Effect of Modified Gegen Qinliantang on FXR/SHP/PPARα Signaling Pathway in Type 2 Diabetic db/db Mice / 中国实验方剂学杂志

ObjectiveTo observe the effect of modified Gegen Qinliantang on the expression levels of proteins related to the farnesoid X receptor/small heterodimer partner/peroxisome proliferator-activated receptor α （FXR/SHP/PPARα） signaling pathway in the liver tissue of db/db model mice with type 2 diabetes mellitus （T2DM） and explore the underlying mechanism of action of modified Gegen Qinliantang. MethodThirty db/db mice were randomly divided into model group， metformin group （0.2 g·kg-1）， and high-， medium-， and low-dose modified Gegen Qinliantang groups （31.9， 19.1， 6.4 g·kg-1）， with 6 mice in each group. An additional six m/m mice were assigned to the blank group. Respective drugs were administered via oral gavage for 12 weeks. Mouse body weight， fasting blood glucose （FBG）， total cholesterol （TC）， triglyceride （TG）， high-density lipoprotein cholesterol （HDL-C）， and low-density lipoprotein cholesterol （LDL-C） levels were measured. Oil red O staining was used to observe hepatic lipid accumulation and periodic acid-schiff （PAS） staining was used to assess hepatic glycogen deposition. Ammonium ferric sulfate staining was used to observe cholesterol deposition in intestinal tissues. Western blot was employed to detect the expression of FXR， cholesterol 7α-hydroxylase （CYP7A1）， SHP， and PPARα proteins in liver tissues， and enzyme-linked immunosorbent assay （ELISA） was used to measure serum free fatty acid （FFA） levels. ResultAt the end of the treatment， compared with the blank group， the model group exhibited significant increases in mouse body weight， FBG， FFA， TC， TG， and LDL-C levels （P<0.01）， along with significant hepatic lipid droplets， reduced hepatic glycogen， noticeable cholesterol accumulation in intestinal tissues， significantly decreased expression of FXR， SHP， PPARα proteins， and significantly increased expression of CYP7A1 protein in liver tissues （P<0.01）. Compared with the model group， the metformin group and the high- and medium-dose modified Gegen Qinliantang groups demonstrated significant reductions in mouse body weight， FBG， FFA， TC， TG， LDL-C levels （P<0.05， P<0.01）， significant increases in HDL-C levels （P<0.05， P<0.01）， decreased hepatic lipid accumulation， increased hepatic glycogen， reduced intestinal cholesterol accumulation， significantly increased expression of FXR， SHP， PPARα proteins， and significantly decreased expression of CYP7A1 protein in liver tissues （P<0.01）. ConclusionModified Gegen Qinliantang may regulate the FXR/SHP/PPARα signaling pathway to suppress FFA levels and improve lipid metabolism in T2DM mice.

Research progress of 5-HT / 中国药理学通报

The serotonin 2A receptor(5-HT

Dihydromyricetin reverses Herceptin resistance by up-regulating miR-98-5p and inhibiting IGF1R/HER2 dimer formation in SKBR3 cells / 南方医科大学学报

OBJECTIVE@#To explore the effect of dihydromyricetin on the expression of miR-98-5p and its mechanism in the development of Herceptin resistance in SKBR3 cells.@*METHODS@#The expression of IGF2 and miR-98-5p and their interaction relationship were analyzed by bioinformatics analysis through TargetScan online databases. SKBR3 cells and drug-resistant SKBR3-R cells were cultured in cell experiments. Xenograft tumor mice were constructed by SKBR3 and SKBR3-R cells. Proteins were detected by western blotting and immunohistochemistry. Transfected cells were constructed by shRNA lentivirus vectors. RT-QPCR was used to detect RNA. Cell proliferation was detected by MTS method. Cell jnvasion was detected by Transwell assay. Luciferase reporting assays were used to verify RNA interactions. IGF-1R/HER2 heterodimer was determined by immunocoprecipitation.@*RESULTS@#The expression of IGF2, p-IGF1R, p-Akt and p-S6K in SKBR3-R cells were significantly higher than those in SKBR3 cells, while the expression of PTEN protein was lower in SKBR3-R cells (P < 0.05). IGF1R/HER2 heterodimer in SKBR3-R cells was significantly increased (P < 0.01).The expression of IGF2 and invasion ability were significantly reduced while transfected with miR-98-5p in SKBR3-R cells (P < 0.05), but the IGF2 mRNA were no difference in both cells (P > 0.05). The expression of miR-98-5p was up-regulated and IGF2 was decreased in drug-resistant xenograft tumor mice after feeding with dihydromyricetin, and the tumor became more sensitivity to Herceptin (P < 0.05).@*CONCLUSION@#Dihydromyricetin could induce the expression of miR-98-5p, which binds to IGF2 mRNA to reduce IGF2 expression, inhibit the IGF-1R/HER2 formation, thereby reversing cell resistance to Herceptin in SKBR3-R cells.

Role of μ-δ heterodimer in down-regulation of expression of excitatory amino acid transporter 3 in hippocampi caused by reinstatement of morphine-induced conditioned place preference in rats / 中华麻醉学杂志

Objective To evaluate the role of μ-δ heterodimer in down-regulation of the expression of excitatory amino acid transporter 3 (EAAT3) in hippocampi caused by reinstatement of morphine-induced conditioned place preference (CPP) in rats.Methods Thirty-two healthy clean-grade male Sprague-Dawley rats,weighing 200-240 g,were assigned into 4 groups (n =8 each) using a random number table method:control group (group C),extinction group (group E),reinstatement group (group R) and reinstatement plus interference plasmid group (group RI).The model of morphine-induced CPP was established,and extinction of CPP was gradually induced by stopping administration.A small dose of morphine 5 mg/kg was intraperitoneally injected again to induce CPP reinstatement,and dwell time around the medicine box was recorded.μ-δ heterodimer interference plasmid 5 μl was injected into the lateral cerebral ventricle after successful establishment of CPP model in group RI.The content of glutamate (Glu) in hippocampi was measured using high-performance liquid chromatography.The EAAT3 expression in hippocampal CA1 and CA3 regions was detected using Western blot.Results Compared with group C,no significant change was found in the dwell time around the medicine box or content of Glu in hippocampi (P＞0.05),and the expression of EAAT3 in hippocampal CA1 and CA3 regions was significantly up-regulated in group E,and the dwell time around the medicine box was significantly prolonged,the content of Glu in hippocampi was increased (P＜0.05),and no significant change was found in the expression of EAAT3 in hippocampal CA1 and CA3 regions in group R (P＞0.05).Compared with group E,the dwell time around the medicine box was significantly prolonged,the content of Glu in hippocampi was increased,and the expression of EAAT3 in hippocampal CA1 and CA3 regions was down-regulated in group R (P＜0.05).Compared with group R,the dwell time around the medicine box was significantly shortened,the content of Glu in hippocampi was decreased,and the expression of EAAT3 in hippocampal CA 1 and CA3 regions was upregulated in group RI (P＜0.05).Conclusion μ-δ heterodimer is involved in down-regulation of EAAT3 expression in the hippocampus caused by reinstatement of morphine-induced CPP in rats.

Therapeutic effects of emodin on acute choiestatic hepatitis and mechanism in rats / 中华实用儿科临床杂志

Objective To investigate the therapeutic effects of emodin on acute cholestatic hepatitis and mec-hanism thereof. Methods Fifty Sprague - Dawley (SD)rats were randomly divided into 5 groups and were treated with emodin,ursodeoxycholic acid,dexamethasone,or normal saline respectively for 4 days. On the fifth day gastric perfusion of alpha - naphthylisothiocyanate(ANIT)was performed to establish models of choiestatic hepatitis. Four to six hours after the establishment of model the above mentioned agents were given continuously. Forty - eight hours after the model establishment blood samples were collected from abdominal aorta to examine the total bilirubin(TB),direct bilirubin (DB),alanine aminotransferase(ALT),total bile acid(TBA),aspartate aminotransferase(AST),alkaline phosphatase (ALP),gamma glutamine transferase(GGT). Specimen of liver was collected to undergo pathological examination. Real - time PCR was used to detect the mRNA expression of farnesoid X receptor(FXR),small heterodimer partner (SHP ),bile salt export pump (BSEP ),uridine diphosphate glucuronosyltransferase 2 family polypeptide B4 (UGT2B4). Results The serum levels of total bilirubin (TB),direct bilirubin (DB),alanine aminotransferase (ALT),total bile acids (TBA),aspartate aminotransferase (AST),and alkaline phosphatase (ALP)of the model group were respectively (68. 1 ± 26. 1)μmol/ L,(46. 3 ± 20. 1)μmol/ L,(483 ± 228)U/ L,(159. 1 ± 57. 9)μmol/L,(2. 0 ± 0. 5)U/ L,(996 ± 382)U/ L,(324 ± 120)U/ L. The levels of TB,DB,ALT,TBA,AST,ALP of the emodin group were respectively (15. 0 ± 8. 7)μmol/ L,(10. 8 ± 3. 9)μmol/ L,(147 ± 71)U/ L,(60. 1 ± 22. 7)μmol/ L, (295 ± 104)U/ L,(222 ± 59)U/ L,and were all significantly lower than those of the model group (all P < 0. 05). The levels of TB,DB,ALT,TBA,AST,GGT,ALP of the emodin group were all significantly lower than those of the ursode-oxycholic acid group (all P < 0. 05). The levels of TB,DB,ALT,TBA,GGT,AST were all significantly lower than those of the dexamethasone group (all P < 0. 01). The expression levels of FXR,SHP,BSEP,UGT2B4 mRNA in the emodin group (1. 087 ± 0. 285,0. 892 ± 0. 390,0. 902 ± 0. 149,1. 785 ± 0. 403)were all significantly higher than those of the model group (0. 152 ±0. 088,0. 559 ±0. 194,0. 561 ±0. 123,0. 177 ±0. 039,all P <0. 05). Conclusions By decreasing the levels of TB,DB,ALT,TBA,AST,ALP and reducing pathological changes,emodin has a protective effect on cholestatic hepatitis. It has better effects than ursodeoxycholic acid and dexamethasone. These effects may be due to promoting FXR,SHP,BSEP and UGT2B4 expression.

Effects of inducible hypothermia on expressions of PGC-1 and SHP in rat model of hemorrhagic shock / 中华急诊医学杂志

Objective To investigate the effects of inducible hypothermia on expressions of peroxisome proliferator activated receptor gamma coactivator-1 (PGC-1) and small heterodimer partner (SHP) in rat model of hemorrhagic shock.Methods SD rats were randomly (random number) divided into three groups:control (C),normothermia (N) and hypothermia (H).Exsanguination was carried out in rats by continuously drawing out venous blood (25 mL/kg) over 15 minutes to establish hemorrhagic shock model.Then,rectal temperatures of rats were maintained by body surface cooling to 32℃ in H group and by body surface warming to 38℃ in N group,respectively.After a shock period of 60 minutes,rats received the infusion of whole blood of their own and lactated Ringer's solution (1 ∶ 2) treatment for 60 min.Rats were warmed to 38℃ by body surface warming and monitored for 3 h after resuscitation.Hematocrit (Hct),base excess (BE),lactate (Lac),and glucose (Glu) were recorded before modeling and after different lengths of hemorrhagic shock period (HSP).The expressions of PGC-1 mRNA and SHP mRNA and the levels of their protein in liver were tested by real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting,respectively.Results The H group had lower lactate levels and higher BElevels than the N group [(6.3±2.1) vs.(10.4±1.5) and (-4.7±2.5) vs.(-9.0±3.2)] (P＜ 0.05).At 72 hours after modeling,there were four survivors in the N group and seven survivors in the H group (P ＜ 0.05,Log Rank).The expressions of PGC-1 mRNA and SHP mRNA increased in N group.Hypothermia resuscitation down-regulated PGC-1 mRNA expression,meanwhile,increased expression of SHP mRNA.Both Hypothermia and Normothermia resuscitation increased SHP protein levels,but decreased PGC-1 protein levels.Conclusions Inducible hypothermia ameliorated acidosis and energy metabolism imbalance through adaptive regulation in PGC-1 and SHP.

Promoted role of bone morphogenetic protein 2/7 heterodimer in the osteogenic differentiation of human adipose-derived stem cells / 北京大学学报(医学版)

Objective:To investigate the role of bone morphogenetic protein 2/7 heterodimer (BMP-2/7)in the osteogenesis of human adipose-derived stem cells (hASCs).Methods:hASCs were exposed to three different treatments in vitro:osteogenic medium with 1 50 μg/L BMP-2/7 (experimental group), osteogenic medium alone (OM group)and proliferation medium (PM group).After 1 ,4 and 7 days of osteogenic induction,the amount of cellular DNA was measured to investigate the cytotoxicity.After 7 and 1 4 days,alkaline phosphatase (ALP)staining and quantification were performed to test the activity of ALP.After 21 and 28 days,the calcification deposition was determined by Alizarin Red S (ARS)stai-ning and quantification.The expressions of the osteoblast-related genes were tested on days 1 ,4,7 and 1 4.In the in vivo study,6 nude mice were used and 4 groups were set and implanted subcutaneously into the back of nude mice:(1 )β-TCP scaffold only (scaffold control group );(2 )β-TCP scaffold with hASCs cultured by PMin vitro for 1 week (PMcontrol group);(3)β-TCP scaffold with hASCs cultured by OM in vitro for 1 week (OM control group);(4)β-TCP scaffold with hASCs cultured by OM with 1 50 μg/L BMP-2/7 in vitro for 1 week (test group).After 4 weeks of implantation,histological staining was performed to evaluate the in vivo osteogenesis of hASCs.Results:After induction for 1 day,there was no significant difference between the experimental group and the PM group on the cellular DNA con-tent (P>0.05 ).After 4 days,the cellular DNA content increased under the stimulation of BMP-2/7 (P0.05).ALP ac-tivity was higher by the induction of BMP-2/7 than in OMalone and PM(P<0.05).More mineraliza-tion deposition and more expressions of osteoblast-related genes such as Runx2,ALP,COL-1 A1 and OC were determined in the experimental group at different time points (P<0.05).HE staining showed that, in the test group and OM control group,the extracellular matrix (ECM)with eosinophilic staining were observed around hASCs,and newly-formed bone-like tissues could be found in ECM around the scaffold materials.Moreover,compared with the OM control group,more bone-like tissues could be observed in ECMwith typical structure of bone tissue in the test group.Masson’s trichrome staining showed that more expression of collagen could be observed in ECM in the test group compared with the other groups.There was small amount of expression of collagen in the OM and PM control groups.No obvious positive results were found in the scaffold group.Conclusion:BMP-2/7 heterodimer plays a significant role in the osteo-genesis of hASCs and is able to enhance the osteogenic differentiation of hASCs in vitro and in vivo.

Thyroid Hormone Regulates the mRNA Expression of Small Heterodimer Partner through Liver Receptor Homolog-1

BACKGROUND: Expression of hepatic cholesterol 7alpha-hydroxylase (CYP7A1) is negatively regulated by orphan nuclear receptor small heterodimer partner (SHP). In this study, we aimed to find whether thyroid hormone regulates SHP expression by modulating the transcriptional activities of liver receptor homolog-1 (LRH-1). METHODS: We injected thyroid hormone (triiodothyronine, T3) to C57BL/6J wild type. RNA was isolated from mouse liver and used for microarray analysis and quantitative real-time polymerase chain reaction (PCR). Human hepatoma cell and primary hepatocytes from mouse liver were used to confirm the effect of T3 in vitro. Promoter assay and electrophoretic mobility-shift assay (EMSA) were also performed using human hepatoma cell line RESULTS: Initial microarray results indicated that SHP expression is markedly decreased in livers of T3 treated mice. We confirmed that T3 repressed SHP expression in the liver of mice as well as in mouse primary hepatocytes and human hepatoma cells by real-time PCR analysis. LRH-1 increased the promoter activity of SHP; however, this increased activity was markedly decreased after thyroid hormone receptor beta/retinoid X receptor alpha/T3 administration. EMSA revealed that T3 inhibits specific LRH-1 DNA binding. CONCLUSION: We found that thyroid hormone regulates the expression of SHP mRNA through interference with the transcription factor, LRH-1.

Sodium nitroprusside and SOD decrease the expression of DNA-PKcs and Ku70/80 of rats cardiomyocytes / 基础医学与临床

Objective To determine the effects of Sodium Nitroprusside ( SNP) , superoxide dismutase ( SOD) and catalase ( CAT ) on the expression of catalytic subunit of the DNA-dependent protein kinase ( DNA-PKcs ) and Ku70/80 heterodimer in cardiomycyte H9C2, as well as their expression in the myocardial tissues of SD rats . Methods H9C2 cells were co-cultured with SNP at concentreations of 10, 20 and 40 mmol/L for 6 hours, SD rats were injected with normal saline , SNP, SNP+SOD, SNP+CAT or SNP+SOD+CAT.Western blot and immuno-histochemistry assay were used to examine DNA-PKcs and Ku70/80 protein expression respectively .Results The expression of DNA-PKcs and Ku70/80 increased in H9C2 cells co-cultured with SNP when compared with control group, but they were be decreased when treated with SOD or/and CAT.The expression of DNA-Pkcs and Ku70/80 in myocardial tissues of experimental groups were higher than the control .Conclusions Radical scavengers may play a role as a protective effect for sodium nitroprusside related injury in cardiac myocytes .

Effect of Resveratrol, a SIRT1 Activator, on the Interactions of the CLOCK/BMAL1 Complex

BACKGROUND: In mammals, the CLOCK/BMAL1 heterodimer is a key transcription factor complex that drives the cyclic expression of clock-controlled genes involved in various physiological functions and behavioral consequences. Recently, a growing number of studies have reported a molecular link between the circadian clock and metabolism. In the present study, we explored the regulatory effects of SIRTUIN1 (SIRT1), an NAD+-dependent deacetylase, on CLOCK/BMAL1-mediated clock gene expression. METHODS: To investigate the interaction between SIRT1 and CLOCK/BMAL1, we conducted bimolecular fluorescence complementation (BiFC) analyses supplemented with immunocytochemistry assays. BiFC experiments employing deletion-specific mutants of BMAL1 were used to elucidate the specific domains that are necessary for the SIRT1-BMAL1 interaction. Additionally, luciferase reporter assays were used to delineate the effects of SIRT1 on circadian gene expression. RESULTS: BiFC analysis revealed that SIRT1 interacted with both CLOCK and BMAL1 in most cell nuclei. As revealed by BiFC assays using various BMAL1 deletion mutants, the PAS-B domain of BMAL1 was essential for interaction with SIRT1. Activation of SIRT1 with resveratrol did not exert any significant change on the interaction with the CLOCK/BMAL1 complex. However, promoter analysis using Per1-Luc and Ebox-Luc reporters showed that SIRT1 significantly downregulated both promoter activities. This inhibitory effect was intensified by treatment with resveratrol, indicating a role for SIRT1 and its activator in CLOCK/BMAL1-mediated transcription of clock genes. CONCLUSION: These results suggest that SIRT1 may form a regulatory complex with CLOCK/BMAL1 that represses clock gene expression, probably via deacetylase activity.

Changes of cholesterol 7alpha-hydroxylase, farnesoid X receptor, and small heterodimer partner expression in liver tissues of rats with obstructive cholestasis / 第二军医大学学报

Objective: To investigate the changes of cholesterol 7alpha- hydroxylase(CYP7A1) ,farnesoid X receptor(FXR), and small heterodimer partner (SHP) expression in liver tissues of bile duct ligated (BDL) rats,and to analyze the relationship between their expression. Methods: Obstructive cholestasis rat model was induced by bile duct ligation,and rats were sacrificed on day 1,3,and 14 after operation. The hepatic tissues were collected and the total mRNA was extracted. The CYP7A1 mRNA expression was determined by real-time RT-PCR. The protein expression of FXR and SHP in the nuclei was determined by Western blotting analysis. Results: Compared with sham-operated group, experimental obstructive cholestasis group had significantly enhanced CYP7A1 mRNA expression and decreased FXR,SHP expression(P<0. 001,P<0. 05). Conclusion: The up-regulation of CYP7A1 may be associated with the down-regulation of FXR and SHP.

Roles of Orphan Nuclear Receptor Small Heterodimer Partner in Bone Development: Microcomputed Tomographic Analysis of Bone Microarchitecture in SHP Knockout Mice / 대한골다공증학회지

OBJECTIVES: Orphan nuclear receptor small heterodimer partner (SHP) is involved in osteoblastic differentiation. This study was undertaken to demonstrate a role of SHP in in vivo bone development using microcomputed tomographic (microCT) analysis of SHP knockout (KO) mice. MATERIAL & METHODS: Tibia bones were harvested from 1-, 4-, 8- and 20-week-old wild type (WT) and SHP KO mice. The microarchitecture of tibial bone was analyzed using a microCT (Skyscan 1172; Skyscan, Kontich, Belgium). Samples were scanned at a resolution of 17 microm (isotropic). The X-ray was operated with 50 kV, 200 microA of energy, 1.2 sec of exposure time, and a 0.5 mm thick aluminum filter. Projections were acquired over an angular range of 180degrees. For quantification of the bone mineral density (BMD), the microCT was calibrated using 2 standard phantoms with densities of 0.25 and 0.75 g/cm3. The image slices were reconstructed and analyzed using CT analyzer software (CTan, Skyscan). RESULTS: The CT values of tibial trabecular bone were significantly decreased in SHP KO compared to WT at 20-week-old mice determined by microCT; (bone volume / tissue volume [BV/TV, 40%], BMD [80%], and trabecular number [Tb.N, 50%]). However, the CT values were not significantly different between WT and SHP KO in cortical bone. Furthermore, the qualitative indices of trabecular bone such as the structure model index (SMI) and the polar moment inertia (PMI) did not differ between WT and SHP KO mice. CONCLUSION: These microCT results supports that SHP may act as a positive regulator of trabecular bone formation.

Association between small heterodimer partner gene and high birth weight infants / 中华围产医学杂志

Objective To evaluate the relationship of small heterodimer partner(SHP) gene and birth weight in China. Methods A cohort study of 191 normal pregnant women was conducted. Both maternal and cord blood samples were collected. PCR-RFLP was used to detect the polymorphism of SNP-rs7504 of SHP. Results (1) The frequency of both neonatal and maternal C allele and (TC+CC) genotype increased significantly with birth weight (P=0.004, OR=3.168; P=0.005, OR=3.315; P=0.013, OR=2.495; P=0.013, OR=2.495). (2) The babies were heavier if they were C allele carrier. The average increase of birth weight was 246.3 g comparing the neonates with TC+CC genotype with those with TT genotype [(3658.7?400.94)g vs (3412.4?444.4)g, P=0.005]. The average birth weight of those maternal C allele carriers was 210.3 g heavier than those non-C allele carriers[(3628.9?405.5) g vs (3418.6?449.0 g]. (3) The fetal C allele was associated with maternal weight in pregnancy, prepregnant BMI, paternal height and weight. Women with C allele were heavier and had higher BMI without statistical significance comparing with those non-C allele carriers. Neither neonatal nor maternal SHP gene was associated with blood glucose and insulin level. (4) Multiple factors analysis showed that birth weight was related to maternal height, weight gain during pregnancy, prepregnant BMI, maternal and cord blood insulin level. After adjustment, the neonatal birth weight remained significantly correlated with cord blood SHP (P=0.0354), but not with maternal SHP gene (P=0.0711). Conclusions SHP gene is associated with newborns birth weight and may affect fetal growth.

The Analysis of SHP (Small Heterodimer Partner) Gene Mutation in Infertile Patients with Polycystic Ovary Syndrome (PCOS) in Korea / 대한불임학회지

OBJECTIVE: We inversigated Small Heterodimer Partner (SHP) gene mutation in Korean Polycystic Ovarian Syndrome (PCOS) patients. SHP protein regulates the activity of nuclear receptors which regulate the cellular development and differentiation. Recently, the mutation of SHP gene was found in the obesity and diabetes patients in Japanese group, and suggested that its mutation may involved in pathogenic mechanism of PCOS. METHODS: This study was performed in 20 PCOS patients and 20 normal women. The DNAs were extracted from the peripheral bloods, and amplified at each exon (1 and 2) of SHP gene by PCR method. Subsequently, each PCR product was digested with the restriction enzyme indicated below for studying restriction fragment length polymorphism (RFLP). After enzyme digestion, the results of RFLP were compared PCOS patients with control women to find any sequence variation. RESULTS: We examined 9 regions of exon 1 with Msp I, Pvu II, Dde I and 3 regions of exon 2 with Pst I, Dde I. There is no heterozygous or homozygous mutation in patients and control women at these restriction sites. CONCLUSION: The genetic analysis at our restriction sites in the SHP gene did not show any genetic variation in Korean PCOS patients. Our PCR-RFLP analysis was not covered the entire SHP gene (68 bp/ 1,006 bp), we need to further analysis of the entire SHP gene.