or quick pulse, sweating, inability to chew, face stiff, four limbs weakness, gloomy complexion, tired, thirst with inclination to drink.The number of SKD cases was only 5, so the data cannot be analyzed.3. The results of sAA activityBy examining the stability and intra-day precision, The Bernfrld method is stable and reliable, and it can be used to measure sAA activity. After acid stimulation, sAA activity of healthy objects significantly increased (P<0.05), patients with PDS and PDH of CSG showed a decline of sAA activity (P<0.05), patients with DSLT showed a increase of sAA activity (P<0.05). Compared with the healthy group, each group sAA activity before and after acid stimulation do not have difference (P>0.05). patients with PDS and PDH of MG showed a decline of sAA activity (P<0.05). sAA activity before acid stimulation expressed not difference between the two syndromes groups and the healthy group(P>0.05), but after acid stimulation, sAA activity were significantly lower than the healthy group(P<0.05). Compared with healthy group, sAA activity ratio in PDS and PDH of CSG and MG showed a significantly decline(P <0.05).4. Relationship between clinical symptoms and sAA activity ratioGasterectasis, abdominal pain and belch in PDS patients with CSG has negatively correlated with sAA activity ratio; Sweating, postural changes difficulty and ptosis in PDS patients with MG has negatively correlated with sAA activity ratio. The reason is autonomic dysfunction(except for specific symptoms of myasthenia gravis)5. The study of sAA N-glycosylation5.1 Study the degree of N-glycosylation of sAAsAA N-glycosylated protein expression were loss in 56.7% PDS patients in MG and 36.4% PDH patients in MG, Significantly higher than the healthy group (P<0.05), DSLT patients and healthy group have no difference in sAA N-glycosylated protein expression, suggesting N-glycosylation expression loss is one pathological factors of sAA salivary amylase activity decreased.5.2 Analysis of different types of sAA N-linked glycanThe binding ratio of sAA with conA in PDS and PDH patients significantly higher than the healthy group(P<0.05), suggesting high mannose type and hybrid type higher than healthy group; The binding ratio of sAA with WGA in PDS and PDH patients lower than the healthy group, suggesting complex-type oligosaccharide is lower than the healthy group; DSLT patients and healthy group have no difference in sAA with conA and WGA, suggesting sugar chain structure of sAA in DSLT patients do not change significantly.The results of affinity chromatography sAA with conA suggesting sAA of PDS patients in CSG and CSG high mannose type and hybrid type sugar chains is greater than complex-type oligosaccharide, affinity chromatography sAA with WGA suggesting complex-type oligosaccharide reduced in sAA of PDS patients; The results of affinity chromatography sAA with WGA suggesting sAA of PDH patients in CSG and CSG complex-type oligosaccharide composition ratio higher than the high mannose type and hybrid type sugar chain, affinity chromatography sAA with WGA suggesting complex-type oligosaccharide increased in sAA of PDH patients. Suggesting the change of sugar chain structure is the direct factors of sAA activity decreases.6. Results of screening plant lectins6.1 LectinTM microarray resultsThe LEL, STL and WGA lectin binding have ability to distinguish between healthy group, PDS and PDH patients with MG, in which WGA showed a greater difference among the three groups. So LEL, STL and WGA can be used to the next affinity chromatography.6.2 Verify the lectin microarray resultsPDH patients sAA protein expression higher than the healthy group in WG、 LEL、STL by using Lectin blot experiments, suggesting GlcNAc sugar chain contained in sAA of PDH patients and sympathetic compensatory hyperactivity, and increasing the amount of acinar cells synthesize glycoproteins.conelusionMG and CSG patients with Pi deficiency syndrome and Pi deficiency-heat showed a decline of sAA activity after citric acid stimulation and sAA activity ratio, one of main factors is incomplete N-glycosylation and changes of N-glycosylation sugar chain subtypes.